KR20000037116A - Insecticidal Protein Toxins from Photorhabdus - Google Patents

Abstract

PURPOSE: An easily administered insecticidal protein as well as the expression of toxin in a heterologous system, and a method for delivering insecticidal toxins that are functional active and effective against many orders of insects, are provided. CONSTITUTION: Native toxins are protein complexes that are produced and secreted by growing bacteria cells of the genus Photorhabdus, the proteins is produced by the species Photorhabdus luminescens. The protein complexes, with a molecular size of approximately 1,000 kDa, can be separated by SDS-PAGE gel analysis into numerous component proteins. The toxins contain no hemolysin, lipase, type C phospholipase, or nuclease activities. The toxins exhibit significant toxicity upon exposure administration to a number of insects.

Description

Insecticidal Protein Toxins from Photorhabdus}

Related Applications

This application is a US patent application filed on Feb. 28, 1996, filed February 28, 1993, filed on Feb. 28, 1993, filed on Feb. 28, 1995, filed on Feb. 28, 1995, filed on Feb. 28, 1995. Continuing application of US patent application Ser. No. 08 / 743,699, filed Nov. 6, 1999, filed Continuing Application of U.S. Patent Application No. 08 / 705,484, filed Aug. 28, 1996, filed 08 / 608,423. This application is also a continuation of US Provisional Application No. 60 / 007,255, filed Nov. 6, 1995.

Many insects are widely considered pests to homeowners, picnickers, gardeners and farmers, and those who invest in agricultural products that are often destroyed or reduced as a result of insect damage to field crops. In particular, in areas with short growing seasons, significant insect damage can mean a loss of all profits to growers and a dramatic reduction in crop harvest. If there is a shortage of certain agricultural supplies, food processors must pay higher costs, and therefore higher costs for edible plants and ultimate consumers of the products obtained from those plants.

The prevention of insect damage to crops and flowers and the elimination of insect pest pests has typically relied on strong organic pesticides and insecticides with a wide range of toxicity. These synthetic products are so harmful to the environment and to people exposed to such drugs that they have been attacked by the general public. Similarly, in a non-cultivated environment homeowners may be content only with avoiding insects without having to kill them in their home or outdoor meals.

The widespread use of chemical pesticides has increased interest in the environment and health of farmers, companies producing pesticides, government agencies, public interest groups and the general public. The development of less invasive insect management methods has been stimulated by the social concern for the environment and the development of biological tools that develop mechanisms of insect management. Biological control agents are suggested as promising substitutes for chemical pesticides.

At each stage of evolutionary development, the creatures found a means to enhance their own success and survival. The use of biological molecules as a tool of defense and attack is known throughout the animal and plant kingdoms. In addition, relatively new tools in genetic engineering have made it possible to modify biological pesticides to achieve specific solutions to specific problems.

One such agent, Bacillus thuringiensis (Bt), is widely used commercially as an effective insecticide. Indeed, insecticides of Bt bacteria are proteins with such limited toxicity and can be used in human edible crops even at harvest time. For nontarget organisms, Bt toxins are digestible nontoxic proteins.

Another known class of biological insect control agents is nematodes of a particular genus known as transfer vectors for pesticidal bacterial commensals. Nematodes, including insecticidal bacteria, attack insect larvae. The bacteria then kill the larvae. Nematodes multiply in the larva's body. Subsequently, the nematode progeny eat the carcass from the inside. The bacterial containing nematode progenitors can then attack another larva.

In the past, insecticidal nematodes of the genus Steinernema and Heterorhabditis have been used as insect control agents. Obviously, nematodes in these genera are each host to bacteria of a particular species. In the nematodes of the genus Helobaltis, the symbiotic bacterium is photoractose luminescence.

While these nematodes are effective as insect control agents, it is presently difficult, expensive and inefficient to produce, maintain and distribute insect control nematodes.

It is known in the art that it is possible to isolate insecticidal toxins from photolapus luminescence which is active only when injected into the larvae of Lepidoptera and Coleoptera insects. This prevented the effective development of the insecticidal properties of the nematode or its bacterial symbiosis. A more practical, less labor intensive, widespread method of delivery of pesticidal toxins that will retain their biological properties after delivery would be useful. It was highly desirable to find toxins having oral activity produced by the genus Photorapdus. Isolation and use of these toxins has been desired for reasons of efficacy. It was only by the applicant's invention that these toxins were isolated and characterized.

<Summary of invention>

Natural toxins are protein complexes produced and secreted by the growing bacterial cells of the genus Photorapdus, and of interest are proteins produced by the photolapus luminescence species. Protein complexes having a molecular size of about 1,000 kDa can be separated into many component proteins by SDS-PAGE gel analysis. The toxin does not have hemolysin, lipase, type C phospholipase or nuclease activity. Toxins show significant toxicity upon exposure to many insects.

The present invention provides expression of toxins in heterologous systems as well as easily administered pesticidal proteins.

The present invention also provides a method for delivering functionally active and effective insecticidal toxins against many neck insects.

The objects, advantages and features of the present invention will become apparent from the following detailed description.

The present invention relates to toxins isolated from bacteria and their use as insecticides.

1 is a diagram of a pair of cloned DNA isolates used as part of a sequence gene for toxins of the invention.

2 is a map of three plasmids used in the sequencing method.

3 is a map showing the internal-relationships of several partial DNA fragments.

4 is a diagram of homology analysis between protein sequences of TcbA _ii and TcaB _ii proteins.

FIG. 5 is a phenogram of the Photolapdus strain. FIG. The relationship of the photolapidus strain is defined by rep-PCR. The upper axis of FIG. 5 measures the% similarity of the strains based on scoring of the rep-PCR product (ie 0.0 [no similarity] to 1.0 [100% similarity]). On the right axis, numbers and letters represent the various strains tested: 14 = W-14, Hm = Hm, H9 = H9, 7 = WX-7, 1 = WX-1, 2 = WX-2, 88 = HP88 , NC-1 = NC-1, 4 = WX-4, 9 = WX-9, 8 = WX-8, 10 = WX-10, WIR = WIR, 3 = WX-3, 11 = WX-11, 5 = WX-5, 6 = WX-6, 12 = WX-12, 14 = WX-14, 15 = WX-15, Hb = Hb, B2 = B2, 48 to 52 = ATCC 43948 to ATCC 43952. The vertical line represents the correlation between the groups of strains or groups of strains at the base of the horizontal line (reading the extrapolated intersection of the vertical lines with the upper axis) (eg, strain W-14 is about 60% similar for strains H9 and Hm). Do).

6 is a diagram of a genomic map of the W-14 strain.

6A shows tca and tcb loci and main gene products.

FIG. 7 is a phenogram of Photorabdus strain as defined by rep-PCR. The upper axis in FIG. 7 shows the% similarity of the strains based on scoring of the rep-PCR product (ie 0.0 [no similarity] to 1.0 [100% similarity]). In the right axis, numbers and letters represent the various strains tested. The vertical line separating the horizontal line represents the correlation between the group of strains or strains at the base of the horizontal line (reading the extrapolated intersection of the vertical lines with the upper axis) (for example, strain indices for strains MP1 and HB Oswego). About 30% similar). The photolabose strains on the phenogram are as follows: 14 = W-14, Hm = Hm, H9 = H9, 7 = WX-7, 1 = WX-1, 2 = WX-2, 88 = HP88, NC1 = NC-1, 4 = WX-4, 9 = WX-9, 8 = WX-8, 10 = WX-10, 30 = W30, WIR = WIR, 3 = WX-3, 11 = WX-11, 5 = WX-5, 6 = WX-6, 12 = WX-12, 15 = WX-15, 14 = WX-14, Hb = Hb, B2 = B2, 48 = ATCC 43948, 49 = ATCC 43949, 50 = ATCC 43950 , 51 = ATCC 43951, 52 = ATCC 43952.

The present invention relates to the discovery of a unique class of pesticidal protein toxins from the genus Photorapdus that have oral toxicity against insects. The unique characteristic of Photorabdus is his bioluminescence. Photolabdus can be isolated from a variety of sources. One such source is a nematode, more particularly a nematode of the genus heterolapidis. Another such source is human clinical samples from wounds (see Farmer et al., 1989, J. Clin, Microbiol. 27, pp 1594-1600). These decay strains have been deposited as ATCC # 43948, 43949, 43950, 43951, and 43952 in the American Type Culture Collection (Rockville, MD) and are incorporated herein by reference. Other sources may include photolabose bacteria that produce insecticidal toxins. The environment of such sources can be terrestrial or aquatic.

The genus Photolapus is taxonomically defined as the Enterobacteriaceae family, but has atypical specific properties. For example, strains of this genus are nitrate reduction negative, produce yellow and red pigments and are bioluminescent. On the other hand, the latter property is not known within Enterobacteriaceae. Photorabdus was only recently described as a genus separated from Xenorhabdus (Boemare et al., 1993, Int. J. Syst. Bacteriol. 43, 249-255). This distinction is due to DNA-DNA hybridization studies, differences in phenotypes (eg, the presence or absence of catalase and bioluminescence (photorabdus) or absence (xenorabdus), and the family of nematode hosts (Xenorabdus: Steiner). Steinernematidae, Photorabdus: Heterorhabditidae. Comparative analysis of cytoplasmic fatty acids (Janse et al., 1990, Lett. Appl. Microbiol. 10, 131-135: Suzuki et al., 1990, J. Gen. Appl. Microbiol., 36, 393-401) was performed by Genolabdus. Support Doo's distinction.

In order to confirm that the strain collections disclosed herein consist of Photolabduus strains, the strains were characterized based on the recognized properties defining Photorabdus and distinguished them from other Enterobacteriaceae and Genorabdus species. (Farmer, 1984, Bergey's Manual of Systemic Bacteriology, Vol. 1, pp. 510-511; Akhurst and Boemere 1988, J. Gen. Microbiol. 134, pp 1835-1845; Boemare et al., 1993, Int. J. Syst. Bacteriol. 43, pp 249-255, incorporated herein by reference). The properties studied were as follows: Gram staining negative bacilli, organism size, colony pigmentation, inclusion bodies, the presence of catalase, nitrate reducing ability, bioluminescence, dye uptake, gelatin hydrolysis, growth on selective media, growth temperature, anaerobic Survival and motility under conditions. Fatty acid analysis was used to confirm that all of the strains herein belonged to one photolapidus.

Currently, the bacterial photorapdus genus consists of one defined species, photolapus luminescence (ATCC type strain # 29999, Poinar et al., 1977, Nematologica 23, 97-102). Various related strains are described in the literature (see, eg, Akhurst et al., 1988, J. Gen. Microbiol., 134, 1835-1845; Boemare et al., 1993, Int. J. Syst. Bacteriol. 43 pp. 249- 255; Putz et al., 1990, Appl. Environ.Microbiol., 56, 181-186). Many of the photolabose strains have been characterized herein. Such strains are listed in Table 18 in the Examples. Since only one species (luminescence) has been defined in the genus Photolapus at present, the properties of the luminescence species have been used herein to characterize the strain. As can be seen in Figure 5, these strains are very diverse. In the future, it can be predicted that not only some properties of the luminescence species, but also other photolabduus species may have some other properties not currently defined by the properties of photolabduus luminescence. However, the scope of the present invention herein is any photolabduus species or strain that produces a protein having functional activity as an insect control agent without considering other properties and characterizations.

In addition, as demonstrated herein, bacteria of the genus Photorapdus produce proteins with the functional activity defined herein. Of particular interest is the protein produced by the photolapid luminescence species. The invention should not be limited to the strains disclosed herein. These strains demonstrate for the first time that the proteins produced by the various isolates of photolabose are toxic upon exposure to insects. Thus, the strains listed herein and any mutants thereof, as well as any strain or species of the genus Photorapdus having the functional activity described herein, are included in the invention described herein.

Some terms used herein have the following specific meanings:

"Agonistic activity" as used herein means that a protein toxin may act as an orally active insect control agent, have a toxic effect, interfere with or disrupt food intake, and may cause or cause death of an insect. It means you can't. When an insect comes into contact with an effective amount of toxin delivered by expression of the transgenic plant, formulated protein composition (s), sprayable protein composition (s), bait matrix or other delivery system, typically it causes death of the insect or This prevents insects from ingesting sources that make toxins available to insects.

The use of the term "genetic material" herein is meant to include all genes, nucleic acids, DNA and RNA.

The term “homolog” herein refers to an amino acid sequence that has been identified as having homology to the control W-14 toxin polypeptide amino acid sequence.

The term “homology” herein refers to a control W-14 toxin polypeptide amino acid sequence as measured using the GAP algorithm using the B10sum 62 protein scoring matrix (Wisconsin Package Version 9.0, Genetic Computer Group (GCG), Madison, Wisconsin). An amino acid sequence having a similarity index of at least 33% and / or at least 26% of an identity.

The term “identity” herein refers to an amino acid sequence containing the same residue at a given position after alignment of the control W-14 toxin polypeptide amino acid sequence by the GAP algorithm.

Protein toxins discussed herein are typically referred to as "pesticides." Pesticides herein means that the protein toxin has a "functional activity" as described further herein and is used as an insect control agent.

The use of the term "oligonucleotide" refers to a macromolecule consisting of short chains of nucleotides of RNA or DNA. Such length may be at least one nucleotide, but is typically in the range of about 10 to about 12 nucleotides. Measurement of the length of an oligonucleotide is well known within the skill of the art and is not limited herein. Thus, oligonucleotides may be less than 10 or more than 12.

The term “photolapus toxin” refers to any protein produced by a photolabose microbial strain that has agonistic activity against insects, wherein photolapus toxin is formulated in a sprayable composition or expressed by a transgenic plant It may be formulated as a bait matrix, delivered via baculovirus, or delivered by any other available host or delivery system.

The use of the term "toxic" or "toxic" herein means that the toxin produced by Photorabdus has "functional activity" as defined herein.

The term "truncated peptide" herein is meant to include all peptides that are fragments of the peptide that have been observed to have functional activity.

As used herein, the term “substantial sequence homology” is sufficiently similar to a DNA fragment having a nucleotide sequence sufficiently similar to another DNA fragment, or another polypeptide to exhibit similar biochemical properties to produce a moiety with similar biochemical properties. A polypeptide having an amino acid sequence is meant.

Fermentation broth from selected strains reported in Table 20 was used to determine the breadth of pesticidal toxin production by the genus Photorapdus and the pesticidal spectrum of these toxins, and to provide a source material for purifying the toxin complex. The strains characterized herein have been shown to have oral toxicity against various insect necks. Such insect trees include, but are not limited to, Coleoptera, Homoptera, Lepidoptera, Diptera, Acarina, Hymenoptera, and Dictoptera .

As with other bacterial toxins, there are many related toxins that differ slightly in sequence due to the mutation rate of the bacteria in the population. Toxins of interest herein are those that produce protein complexes that are toxic to various insects upon exposure as described herein. Preferably, the toxin is active against Lepidoptera, Coleoptera, Simulaceae, Fly, Logs, Scrub and Mite. The present invention is intended to include protein toxins homologous to the protein toxins produced by the strains herein and any of the variant strains thereof, as well as any protein toxins produced by Photorapdus. These homologous proteins may differ in sequence but are not different in function from the toxins described herein. Homologous toxin is meant to include a protein complex of 300 kDa to 2,000 kDa, consisting of at least two subunits, the subunit being a peptide that is the same or not the same as the other subunits. Various protein subunits have been identified and taught in the Examples herein. Typically, protein subunits range from about 18 kDa to about 230 kDa; About 160 kDa to about 230 kDa; About 100 kDa to about 160 kDa; About 80 kDa to about 100 kDa; And about 50 kDa to about 80 kDa.

As mentioned above, some photolabose strains can be isolated from nematodes. Some nematodes, the long cylindrical parasites of Nematoda, have evolved the ability to use insect larvae as an advantageous growth environment. Insect larvae provide a food source for growing nematodes and an environment for reproduction. As long as certain nematodes invade the larvae, the dramatic effect is the death of the larvae. The death of the larvae is caused by the presence of bacteria in certain nematodes that produce insecticidal toxins that stop larval growth and inhibit feeding activity.

Interestingly, each genus of insect parasitic nematodes appears to host a particular species of bacteria that has been uniquely adapted for symbiotic growth with nematodes. During the start of this study, the genus Bacterium genodudus was renamed Genorabdus and Photorabdus. Bacteria in the genus Photorabdus are characterized as symbiosis of heterorabditus nematodes, and genorabdus are symbiosis of Stainnerema species. While such changes in names are reflected herein, changes in names should not in any way alter the scope of the invention described herein.

Peptides and genes disclosed herein are described in the Journal of Bacteriology "Instructions to Authors" p. i-xii (Jan. 1996), according to the recently published guidelines. The following peptides and genes were isolated from photolapidus strain W-14.

The sequences in this list are sorted by genomic region. More specifically, Photolab durum luminescens bacteria (W-14) have four or more distinct genomic regions, tca, tcb, tcc and tcd. As can be seen in Table 1, peptide products are prepared in these distinct genomic regions. Moreover, as described in Examples, specifically Examples 15 and 21, each gene product produced from three genomic regions exhibits insect activity in combination. In addition, there is marked homology between these four genomic regions.

As explained in detail in the Examples, the tcbA gene is divided into two possible biologically active protein fragments (TcbA and TcbA _{ii / iii} ). Expressed in collie. In addition, the TcdA gene. Expressed in collie. As described in Example 16, when a natural unprocessed TcbA toxin is treated with the endogenous metalloprotease or the digestive tract content of an insect containing a protease, the TcbA protein toxin is processed into smaller subunits that are larger in size than the native peptide. Decreases, and increases the activity of the southern corn rootworm. The smaller toxin peptide will remain as part of the toxin complex. In some situations it may be desirable to increase the activation of toxins using proteolytic processing or cleavage peptides. Thus, it may be more desirable to use truncated peptides in some applications, ie in commercial transgenic plant applications.

In addition to the W-14 strain, there are other species in the genus Photorapdus with differential action activity (see specifically Tables 20 and 36). Although there is differential activity, in some cases amino acid sequences have substantial sequence homology. Moreover, molecular probes indicate that some genes contained in the strains are homologous to genes contained in the W-14 strain. In fact, all of the strains described herein have one or more homologues for the W-14 toxin gene. The antibody data in Example 26 and the N-terminal sequence data of Example 25 further support the conclusion that there is homology and identity (based on amino acid sequence) between the protein toxins produced by these strains. At the molecular level, the W-14 gene probe indicates that these homologues or W-14 genes (Tables 37, 38 and 39) exist throughout the genus Photorapdus. Moreover, it is also possible for new toxin genes to be present in other strains that are not homologous to W-14 but retain overall protein properties (see, eg, Examples 14 and 25).

Although homology or identity exists between the toxin genes produced by the Photoractus strains, the strains themselves vary. When using the polymerase chain polymerization technique described in Example 22, most of the strains described herein are distinguished. For example, as can be seen in Figure 5, the relative% similarity in some strains, such as HP88 and NC-1, is about 0.8, indicating that the strains are similar, with HP88 and Hb being 0.1, which is substantially Indicates a difference. Thus, although the toxin genes of insects or the gene products produced by these strains are the same or similar, the strains are of different kinds.

It is clear from the examples and the data further disclosed herein that a novel and unique group of pesticidal protein toxins has been found. It is described herein that these toxins are widely present in bacterial strains of the genus Photorapdus. It also shows that these toxin genes are widely present in the Enterobacteraceae family. Antibodies prepared as described in Example 21 or gene probes prepared as described in Example 25 can be used to further screen bacterial strains in the Enterobacteraceae family that produce homologous toxins with functional activity. . It may also be used to identify the presence of specific primers that may facilitate the identification of new genes in the genus Photorapus or in the Enterobacteraceae family.

As described above, bacteria of the genus Photoractus or Enterobacteraeaceae can be used for rapid screening for homologous toxin products as described in Example 26. Those skilled in the art are very familiar with using antibodies as assay or screening techniques (see US Pat. No. 5,430,137, incorporated herein by reference). Moreover, the induction of antibodies by 6-20 amino acid residue segments that tend to occupy the exposed surface of the polypeptide is generally described in Current Protocols in Immunology, Coligan et al, National Institutes of Heath, John Wiley & Sons, Inc.). Amino acids are generally composed of contiguous amino acid residues, but in certain cases are also formed by noncontiguous amino acids that are limited by a particular form. The amino acid segments recognized by the antibody are very specific and are generally considered epitopes. Amino acid fragments can be produced by chemical and / or enzymatic cleavage of natural proteins, by automated solid phase peptide synthesis, or by production from genetically engineered organisms. Polypeptide fragments can be isolated by various HPLC or FPLC chromatography methods or combinations thereof known in the art. The selection of polypeptide fragments is described, for example, on the surface of proteins of Kyte and Doolittle's 1982 (Journal of Molecular Biology 157: 105-132) and Chou and Fasman's 1974 (Biochemistry 13: 222-245). This can be helped by an algorithm that represents a sequence that has been modified. For the preparation of immunogens containing significant polypeptide fragments, keyhole lymphets are generally used via chemical reactions via free amino (lysine), sulfhydryl (cysteine), phenolic groups (tyrosine) or carboxyl groups (aspartate or glutamate). The polypeptide is covalently bound to a carrier protein such as hemocyanin. The immunogen along with the adjuvant is injected into rats or rabbits or chickens that elicit an immune response against the immunogen. Analysis of the amount of antibody in the antiserum of injected animals against polypeptide fragments can be measured using several immunological methods such as ELISA and Western blot. In addition, monoclonal antibodies can be prepared using splenocytes of injected animals that produce immortal hybridoma cells in which fusion with tumor cells produces a single antibody group. Hybridoma cells are screened by immunological methods to select cell lines that produce specific antibodies to significant polypeptide fragments. Purification of antibodies from different biological sources can be performed using various antigen affinity or antibody affinity columns or other chromatographic HPLC or FPLC methods.

The toxins described herein are very unique in that the toxins have functional activity, which is the key for developing insect management methods. In developing insect control methods, protein can be injected directly into the organism, avoiding the digestive tract, to delay or avoid proteolytic processes. In such a case, the protein administered to the organism will retain its function until it is denatured, unspecifically degraded, or removed by the immune system in higher organisms. Injecting insecticidal toxins into insects is a possible application only in the laboratory and only for large insects which are thus easily injected. The observation that the pesticidal protein toxins described herein exhibit their toxic activity after oral ingestion or contact with the toxin allows the development of an insect management plan based solely on incorporating the protein toxin into the insect's food. Such a plan can lead to the production of insect baits.

Photolapus toxin can be administered to an insect in purified form. Toxins may also be delivered in an amount of about 1 to about 100 mg / L broth. This may vary depending on formulation conditions, conditions of inoculation source, isolation techniques of toxins, and the like. Toxins can be administered as exudate secretions or intracellular proteins originally expressed in heterologous prokaryotic or eukaryotic hosts. Bacteria are typically hosts in which proteins are expressed. Eukaryotic hosts may include, but are not limited to, plants, insects, and yeasts. In addition, toxins can be produced in insects by transgenic plants of bacteria or fields or by baculovirus vectors. Typically, the toxin will be introduced to the insect by incorporating one or more toxins into the insect's food.

Full lethality is useful for ingestion by insects, but is not required to achieve useful toxicity. If an insect avoids toxins or stops feeding, such avoidance will be useful in some applications, even if the effect is close to lethal. For example, where insecticidal transgenic crop plants are desired, the ultimate goal is to protect the plant rather than kill the insect, so insect reluctance to ingest the plant is as useful as lethal toxicity to insects.

There are many other ways in which toxins can be incorporated into insect food. For example, larval food sources can be mixed with toxic proteins by spraying the protein solution into the food as disclosed herein. In addition, the purified protein may be genetically engineered into other harmless bacteria and then grown in culture, applied to food sources or left in soil in areas where insect killing is required. In addition, proteins can be genetically processed directly into insect food sources. For example, the main food source for many insect larvae is plants.

By incorporating the genetic material encoding the pesticidal properties of the photolabose toxin into the genome of the plant that is feeding the particular insect pest, the adult or larvae can die after consuming the food plant. A large number of monocotyledonous and dicotyledonous plants were transformed. Fruits and vegetables as well as transgenic crops are commercial targets. Such crops include, but are not limited to, corn, rice, soybeans, canola, sunflower, alfalfa, sugar cane, wheat, cotton, peanuts, tomatoes, potatoes and the like. Several techniques exist for introducing exogenous genetic material into plant cells and for obtaining plants that stably maintain and express the introduced genes. Such techniques include facilitating the direct introduction of genetic material coated into particulates into cells (US Pat. No. 4,945,050 (Cornell), and US Pat. No. 5,141,131 (DowElanco)). Plants can be transformed using the Agrobacterium technology [US Patent 5,177,010 (University of Toledo), US Patent 5,104,310 (Texas A & M), European Patent Application 0131624B1, European Patent Application 120516, 159418B1 and 176,112 (Schilperoot), United States Patents 5,149,645, 5,469,976, 5,464,763, 4,940,838, and 4,693,976 (Schilperoot), European Patent Applications 116718, 290799 and 320500 (all MaxPlanck), European Patent Applications 604662 and 627752 (Japan Tobacco), European Patent Applications 0267159 and 0292435, and US Patent 5,231 (All Ciba Geigy), US Pat. Nos. 5,463,174 and 4,762,785 (all Calgene), and US Pat. Nos. 5,004,863 and 5,159,135 (all Agracetus). Other transformation techniques include whiskers techniques (see US Pat. Nos. 5,302,523 and 5,464,765, all of Zeneca). Electroporation techniques are also used to transform plants [International Application Publication 87/06614 (Boyce Thompson Institute), US Patents 5,472,869 and 5,384,253 (all Dekalb), International Application Publications 92/09696 and 93/21335 (all PGS) ). All of these transformation patents and publications are incorporated herein by reference. In addition to many techniques for transforming plants, the types of tissues in contact with exogenous genes may also vary. Such tissues will include, but are not limited to, embryonic tissue, callus tissue types I and II, hypocotyl, meristem, and the like. Almost all plant tissues can be transformed during dedifferentiation using appropriate techniques within the skill of the art.

Another variable is the selection of selectable markers. Preference for a particular marker is at the discretion of the skilled person, but any of the following selectable markers may be used with any other gene not listed herein, which may serve as a selectable marker. Such selectable markers include glyphosate, as well as the aminoglycoside phosphotransferase gene (Aph II) of transposon Tn5 encoding resistance to the antibiotics kanamycin, neomycin and G418; Hygromycin; Methotrexate; Phosphinothricin (bialophos); Include, but are not limited to, genes encoding tolerance or tolerance to imidazolinone, sulfonylureas, and triazolopyrimidine herbicides (e.g., chlorosulfuron; bromoxynil, dalapon, etc.) .

In addition to selection markers, the use of reporter genes may be desirable. In some cases, reporter genes can be used without selection markers. Reporter genes are typically genes that are present or not expressed in a recipient organism or tissue. Reporter genes typically encode proteins that provide some phenotypic change or enzymatic properties. Examples of such genes are described in K. Weising et al., Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes are glucuronidase (GUS) genes.

Regardless of the transformation technique, the genes are preferably introduced into a gene transfer vector adapted for expressing photolabduus toxin in plant cells by including the plant promoter in the vector. In addition to plant promoters, promoters from various sources can be used efficiently in plant cells to express exogenous genes. For example, promoters of bacterial origin (e.g., octopin synthase promoter, nopaline synthase promoter, mannofine synthase promoter), promoters of viral origin (known as cauliflower mosaic virus (35S and 19S), 35T) Reprocessed 35S) and the like can be used (see PCT / US96 / 16582, WO97 / 13402, published April 17, 1997, incorporated herein by reference). Plant promoters include small subunits of ribulose-1,6-bisphosphate (RUBP) carboxylase, beta-conglycinin promoter, pazeolin promoter, ADH promoter, heat shock promoter and tissue specific promoter But it is not limited thereto. Promoters can also include certain enhancer sequence elements that can improve transcriptional efficacy. Typical enhancers include, but are not limited to, Adh-Intron 1 and Adh-Intron 6. Configuration promoters can be used. Constitutive promoters produce continuous gene expression at all times in all cell types (eg, actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types such as leaves or seeds (eg, zein, oleosin, napin, ACP), and these promoters may also be used. Promoters are also active during certain stages of plant development and may also be active in plant tissues and organs. Examples of such promoters include, but are not limited to, pollen specificity, pear specificity, corn beard specificity, cotton fiber specificity, root specificity, seed endoplasmic specificity promoter and the like.

Under certain circumstances, it may be desirable to use an inducible promoter. Inducible promoters are responsible for the expression of genes that respond to specific signals (eg, physical stimuli (heat genes); light (RUBP carboxylase); hormones (Em); metabolites; and stress). Other preferred transcription and translation elements that function in plants can be used. Many plant-specific gene transfer vectors are known in the art.

In addition, in order to obtain high expression of bacterial genes in plants, it is known that it is desirable to reengineer bacterial genes so that they can be expressed more efficiently in the cytoplasm of plants. It is one such plant in which corn is desirable to reengineer bacterial gene (s) prior to transformation to increase the expression level of toxins in the plant. One reason for reengineering is due to the very low G + C content (and consequently bias towards high A + T content) of the native bacterial gene (s). This produces sequences that mimic or replicate plant gene control sequences that are known to be very rich in A + T. The presence of some A + T rich sequences in the DNA of the gene (s) introduced into the plant (eg, the TATA box region normally found in the gene promoter) can cause aberrant transcription of the gene (s). In contrast, the presence of other regulatory sequences present in the transcribed mRNA (eg, polyadenylation signal sequence (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability. Can be. Thus, one purpose in the design of reengineered bacterial gene (s), more preferably referred to as plant optimized gene (s), is to produce DNA sequences with higher G + C content, preferably metabolic. It produces something close to that of the plant gene that encodes the enzyme. Another goal in the design of plant optimized gene (s) is to produce DNA sequences that must not only have higher G + C content but also be prepared so that translation is not inhibited by altering sequence changes.

An example of a plant with a high G + C content is corn. Table 2 below describes the high degree of G + C content in corn. As in corn, the G + C content in other plants is also believed to be high.

Collection of G + C Content in Protein Coding Regions of Corn Genes Protein class ^a Range of% G + C Average ^b of% G + C Metabolic Enzymes (40) 44.4-75.3 59.0 (8.0) Storage protein Group I (23) 46.0-51.9 48.1 (1.3) Group II (13) 60.4-74.3 67.5 (3.2) Group I + II (36) 46.0-74.3 55.1 (9.6) ^c Structure Proteins (18) 48.6-70.5 63.6 (6.7) Regulatory Proteins (5) 57.2-68.9 62.0 (4.9) Uncharacterized Protein (9) 41.5-70.3 64.3 (7.2) Total Protein (108) 44.4-75.3 60.8 (5.2) Provide the number of genes in type ^a in parentheses. ^b Provide the standard deviation in parentheses. ^c The mean of the combined group is ignored in the calculation of the total mean.

In the data in Table 2, coding regions of genes were extracted from GenBank (Release 71) registration and basal compositions were calculated using the MacVector ^™ program (IBI, New Haven, CT). Intron sequences are ignored in the calculations. Group I and Group II storage protein gene sequences are distinguished by their marked differences in basal composition.

Due to the flexibility provided by the redundancy of the genetic code (ie, some amino acids are designated by one or more codons), the evolution of different organisms or types or organisms results in different uses of the extra codons. This "codon bias" is reflected in the average basal composition of the protein coding region. For example, a creature with a relatively low G + C content uses a codon with A or T at the third position of the extra codon, while an organism with a higher G + C content has a G or C at the third position. Use a codon having It is believed that the presence of "minor" codons in the mRNA of a gene can reduce the complete translation rate of the mRNA, especially if the relative abundance of the charged tRNA corresponding to the minor codon is low. Expanding this, the reduction in translation rate by individual minor codons will be at least additive for multiple minor codons. Thus, mRNAs with relatively high content of minor codons will have correspondingly low translation rates. This rate of translation will be reflected by the low level of synthesis of the protein being encoded.

To reengineer the bacterial gene (s), the codon bias of the plant is measured. Codon bias is the statistical codon distribution that a plant uses to encode its protein. After measuring the bias, the percentage of codons in the gene (s) of interest is determined. The first codon preferred by the plant should be determined with the second and third choice of preferred codons. The nucleic acid sequence generated by reverse translation of the amino acid sequence of the protein of interest encodes the same protein as in the native bacterial gene, but this generated nucleic acid sequence corresponds to the preferred first codon of the desired plant. New sequences are analyzed for restriction enzyme sites that can be generated by alterations. The identified site is further modified by replacing the codon with the preferred codon of the second or third choice. Other sites in the sequence that may affect the transcription or translation of the gene of interest are exon: intron 5 'or 3' binding, poly A addition signals, or RNA polymerase termination signals. The sequence is further analyzed and altered to reduce the frequency of TA or GC doublets. In addition to the doublet, G or C sequence blocks having about 4 or more identical residues may affect the transcription of the sequence. Thus, these blocks are also modified by replacing a codon, such as a first or second selection, with a codon of the next preferred selection. Preferably, the plant optimized gene (s) comprises about 63% of the first selection codon, about 22% to about 37% of the second selection codon, and 15% to 0% of the third selection codon, total% Is 100%. The most preferred plant optimized gene (s) comprises about 63% of the first selection codon, at least about 22% of the second selection codon, about 7.5% of the third selection codon and about 7.5% of the fourth selection codon, The total percentage is 100%. By the methods described above, one skilled in the art can alter the gene (s) exogenous to a particular plant so that the gene is optimally expressed in the plant. This method is further described in International Patent Application No. PCT / US96 / 16582, published April 17, 1997, WO97 / 13402.

Thus, in order to design plant optimized gene (s), the amino acid sequence of the toxin is reversed into the DNA sequence, using the extra gene code determined from the codon bias table identified for the gene DNA sequence of the particular plant to be transformed. Translate. The resulting DNA sequence, which is completely homologous in codon use, has a restriction enzyme recognition site and a preferred base composition that is strategically placed in addition to a higher degree of codon diversity and that may interfere with transcription of the gene or translation of the product mRNA. Further modifications are made to establish this missing DNA sequence.

When a bacterial gene is expressed in a plastid, in theory the bacterial gene can be more easily expressed in the plant. In this case, bacterial genes can be expressed in plants without optimizing the genes for plant expression, and proteins can be obtained at high expression rates. See US Pat. Nos. 4,762,785, 5,451,513 and 5,545,817, which are incorporated herein by reference.

One of the problems with commercial use of transgenic plants is resistance management. This is especially true for Bacillus thuringiensis toxins. Many companies use Bacillus terringiensis commercially and are very concerned about the resistance of Bt toxins. One method of managing insect resistance is to combine toxins produced by photolapids with toxins such as Bt, plant insect proteins (Ciba Geigy) or other toxins. This binder may be formulated for sprayable applications or may be a molecular binder. Plants can be transformed with the photolapose gene, which produces insect toxins, and other insect toxin genes such as Bt.

European patent application 0400246A1 describes the transformation of two Bt in plants, which can be any two genes. Another method for producing transgenic plants comprising one or more insect resistance genes is that each plant produces two plants containing insect resistance genes. These plants can be backcrossed using traditional plant breeding techniques to produce plants comprising one or more insect resistance genes.

In addition to producing transgenic plants containing plant optimized gene (s), there are other delivery systems that may be desired to reengineer bacterial gene (s). Along the same lines, genetically engineered and easily isolated protein toxins, which fuse together the pesticidal activity of molecules and toxins that attract insects as a food source, can be engineered and expressed in bacteria or eukaryotic cells using well known standard techniques. Can be. After purification in the laboratory, such toxic agents with "built-in" baits can be packaged in a standard insect trap housing.

Another delivery system is the incorporation of the toxin's genetic material into the baculovirus vector. Baculoviruses infect certain insect hosts that include the desired targets of photolabose toxin. Infectious baculoviruses containing expression constructs for the photolaptoxin can be introduced into an insect rampant area to infect and poison or poison the insect.

In order to deliver pesticidal properties, the nucleic acid sequence encoding the coding amino acid sequence of the photolabose toxin needs to be integrated into a protein expression vector suitable for the host where the vector will reside. One method for obtaining nucleic acid sequences encoding proteins with pesticidal properties is to isolate natural genetic material that produces toxins in Photorapdus using information estimated from the amino acid sequences of the toxins, most of which are described below. It is. As described below, a method of purifying a protein involved in toxin activity is also disclosed.

Using the N-terminal amino acid sequence data as described below, oligonucleotides complementary to all or part of the DNA base encoding the first amino acid of the toxin can be prepared. These oligonucleotides can be radiolabelled and then used as molecular probes for isolating genetic material from genomic gene libraries made from genetic material isolated from strains of photolapose. Gene libraries can be cloned into plasmids, cosmids, phage or phagemid vectors. The library can be used to transform Escherichia coli and screen it using antibodies generated against this toxin to determine whether the transformed cells produce a toxin, or directly analyze for insect toxicity. have.

Since the synonymous genetic code allows amino acids to be encoded in DNA by any of several 3-nucleotide combinations, this study requires the production of a set of oligonucleotides. For example, the amino acid arginine can be encoded by nucleic acid triplets CGA, CGC, CGG, CGT, AGA and AGG. Since it is not possible to predict which triplet is used at that position in the toxin gene, oligonucleotides should be prepared with each possible triplet presented. One or more DNA molecules corresponding to protein subunits may be necessary to construct a sufficient number of oligonucleotide probes to recover all of the protein subunits necessary to achieve oral toxicity.

Any of several techniques well known to those skilled in the molecular biology field can be easily isolated from the amino acid sequence of the purified protein to clone all or part of the genetic material responsible for the production of toxins into an expression vector. . Typical expression vectors are DNA plasmids, but other means of delivery can also be envisioned, including but not limited to cosmids, phagemids and phages. Features required or required for plasmid replication, for example replication origin and other forms of antibiotic resistance or selectable markers, for example Streptomyces hygroscopicus or viridochromogene In addition to the bar gene of, protein expression vectors generally additionally require an expression cassette that incorporates the cis-acting sequences required for transcription and translation of the gene of interest. The cis-acting sequences required for expression in prokaryotic cells differ from those required in eukaryotic cells and plants.

Eukaryotic expression cassettes require a 5 ') transcriptional promoter upstream to the gene of interest, a transcription termination region such as a poly-A addition site and a ribosomal binding site upstream of the first codon of the gene of interest. In bacterial cells, a useful transcriptional promoter that can be incorporated into a vector is the T7 RNA polymerase-binding promoter. Promoters as described herein above are known to effectively advance the transcription of mRNA. In addition, upstream of the gene of interest, the vector may comprise a nucleotide sequence that encodes a signal sequence known to covalently bind the protein to a specific portion of the host cell, such as the cell surface.

Insect viruses or baculoviruses are known to infect certain insects and cause side effects. The effect of the virus on insects is slow, and the virus does not stop insect feeding. Thus, viruses do not appear to be useful as insect pest control agents. Incorporation of the photolapus toxin gene into a baculovirus vector can provide an effective method of delivery of toxins while increasing the lethality of the virus. In addition, since different baculoviruses are specific to different insects, it may be possible to use certain toxins to selectively target particularly harmful insect pests. Particularly useful vectors for toxin genes are nuclear polyhedrosis viruses. Delivery vectors using this virus have been described previously and are currently the selection vectors for delivering foreign genes to insects. Virus-toxin gene recombinants may be constructed in oral deliverable form. Baculoviruses generally infect the insect's midgut membranes. Toxin genes inserted behind the strong viral coat protein promoter will be expressed and will quickly kill infected insects.

In addition to transgenic plant delivery systems for insect viruses or baculoviruses or protein toxins of the present invention, proteins are also known as Bacillus terringiensis encapsulation techniques (e.g., US Pat. Nos. 4,695,455, 4,695,462, all of which are incorporated herein by reference). As in, but not limited to, US Pat. No. 4,861,595. Another delivery system for the protein toxins of the present invention is the preparation of proteins into the bait matrix, which can then be used at ground or underground insect bait stations. Examples of such techniques include, but are not limited to, the PCT patent application WO 93/23998, which is incorporated herein by reference.

As mentioned above, codon preferences of other hosts may be different from photolapidus, so when expressed in non-natural hosts, it may be necessary to alter the sequence encoding the protein. In such cases, without making supplemental changes to the coding sequence, the translation in the new host may be very insufficient. In addition, modifications to amino acid sequences may be desired to avoid inhibitory cross-reactivity with proteins of the new host or to improve the pesticidal properties of the protein in the new host. Genetically modified toxin genes will encode toxins that exhibit, for example, enhanced or reduced toxicity, transformed insect resistance development, transformed stability, or modified target species specificity.

In addition to photolabose genes encoding toxins, the present invention encompasses related nucleic acid sequences that encode amino acid biopolymers homologous to toxin proteins and retain the toxic effects of photolabose protein after oral ingestion in insects.

For example, the toxin used in the present invention seems to inhibit the larva's food intake before killing the larva. By manipulating the nucleic acid sequence of the photolabose toxin or its control sequence, the genetic engineer placing the toxin gene in the plant can, for example, remove its full toxicity against larvae and maintain its ingestion-inhibiting activity while maintaining its ingestion-inhibiting activity. You can adjust the mode of action. This change allows the transformed plant to survive till harvest without the unnecessary dramatic effect of removing all target insects from the ecosystem. All such alterations of the gene encoding the toxin or the protein encoded by the gene are envisioned to be within the scope of the present invention.

Other planned alterations of nucleic acids include the addition of target sequences that deliver toxins to specific sites of insect larvae to improve their efficacy.

Strains W-14, ATCC 55397, 43948, 43949, 43950, 43951 and 43952 have been deposited in the American Type Culture Collection (Rockville Parknrow Drive 12301, Maryland, USA 20852). Amino acid and nucleotide sequence data for W-14 natural toxin (ATCC 55397) are shown below. Isolation of genomic DNA for toxins from bacterial hosts is also exemplified herein. Other strains herein have been deposited with the US Department of Agriculture (Feoria North University Drive 1815, Illinois, USA).

Standard and molecular biology techniques are described and taught herein below in the specification. Additional information can be found in Sambrook, J., Fritsch, EF, and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Current Protocalsin Molecular Biology, ed . F. M. Ausubel et al., (1997).

The following abbreviations are used throughout the examples: Tris = tris (hydroxymethyl) amino methane; SDS = sodium dodecyl sulfate; EDTA = ethylenediaminetetraacetic acid; IPTG = isopropylthio-B-galactoside; X-gal = 5-bromo-4-chloro-3-indoyl-B-D-galactoside; CTAB = cetyltrimethylammonium bromide; kbp = kilobase pair; dATP, dCTP, dGTP, dTTP, I = 2'-deoxynucleoside 5'-triphosphate of adenine, cytosine, guanine, thymine and inosine, respectively; ATP = Adenosine 5'-triphosphate.

<Example 1>

Purification of toxins derived from photolapus luminescence and demonstration of toxicity after oral delivery of purified toxins

Insecticidal protein toxins of the present invention were purified from photolabduum luminescence strain W-14, ATCC deposit no. 55397. The original culture of photolapus luminescence was maintained on a Petri dish containing 2% Proteose Peptone # 3 (ie PP3, Difco Laboratories, Detroit MI) in 1.5% agar, incubated at 25 ° C. and weekly Moved. Colonies of the first form of bacteria were inoculated into 200 ml PP3 broth supplemented with 0.5% polyoxyethylene sorbitan monostearate (Tween 60, Sigma Chemical Company, St. Louis MO) in a 1 L flask. It was. Broth cultures were grown for 72 hours at 30 ° C. on a rotary shaker. Toxin proteins can be recovered from cultures grown in the presence or absence of tweens; The absence of tweens can affect the shape of the grown bacteria and the profile of the proteins produced by the bacteria. In the absence of tweens, as long as the molecular weight of at least one identified toxin subunit shifts from about 200 kDa to about 185 kDa, variant shifts occur.

The 72 hour culture was centrifuged at 10,000 × g for 30 minutes to remove cells and debris. The supernatant fractions containing pesticidal activity were decanted and adjusted to 50 mM K ₂ HPO ₄ by addition of 1.0 MK ₂ HPO ₄ in the appropriate volume. The pH was adjusted to 8.6 by adding potassium hydroxide. This supernatant fraction was then mixed with DEAE-Sephacel (Pharmacia LKB Biotechnology) equilibrated with 50 mM K ₂ HPO ₄ . Toxic activity was adsorbed on DEAE resin. The mixture was then poured into a 2.6 × 40 cm column and washed with 50 mM K ₂ HPO ₄ at room temperature at a flow rate of 30 ml / hr until the effluent reached steady baseline UV absorption at 280 nm. The column was then washed with 150 mM KCl until the effluent reached the normal 280 nm baseline again. Finally, the column was washed with 300 mM KCl and fractions collected.

Fractions containing toxins were pooled and filtered sterilized using a 0.2 micron pore membrane filter. The toxin was then concentrated and equilibrated to 100 mM KPO ₄ , pH 6.9 using an ultrafiltration membrane (Centriprep 100, Amicon Division-WR Grace and Company) having a molecular weight cutoff of 100 kDa at 4 ° C. A 3 ml sample of toxin concentrate was applied on top of a 2.6 × 95 cm Sephacryl S-400 HR gel filtration column (Pharmacia LKB Biotechnology). The elution buffer was 100 mM KPO ₄ , pH 6.9, which was developed at 4 ° C. at a flow rate of 17 ml / hr. Eluate was monitored at 280 nm.

Fractions were pooled and tested for toxic activity. The toxicity of the chromatographic fractions was tested by biological assays using Manduca sexta larvae. Fractions can be applied directly to insect food (Cornabis wheat germ food, ICN Biochemicals Division-ICN Biomedicals, Inc.) or 5 μL of samples can be obtained from 4 or 5 instar larvae using a 30 gauge needle. Administration was by intrarahemocelic injection via the first proleg. The weight of each larvae in the treatment group was recorded at 24 hour intervals. Toxicity is estimated if the insect stops eating and dies within 24 days of consuming the treated insect food, or dies within 24 hours after injection of the fraction.

Toxic fractions were collected and concentrated using Centriprep-100, then 7.5 mm × 60 cm TSK-GEL G-4000 SW gel with 100 mM potassium phosphate, pH 6.9 elution buffer running at 0.4 ml / min. Analysis by HPLC using an osmosis column. This analysis revealed that the toxin protein was contained within one sharp peak eluted from the column with a retention time of about 33.6 minutes. This retention time corresponds to an estimated molecular weight of 1,000 kDa. Peak fractions were collected for further purification and fractions not containing this protein were discarded. The peak eluted from HPLC absorbed UV light at 218 and 280 nm but not at 405 nm. Absorption at 405 nm was shown to be caused by the genorabine antibiotic compound.

The collected peak fractions were electrophoresed in non-modified agarose gel (Metaphor Agarose, FMC BioProducts), indicating that two protein complexes were present in the peak. Peak material buffered in 50 mM Tris-HCl, pH 7.0 was transferred to 100 mM Tris-HCl, pH 7.0, under standard buffer conditions (anode buffer 1M Tris-HCl, pH 8.3); cathodic buffer 0.025M Tris, 0.192M glycine). It was isolated on a buffered 1.5% agarose stacking gel and a 1.9% agarose lysis gel buffered with 200 mM Tris-borate, pH8.3. The gel was developed at 15 ° C. at a constant current of 13 mA until the phenol red tracking dye reached the end of the gel. Two protein bands were visualized on the agarose gel using Coomassie brilliant blue staining.

Slow moving bands were referred to as "protein band 1" and fast moving bands were referred to as "protein band 2". The two protein bands were present in approximately equal amounts. In order to accurately excise two protein bands from the unstained portion of the gel, a Kumaji stained agarose gel was used as a guide. The excised pieces containing the protein bands were placed in a solvent to soften and a small amount of sterile water was added. As a control, a portion of the gel containing no protein was also excised and treated in the same manner as in the gel piece containing the protein. Proteins were recovered by electroelutation from gel pieces at 100 V (constant voltage) in 100 mM Tris-borate, pH 8.3 for 2 hours. Alternatively, the protein was manually eluted from the gel piece by adding an equal volume of 50 mM Tris-HCl (pH 7.0) to the gel piece and then incubated at 30 ° C. for 16 hours. Thereby, the protein was dispersed from the gel into the buffer, which was then collected.

The results of the insect toxicity test using HPLC-purified toxin (33.6 min, peak) and agarose gel purified toxin demonstrated the toxicity of the extract. Injecting 1.5 μg HPLC purified protein killed the insect within 24 hours. Both Protein Band 1 and Protein Band 2 recovered from the agarose gel by manual or electroelution killed the insect when injected. The estimated protein concentration for these samples was less than 50 ng / larva. Comparing the weight gain and mortality between larva groups injected with either protein band 1 or protein band 2, it was shown that protein band 1 is more toxic by injection delivery.

When HPLC-purified toxins were applied to the larvae at a concentration of 7.5 μg / larva, this stopped larval weight gain (24 larvae tested). The larvae start feeding, but after consuming only a very small amount of toxin-treated food, they again begin to develop pathological symptoms induced by toxins and stop feeding. Larvae began to fade, and most of the larvae had diarrhea. Significant insect lethality occurred when several 5 μg toxin doses were applied to the food over a 7-10 day period.

Agarose-isolated protein band 1 significantly inhibited larval weight gain at a dose of 200 ng / larva. Larvae fed a similar concentration of protein band 2 were not inhibited and gained weight in the same proportion as the control larvae. Twelve larvae consumed the protein and 45 larvae consumed the protein-containing agarose slices. These two sets of data indicate that protein band 1 is orally toxic to manduka sexta. In this test, it is evident that protein band 2 is not toxic to manduka sexta.

Further analysis of protein bands 1 and 2 by SDS-PAGE under denaturing conditions showed that each band consisted of several smaller protein subunits. Proteins were visualized by silver staining followed by Kumazi Brilliant Blue staining for maximum sensitivity.

The protein subunits in the two bands were very similar. Protein band 1 contained eight protein subunits of 25.1, 56.2, 60.8, 65.6, 166, 171, 184 and 208 kDa. Protein band 2 had the same profile except that 25.1, 60.8 and 65.6 kDa proteins were not present. The 56.2, 60.8, 65.6 and 184 kDa proteins were present at approximately the same concentrations in the complex of protein band 1 and showed at least 80% of the total protein content of the complex.

Natural HPLC-purified toxins were further characterized as follows. Toxins are thermally unstable and after heating to 60 ° C. for 15 min. Injecting or ingesting sexta larvae lost their ability to kill or inhibit weight gain. Assays were designed to detect lipase, type C phospholipase, nuclease or erythrocyte hemolytic activity and performed with purified toxins. None of these activities were present. Antimicrobial zone inhibition assays were also performed and purified toxins did not inhibit the growth of gram negative or gram positive bacteria, yeast or filamentous fungi, indicating that the toxicity is not genolabin antibiotic.

Natural HPLC-purified toxins were tested for their ability to kill insects other than Manduka Sexta. Table 3 lists the insects that are killed by HPLC-purified photolabduum luminescence toxin in this study.

Insects killed by photolapux luminescence toxin Common name neck Genus and species Delivery path Beetle Moth (tobacco hornworm) Lepidoptera Manduca sexta Oral and injection Mealworm Coleoptera Tenebrio molitor oral- Pharaoh Ant felling Monomorium pharaanis oral- German cockroach A tree Blatella germanica Oral and injection mosquito Fly Aedes aegypti oral-

Further Characterization of High Molecular Weight Toxin Complex

In a further analysis, toxin protein complexes were further characterized from W-14 growth medium. Culture conditions and initial purification steps through the S-400 HR column were the same as described above. After the high molecular weight toxin complex was isolated from the S-400 HR column fractions, the toxin fractions were equilibrated with 10 mM Tris-HCl (pH 8.6) and concentrated in a CentriPlus 100 (Amicon) concentrator. The protein toxin complex was then applied to a weak anion exchange (WAX) column, Vydac 301VPH575 (Hesparia, Calif.) At a flow rate of 0.5 ml / min. The protein was eluted with 0-250 mM KCl in linear potassium chloride gradient 10 mM Tris-HCl, pH 8.6 for 50 minutes. As a result of absorbance measurement at 280 nm, eight protein peaks were detected.

Biopsies were performed on all fractions eluted from HPLC columns using newborn southern corn root worms (Diabrotica undecimpunctata howardi, SCR) larvae and tobacco tentacles worms (Manduca sexta, THW). THW was grown by feeding cicadabang wheat pear diet (ICN) at 25 ° C. with a cycle of 16 hours during the day and 8 hours at night. SCR was fed with Insecta-Diet (BioServ) from corn rootworm larvae at 25 ° C. with a 16-day / 8-hour cycle.

Maximum mortality of SCR and THW larvae was observed at peak 6, which eluted with about 112 mM to 132 mM KCl. Analysis of peak 6 by SDS-PAGE revealed prominent peptides of 170 kDa, 66 kDa, 63 kDa, 59.5 kDa and 31 kDa. A polyclonal antibody mixture for the Western blot analysis of the peak 6 protein fraction (described in example _{21) TcaA ii -syn, TcaA iii} -syn, TcaB ii -syn, TcaC-syn, and _ii -syn TcbA peptide And C5F2, a monoclonal antibody to the TcbA _iii peptide. Peak 6 contained immunoreactive bands of 170 kDa, 90 kDa, 66 kDa, 59.5 kDa and 31 kDa. These _{TcaC (166 kDa), TcaA ii} + TcaA iii (92 kDa), TcaA iii (66 kDa), respectively, are very close to the expected size for the TcaB _{i i} (60 kDa) and TcaA _ii (25 kDa). Peak 6 shifted to a single band with similar mobility as that of Band 1, further analyzed by natural agarose gel electrophoresis as described herein.

Protein concentration of purified peak 6 toxin protein was measured using BCA reagent (Pierce). Dilutions of this protein were prepared in 10 mM Tris (pH 8.6) and used for dietary biopsies. After 240 hours, all newborn larvae that received 450 ng or more of the Peak 6 protein fraction via dietary biopsy died. Larvae fed 90 ng of the same fraction had a mortality of 40%. After 240 hours, 90 and 20 ng of peak 6 protein fractions were administered and survived about 10% and 70% of the control weight, respectively.

<Example 2>

Insecticide Availability

The usability and toxicity of photolapux luminescence was further characterized. Photolabduus lumenense (strain W-14) culture broth was produced as follows. The production medium was 2% Baekto Proteose Peptone® # 3 (PP3, Difco Laboratories, Detroit, Michigan) in Milli-Q® deionized water. Seed culture flasks consisting of 175 ml of medium were placed in a 500 ml tribaffle flask with a Delong neck covered with Kaput and autoclaved at T = 121 ° C. (250 ° F.) for 20 minutes. . The production flask consisted of 500 ml in 2.8 L in a delon necked 500 ml tribaffle flask covered with Shin-etsu silicone foam stopper. They were autoclaved at T = 121 ° C. (250 ° F.) for 45 minutes. Seed cultures were incubated at 28 ° C. at 150 rpm in a rotary shake incubator with a 6.08 cm (2 inch) operating radius. After growing for 16 hours, 1% seed culture was placed in the production flask and grown for 24 hours before recovery. Toxin production appeared during log phase growth. The microbial broth was transferred to 1 L centrifuge and the cell biomass pelleted (30 min [R.C.F = 1600] HG-4L Rotor RC3 Sorval centrifuge, Dupont, Wilmington, Delaware at 2500 RPM 4 ° C). The broth was further clarified by cooling the first broth at 4 ° C. for 8-16 hours and recentrifuging for at least 2 hours (the above conditions) to remove the presumed mucopolysaccharides that settle upon standing. (Alternative processing methods combine the two steps and include the use of 16 hours of clarification centrifugation under the same conditions as above.) The broth was then stored at 4 ° C. before bioanalysis or filtration.

Photolabose culture broth, and protein toxin (s) purified from the broth, showed activity (lethal and / or growth inhibition, reduced adult appearance) against many insects. More specifically, activity has been shown for corn rootworms (larvae and adults), Colorado potato beetles, and turf grubs, which are members of the insects of the Coleoptera. Other members of the coleoptera include wireworms, pollen beetles, flea beetle, seed beetles and weevils. Activity was also observed for aster leafhoppers, members of the same tree. Other members of the same tree include many host specific aphid species, as well as planthoppers, pysllas, sock, scale insects, whiteflies and spider bugs. . The broth and purified fractions are also found in sugar beet matrix, cabbage beetle, black cutworm, gray tobacco moth, European grain borer, corn earworm and codling moth (all of which are Lepidoptera) Being a member). Other typical members of this neck include moths, Indian mealmoths, leaf rollers, cabbage worms, cotton ballworms, bagworms, eastern tent caterpillers, Grass webworms, and fall armyworms. Activity has also been shown for fruit flies and mosquito larvae that are members of the fly. Other members of the flyfly include pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly, crane fly, housefly and various mosquito species. It also showed activity against carpenter ants and Argentine ants, members of the neck, including fire ants, oderous ants and small black ants.

Broth / fractions are useful for reducing the population of insects and have been used in methods of inhibiting insect communities. The method may comprise applying the effective amount of the insect inactivation described above to the insect's area. The results are reported in Table 4.

Activity against corn rootworm larvae was tested as follows. Photolabose culture broth (filtration sterilized, no cells) or purified HPLC fractions were applied directly to 0.25 ml surface (˜1.5 cm 2) of artificial feed in 30 μl aliquots, respectively, in control medium or 10 mM sodium phosphate buffer ( pH 7.0). The food plate is air dried in a sterile flow hood, and the wells are filled with a single, newborn Diabrotica undecimpunctata howardi (Southern Corn Root, SCR) hatched from sterile eggs SCRs can be grown in artificial diets or grown on seedlings grown in Metromix® (Western Corn Root Beetle, WCR) in two-year-old Diabrotica virgifera virgifera (WCR). It was. Two-year-old larvae were weighed before adding food. The plate is sealed, placed in a humidified growth chamber and appropriately at 27 ° C. (4 days for newborn and adult SCR, 2 to 5 days for WCR larvae, 7 to 14 days for two age SCR). Lethality and weight measurements were recorded as indicated. In general, 16 insects were used per treatment in all studies. Control mortality was as follows: neonatal larvae: <5%, adult beetles: 5%.

The activity on the Colorado potato beetle was tested as follows. Photolabose culture broth or control medium was added to a 1.5 mL surface (˜2.0 cm 2) of standard artificial feed maintained in the wells of a 24-well tissue culture plate. Each well was treated with 50 μl and left to air dry. Individual two-year-old Colorado potato beetle (Leptinotarsa decemlineata, CPB) larvae were then placed on the feed and mortality was recorded 4 days later. Ten larvae per treatment were used for all cases of study. The mortality rate of the control group was 3.3%.

The activity against Japanese beetles slugs and beetles was tested as follows. Jang slugs (Popillia japonica, 2-3 ages) were collected from infiltrated mounds and kept in the laboratory in soil / peat containing carrot pieces added as additional feed. Small beetles were trapped locally with pheromones and kept in the laboratory in plastic containers with maple leaves added as feed. Diluted photolabose culture broth or control corn medium was added to the rootworm artificial feed (30 μl / 1.54 cm 2) or carrot slices (larvae), and then the two steps alone were placed in the feed wells and any mortality and uptake was achieved. Observed. In all cases there was a clear decrease in the intake observed (dung production).

Activity against mosquito larvae was tested as follows. Assays were performed in 96-well microtiter plates. Each well contained 200 μl of an aqueous solution (photolapdus culture broth, control medium or H ₂ O) and approximately 20, 1 day old larvae (Ades agetti). There were six wells per treatment. The results were read 2 hours after invasion and did not change during the 3 day observation period. No control mortality was observed.

Activity against fruit flies was performed as follows. Purchased Drosophilia melanogester medium was prepared using 50% dry medium and 50% solution of either water, control medium, or photolabose culture medium. This was done by placing 8 ml of dry media in each of three breeding vials and adding 8 ml of the appropriate solution. Ten late Drosophila maggots were then added to each vial. The vials were kept on a laboratory bench at room temperature under the illumination of a fluorescent ceiling. Pups or adult counts were done 3, 7 and 10 days after exposure. The incorporation of photolapus culture broth into Drosophila maggot feed medium resulted in a slight (17%) decrease in adult appearance on day 10, but caused a significant reduction compared to water and control medium (3%). .

The activity against star cicada was tested as follows. Ingestion analysis for the stellar cicada (Macrosteles severini) was designed to allow for active intake without any external contact. A reservoir of active / "feed" solution was made by drilling two holes in the center of the bottom portion of a 35 x 10 mm Petri dish. A 6.08 cm (2 inch) Parafilm M® square was placed across the top of the dish and closed with an "O" ring. Approximately seven cicada larvae were then placed in a 28.3 g (1 ounce) plastic cup and the reservoir was placed on top of the cup with the parafilm facing down. Test solution was then added to the reservoir through the hole. In tests using dilution-free photorabose culture broth, the broth and control medium were dialyzed against water to reduce control lethality. The lethality was reported on day 2, with a control mortality of 26.5%. In tests with purified fractions (200 mg protein / ml), a final concentration of 5% sucrose was used for all treatments to improve the viability of star larvae. This analyte was maintained at 28 ° C., 70% RH in a 16/8 light irradiation period in the incubator. Analytes were rated for 72 hour viability. Control mortality was 5.5%.

Activity against Argentine ants was tested as follows. 1.5 ml aliquots of 100% photolabose culture broth, control medium or water were pipetted into 2.0 ml clear glass vials. These vials were plugged with pieces of cotton dental wick wetted by proper treatment. Each vial was placed in a separate 60 x 16 mm Petri dish containing 8-12 adult Argentine ants (Linepithema humile). Three replicates were performed per treatment. Biological test plates were kept on laboratory benches at room temperature under the illumination of fluorescent ceilings. Lethality readings were made 5 days after exposure. Control mortality was 24%.

Activity against Carpenter ants was tested as follows. Black Carpenter ants (Camponotus pennsylvanicus) were collected from trees owned by DowElanco, Indianapolis, Indiana. Tests using photolabduose culture broth were performed as follows. Each plastic biological test container (7 1/8 "x 3") contained 15 workers, paper shelters, and 10 ml of broth or control medium in a plastic short glass. Cotton wicks were delivered to the treatment for the ants through holes in the short glass cover. All treatments contained 5% sucrose. Biological specimens were maintained in the dark at room temperature and scored for 19 days. Control mortality was 9%. Analytes carrying the purified fractions used artificial ant feed mixed with the treatment (purified fraction or control solution) at a rate of 0.2 ml treatment / 2.0 g feed in a plastic test tube. The final protein concentration of the purified fraction was less than 10 μg / g feed. Ten ants, water source, shelter and treated feed per treatment were placed in sealed plastic containers and kept in a dark at 27 ° C. in a humid incubator. The mortality rate was recorded on day 10. Control mortality was not shown.

Tests for various Lepidoptera larvae were tested as follows. The photolabose culture broth or purified fraction is diluted directly in control medium or 10 mM sodium phosphate buffer, pH 7.0 and directly added to the surface of 0.25 ml of surface (˜1.5 cm 2) of standard artificial feed in a 30 μl aliquot. It was. The feed plate was air dried in a sterile flow hood and the wells were taken up as stand alone larvae. European corn borer (Ostrinia nublilalis) and corn beetle (Helicoverpa zea) eggs were hatched from us by commercial sources, while sugar beetles (Spodoptera exigua), cabbage bug (Trichoplusia ni), and gray tobacco moth (Heliothis virescens) ), Apple moths (Laspeyresia pomonella) and black larvae (Agrotis ipsilon) larvae were internally supplied. After infestation with the larvae, the feed plates were sealed and placed in a humid growth chamber and kept in the dark at 27 ° C. for an appropriate period. Lethality and gravimetric measurements were recorded at 5-7 days for photolabdus culture medium and 4-7 days for purified fractions. In general, 16 insects per treatment were used in all studies. Control mortality ranged from 4-12.5% for control medium and less than 10% for phosphate buffer.

Effect of Photolapux luminescence (strain W-14) culture medium and purified toxin fraction on mortality and growth inhibition of different insect species / species Insect neck / species Broth Purified fraction Dead% G.I.% Dead% G.I.% Coleoptera Corn root worm Southern / newborn larva Southern / 2 age Southern / adult Western / 2 age 100na45na na38.5nt35 100ntntnt nantntnt Colorado potato beetle 93 nt nt nt Jang-sug slug 2 nana a.f.a.f. ntnt ntnt Mosquito larvae 17100 ntna ntnt ntnt Cicada 96.5 na 100 na Argentinian Ant Carpenter Ant 7571 nana nt100 nana ButterfliesBlood procession maggotsblack Biloba cabbage cabbagewormapple mothcorn beetleEuropean corn drillingworm 12.5ntntnt56.396.713.5 36ntntnt94.298.452.5 18.75021.96.2597.910019.4 41.471.266.845.9nana85.6 G. I. = growth inhibition, na = not applicable, nt = not tested, a.f. = Anti-feeding agent

<Example 3>

Insecticide Availability in Soil Applications

Photolabduum luminescence (strain W-14) culture broth has been shown to be active against corn rootworms when added directly to soil or soil mixture (Metromix®). The activity against newborn SCR and WCR in Chinese was tested as follows (Table 5): The test was carried out using corn seedlings (United Agriseeds brand CL614) germinated for 6 days in light on wet filter paper. At cm length, single phosphorus / plants were planted in 591 ml of clear plastic cups containing 50 g of anhydrous metromix® 20 Twenty new SCRs or WCRs were then placed directly on the roots of the seedlings and metromixed (registered). Upon invasion, the seedlings were moistened with a total volume of 50 ml of dilute broth solution, after wetting, the cups were sealed and left for 7 days at room temperature in the light. The seedlings were then washed to remove metromix® and the roots cut and weighed Activity was assessed as leaf damage induced by uptake and percentage of percentage of corn roots remaining relative to control plants. Leaf damage was visually observed and evaluated as one of-, +, ++, or +++,-indicating no damage and +++ indicating severe damage.

The activity for neonatal SCR in soil was tested as follows (Table 6). Testing was conducted using corn seedlings (United Agriseeds brand CL614) that germinated for 6 days in light on wet filter paper. When the roots were approximately 3-6 cm long, single phosphorus / plants were seeded in 591 ml of clear plastic cups containing 150 g of outdoor soil of Lebanon, Indian, where corn was seeded the previous year. This soil was not previously treated with pesticides. 20 newborn SCRs were then placed directly on the roots of the seedlings and covered with soil. After invasion, the seedlings were moistened with 50 ml total volume of dilute broth solution. After soaking, the unsealed cups were incubated in a chamber of high relative humidity (80%) at 26.7 ° C. (78 ° F.). The seedlings were then washed to remove all soil and the roots cut and weighed. Activity was assessed as a percentage of the percentage of corn root remaining relative to the control plant and leaf damage induced by uptake. Leaf damage was visually observed and evaluated as one of-, +, ++, or +++,-indicating no damage and +++ indicating severe damage.

Effects on Root Beetle Larvae after Soaking After Invasion of Photorabactus Luminesense (Strain W-14) Culture Broth (Metromix®) process larva Leaf damage Root Weight (g) % Southern Corn Roots water - - 0.4916 ± 0.023 100 Badge (2.0% v / v) - - 0.4416 ± 0.029 100 Broth (6.25% v / v) - - 0.4641 ± 0.081 100 water + +++ 0.1410 ± 0.006 28.7 Badge (2.0% v / v) + +++ 0.1345 ± 0.028 30.4 Broth (1.56% v / v) + - 0.4830 ± 0.031 104 Western Corn Roots water - - 0.4446 ± 0.019 100 Broth (2.0% v / v) - - 0.4069 ± 0.026 100 water + - 0.2202 ± 0.015 49 Broth (2.0% v / v) + - 0.3879 ± 0.013 95

Effects on Southern Root Beetle Larvae after Soaking after Invasion of Photorabactus Luminense (Strain W-14) Culture Broth (Soil) process larva Leaf damage Root Weight (g) % water - - 0.2148 ± 0.014 100 Broth (50% v / v) - - 0.2260 ± 0.016 103 water + +++ 0.0916 ± 0.009 43 Broth (50% v / v) + - 0.2428 ± 0.032 113

The activity of the second-stage Zung slugs in the Metromix® of the Photoractose luminescence (strain W-14) culture broth was observed in the test performed as follows (Table 7). Approximately 50 g of anhydrous metromix® was added to 591 mL of a clear plastic cup. This Metromix® was then wetted to a total volume of 50 ml of 50% (v / v) of diluted Photolab Dux Broth solution. Dilution of the crude broth was done with water and 50% broth was prepared by adding 25 ml crude broth to 25 ml for a total volume of 50 ml. A 1% (w / v) solution of Proteose peptone # 3 (pp3) at 50% dilution of normal medium concentration was used as the broth control. After soaking, five second-generation Jang slugs were placed on top of the wetted Metromix®. The healthy Jang slug larvae quickly dug and stealed into Metromix®. Within one hour, the larvae that were not hidden were removed and replaced with new larvae. The cup was sealed and placed in a dark incubator at 28 ° C. After 7 days, the larvae were removed from Metromix® and mortality was recorded. Activity was assessed as a percentage of lethality relative to the control.

Effects on Zung Slugs after Invasion of Photolab Dose Luminescence (Strain W-14) Culture Broth (Metromix®) process Dead* Dead% water 7/15 47 Control medium (1.0% v / v) 12/19 63 Broth (50% v / v) 17/20 85 * Expressed as the ratio of lethal / survival larvae

<Example 4>

Insecticide Availability in Leaf Applications

The activity of photolabdus broth against European corn puncture was observed when broth was applied directly to the surface of corn leaves (Table 8). In these assays, photolabose broth was diluted 100-fold with culture medium and manually added to the surface of the cut corn leaves at a rate of ˜6.0 μl / cm 2 of the leaf surface. The leaves were air dried and cut into pieces of equal size approximately 2 × 2 inches. The leaves were rolled up and tied with a paper clip and placed in the bottom surface to provide 0.64 cm (0.25 inch) of 2% agar in 28.3 g (1 oz) plastic short glass. Twelve new European corn punches were then placed on the dried leaves and the cup sealed. After incubation for 5 days in the dark at 27 ° C., samples were recorded for ingestion damage and recovered larvae.

Effect on European Corn Perforated Larvae after Application of Photorapus Luminesense (Strain W-14) Culture Broth to Infused Corn Leaves process Leaf damage Recovered Larva Weight (mg) water Wide range 55/120 0.42 mg Control badge Wide range 40/120 0.50 mg Broth (1.0% v / v) Small 3/120 0.15 mg

The activity of neonatal gray tobacco moth (Heliothis virescens) in culture broth was demonstrated using leaf immersion methodology. French cotton leaves were cut from the plants and the leaf discs were cut with 18.5 mm cork-chisels. Incubate the disks individually with photolapus luminescence (strain W-14), concentrated approximately 10-fold using Amicon (Beverly, Mass.) Proflux M12 transgenetal filtration system with control medium (pp3) or a 10 kDa filter. Immerse in broth. Excess solution was removed and a straightened paper clip was placed through the center of the disc. The paper clip was then pushed into a plastic, 28.3 g (1.0 ounce) single glass containing approximately 2.0 ml of 1% agar. This served to suspend the leaf disks on the agar. After drying of the leaf disk, a single newborn gray tobacco moth larva was placed on the disk and the cup was clamped. The cups were then sealed in plastic bags and placed for 5 days in a darkened 27 ° C. incubator. Residual larvae and leaf materials were then weighed and set as a measure for leaf damage (Table 9).

Effect of Gray Tobacco Moth Neoplasia on Cotton Leaf Soaking Analysis of Photorapid Luminesense (Strain W-14) Culture Broth process Leaf disc Final weight (mg) larva Contrast leaf 55.7 ± 1.3 na * Control badge 34.0 ± 2.9 4.3 ± 0.91 Potorabdus Broth 54.3 ± 1.4 0.0 ** *-Not applicable, **-No live larvae found

<Example 5>

Part A

Characterization of Toxin Peptide Components

In the subsequent assay, the band of toxin protein subunits isolated in Example 1 were digested on a 7% SDS polyacrylamide electrophoretic gel in a ratio of 30: 0.8 (acrylamide: bis-acrylamide). This gel matrix promoted better resolution for larger proteins. The gel system used to measure the band 1 and band 2 subunit molecular weights in Example 1 was an 18% gel of 38: 0.8 (acrylamide: bis-acrylamide), which allowed a wide range of size separations but The resolution was low.

In this assay, ten protein bands were degraded rather than eight. Table 9 shows the theoretical molecular weights of the 10 resolved bands and the molecular weights measured under these conditions were compared directly with those of the previous examples. It is not surprising that additional bands were detected under the different separation conditions used in this example. Deviations between previous and novel measurements of molecular weight can also be predicted according to differences in assay conditions. In the analysis of this example it is believed that the high molecular weight measurement is more accurate than that in Example 1 as a result of the improved resolution. However, these are measurement results based on SDS PAGE analysis, which are typically inaccurate in analysis and result in estimation of the peptide and may be further altered due to post- and co-translational formulas.

Amino acid sequences were determined for 5 N-terminal portions of 10 resolved peptides. Table 10 correlates with the molecular weight and identified sequences of the proteins. In SEQ ID NO: 2, certain analyzes suggest that the proline at residue 5 may be asparagine (asp). In SEQ ID NO: 3, certain analyzes suggest that the amino acid residues of residues 13 and 14 may both be arginine (arg). In SEQ ID NO: 4, specific analysis suggests that the amino acid residue of residue 6 may be either alanine or serine. In SEQ ID NO: 5, certain analyzes suggest that the amino acid residue of residue 3 may be aspartamic acid (asp).

Example 1 Measurement New measurement * Sequence list 208 200.2 kDa SEQ ID NO: 1 184 175.0 kDa SEQ ID NO: 2 65.6 68.1 kDa SEQ ID NO: 3 60.8 65.1 kDa SEQ ID NO: 4 56.2 58.3 kDa SEQ ID NO: 5 25.1 23.2 kDa SEQ ID NO: 15 * New measurements are based on SDS PAGE and not on gene sequence. SDS PAGE is not analytically accurate.

<Example 5>

Part B

Characterization of Toxin Peptide Components

The new N-terminal sequence, SEQ ID NO: 15, Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr, is a further N-terminal sequencing of peptide isolated from the native HPLC purified toxin described in Example 5, Part A above. Got by This peptide is derived from the tcaA gene. This peptide label TcaA _ii begins at position 254 and extends to position 491, where the TcaA _iii peptide, SEQ ID NO: 4 begins. The estimated size of the peptide based on the gene sequence is 25,240 Da.

<Example 6>

Characterization of Toxin Peptide Components

In a further assay, the toxin protein complex was subjected to 10% -80% ammonium sulphate precipitation followed by ion exchange chromatography step (mono Q) and two molecule sizing from Photolabduus luminescence growth medium (after incubation in the absence of Tween). Isolation by performing a chromatography step. These conditions are similar to those used in Example 1. During the first molecular sizing step, the second biologically compatible peak was found at about 100 ± 10 kDa. Based on protein measurements, this fragment was 20-50 times less active than the larger or first active peak of about 860 ± 100 kDa (natural). During this isolation experiment, less active peaks of about 325 ± 50 kDa, which contained much of the starting biological activity, were also resolved. The 325 kDa peak is believed to be related to or derived from the 860 kDa peak.

56 kDa protein was resolved in this assay. The N-terminal sequence of this protein is shown in SEQ ID NO: 6. It is noteworthy that this protein shows significant identity and conservatism at the N-terminus with SEQ ID NO: 5, both of which can be encoded by different members of the gene population and the protein produced by each gene is the two as a toxin complex of insecticide It suggests that they are sufficiently similar to each other to work.

Second, the prominent 185 kDa protein was consistently present in an amount comparable to that of protein 3 from Table 10 and would be the same protein or protein fragment. The N-terminal sequence of this 185 kDa protein is shown in SEQ ID NO: 7.

Additional N-terminal amino acid sequence data was also obtained from the isolated proteins. None of the determined N-terminal sequences appeared identical to the proteins identified in Table 10. Other proteins were present in the isolated formulation. One such protein had an estimated molecular weight of 108 kDa and had an N-terminal sequence as shown in SEQ ID NO: 8. The second such protein had an estimated molecular weight of 80 kDa and had an N-terminal sequence as shown in SEQ ID NO: 9.

When protein material was analyzed by size at approximately 325 kDa activity peaks, bands of approximately 51, 31, 28 and 22 kDa were observed. As in all cases where molecular weights were determined by analysis of electrophoretic shifts, these molecular weights received error effects caused by buffer ion intensity differences, electrophoretic power differences, and the like. Those skilled in the art will understand that the critical molecular weight values cannot be determined by these standard methods and each will have a deviation. Proteins of these sizes were assumed to be degradation products of the larger protein species (approximately 200 kDa in size) observed in the larger initial toxin complexes.

Finally, some formulations included a protein with the N-terminal sequence shown in SEQ ID NO: 10. This sequence was strongly homologous to the caffeonin protein, an auxiliary protein known to function in the assembly of large protein complexes. Applicants cannot prescribe such assembly functions for the protein identified in SEQ ID NO: 10, but is consistent with the presence of the described toxin protein complex that such caffeonine protein may be involved in its assembly. Moreover, such proteins cannot be directly suggested to have toxic activity, but these proteins may be important in determining the overall structural properties of protein toxins and thus will contribute to the in vivo viability of the complex after toxic activity or oral delivery. .

Subsequent analyzes of the stability of the protein toxin complex against proteinase K were performed. After 24 hours of incubation of the complex in the presence of 10 molar excess of proteinase K, it was confirmed that activity was ultimately eliminated (mortality dropped to about 5% upon oral application). These data demonstrate the proteolytic enzymatic properties of the toxin.

Toxic activity was also maintained by the dialysis membrane, which again identified a large size natural toxin complex.

<Example 7>

Isolation, Characterization, and Partial Amino Acid Sequencing of Photolapus Toxin

Isolation and N-terminal Amino Acid Sequencing: In a set of experiments similarly performed in Examples 5 and 6, ammonium sulfate precipitation of photolabdus protein was added slowly to the photolabdus broth, typically 2-3 liters of ammonium sulfate crystals. By adjusting to a final concentration of either 10% or 20%. After stirring at 4 ° C. for 1 hour, the material was centrifuged at 12,000 × g for 60 minutes. The supernatant was adjusted with 80% ammonium sulfate and stirred at 4 ° C. for 1 hour and then centrifuged at 12,000 × g for 60 minutes. The pellet was resuspended in 1/10 volume of 10 mM Na ₂ · PO ₄ , pH 7.0 and dialyzed overnight at 4 ° C. against the same phosphate buffer. The dialyzed material was centrifuged at 12,000 × g for 1 hour prior to ion exchange chromatography.

HR 16/50 Sepharose (Pharmacia) anion exchange column was equilibrated to 10 mM Na ₂ PO ₄ , pH 7.0. Centrifuged, dialyzed ammonium sulphate pellets were added to the Q Sepharose column at a rate of 1.5 ml / min and vigorously washed with equilibration buffer at 3.0 ml / min until the optical density (OD 280) reached less than 0.100. . Next, a 60 minute NaCl gradient of 3 mL / min over 0 to 0.5 M or a series of step eluents using 0.1 M, 0.4 M and finally 1.0 NaCl, respectively, for 60 minutes were added to the column. Fractions were collected and concentrated using Centriprep 100. Alternatively, the protein may be eluted with 0.4 M alone without prior elution with 0.1 M NaCl.

A 2 ml aliquot of the concentrated Q Sepharose sample was loaded onto an HR 16/50 Sepharose 12 (Pharmacia) gel permeation column equilibrated to 10 mM Na ₂ PO ₄ , pH 7.0 at a rate of 0.5 ml / min. This column was washed with the same buffer at a rate of 0.5 ml / min for 240 minutes and a 2 minute sample was recovered. Empty volumes of material were collected and concentrated using Centriprep 100. A 2 ml aliquot of the concentrated Sepharose 12 sample was loaded onto an HR 16/50 Sepharose 4B-CL (Pharmacia) gel permeation column equilibrated with 10 mM Na ₂ PO ₄ , pH 7.0 at a rate of 0.5 ml / min. This column was washed with the same buffer at a rate of 0.5 ml / min for 240 minutes and a 2 minute sample was recovered.

The excluded protein peak was subjected to the second fraction by applying it to a gel permeation column using Sepharose CL-4B resin separating proteins in the range of -30 kDa to 1000 kDa. This fraction was broken down into two peaks; It was a dominant peak eluting at an empty volume (> 1000 kDa) and an apparent molecular weight of about 860 kDa. Over the course of the week, subsequent gel permeated samples showed a gradual appearance of a third peak (approximately 325 kDa), presumably formed from the main peak by limited proteolysis. Biological tests conducted on these three peaks indicated that the empty peak was inactive, whereas the 860 kDa toxin complex was highly active and the 325 kDa peak was very strong but less active. SDS PAGE analysis of Sepharose CL-4B toxin complex peaks from different fermentation production showed two distinct peptide patterns, labeled "P" and "S". The two patterns showed significant differences in their molecular weight and the concentration of peptide components in their fractions. The most frequently produced "S" pattern had four high molecular weight peptides (> 150 kDa), while the "P" pattern had three high molecular weight peptides. In addition, the "S" peptide fraction has been shown to have 2-3 times higher activity against European maize borer. This shift may be related to variations in protein expression due to other factors based on the age of the inoculum and / or growth parameters of the old culture.

Milligram quantification of peak toxin complex fractions determined with "P" or "S" peptide pattern was performed with preliminary SDS PAGE and 3 with Seprabuff® for TRIS-glycine (PVDF membrane (ProBlott®). Blots were transfected for 4 hours Blots were sent to Harvard MicroChem and Cambridge ProChem for amino acid analysis and N-terminal amino acid sequencing, respectively.Three peptides in the “S” pattern were identified in the sequence identified in the previous example. Had a unique N-terminal amino acid sequence as compared to the 201 kDa (TcdA _ii ) peptide shown in SEQ ID NO: 13, below, shared 33% amino acid identity and showed 50% similarity to SEQ ID NO: 1 (TcbA _ii ) (Table 11, Table 11 In the vertical line indicates amino acid identity and colon indicates conservative amino acid substitutions.The second peptide of 197 kDa (TcdB) shown in SEQ ID NO: 14 had 42% identity and SEQ ID NO: 2 (TcaC). ), The third 205 kDa peptide is represented by TcdA _ii, and the limited N-terminal amino acid sequence (TcbA) of SEQ ID NO: 16 of the peptide of at least 235 kDa is SEQ ID NO: 1). The cloned gene (tcbA) comprising the putative amino acid sequence corresponding to (Tcb _ii ), the amino acid sequence deduced from SEQ ID NO: 11, was consistent with SEQ ID NO: 12. This means that a larger 235+ kDa peptide was added to the 201 kDa peptide (TcbA) during fermentation. _ii ) proteolytically processed into (SEQ ID NO: 1), possibly leading to the activation of the molecule.In another sequence, the sequence reported as the original sequence 5 (TcaB _ii ) reported in Example 5 above It was found to contain aspartamic acid residues (Asp) rather than glycine at the third position and two additional amino acids Gly and Asp at positions 18 and 19. The other two other sequences, SEQ ID NO: 2 (T In caC) and SEQ ID NO: 3 (TcaB _i ), additional amino acid sequences were obtained. Densitometer quantitation was performed using the same sample as the "S" formulation that was sent for N-terminal analysis. This analysis showed that the 201 Kda and 197 kDa peptides represented 7.0% and 7.2% of the total Coomassie Brilliant Blue stained protein in the “S” pattern, respectively, and were present in similar amounts in the other abundant peptides. It is contemplated that these peptides may exhibit protein analogs similar to those found in other bacterial toxins, such as various CryI Bt toxins. These proteins vary in similarity by 40-90% in the N-terminal amino acid sequence, including toxic fragments.

Internal Amino Acid Sequencing: To facilitate cloning of the toxin peptide gene, the internal amino acid sequence of the selected peptide was obtained as follows. Milligram quantitation of peak 2A fractions determined with "P" or "S" peptide pattern was performed by preliminary SDS PAGE, and TRIS-Glycine (3 with Seprabuff® for PVDF membranes (ProBlott®, Applied Biosystems)). -4 hours transblot Blots were sent to Harvard MicroChem and Cambridge ProChem for amino acid analysis and N-terminal amino acid sequencing, respectively TcbA _ii (including SEQ ID NO: 1), TcdA _ii , and TcaB _i (SEQ ID NO: 6) The three peptides referred to as (including 3) were trypsin digested by Harvard MicroChem followed by HPLC chromatography to separate the individual peptides.N-terminal amino acid analysis was performed on selected trypsin peptide fragments. internal peptides were sequenced for the peptide TcdA _ii (205 kDa peptide) referred to as TcdA _ii -PT111 (SEQ ID NO: 17) and _ii TcdA -PT79 (SEQ ID NO: 18). two internal Are suited TcaB _i -PT158 (SEQ ID NO: 19) and was sequenced for a TcaB _i -PT108 (SEQ ID NO: 20) peptide TcaB _i (68 kDa peptide) referred to as the Four internal peptides are TcbA _ii -PT103 (SEQ ID NO: 21), TcbA and peptide TcbA _ii (201 kDa peptide), referred to as _ii- PT56 (SEQ ID NO: 22), TcbA _ii- PT81 (a) (SEQ ID NO: 23) and TcbA _ii- PT81 (b) (SEQ ID NO: 24).

M-terminal amino acid sequence (identity and similarity are manually calculated) 201 kDa (33% identity and 50% similarity to SEQ ID NO: 1) L I Ｇ Ｙ N N O F S G * A Sequence 13: ｜ ｜: ｜ F I Q Ｇ D S D L F G N-A Sequence 1 197 kDa (42% identity and 58% similarity with SEQ ID NO: 2) M Ｑ N S Q T F S V G E L SEQ ID NO: 14 | |: | |:: | M M D S P E V S I T T L Sequence 2

<Example 8>

Screening for the isolation of genes encoding peptides consisting of cosmid library construction of photolapux luminescence (strain W-14) genomic DNA and toxin protein preparations

As a prerequisite for the production and other uses of photolabdus insect toxin proteins in heterologous hosts, it is necessary to isolate and characterize the genes encoding such peptides. It is both a purpose and a pursuit. One approach described below is based on the use of monoclonal clones or polyclonal antibodies against purified toxins and use this to isolate clones from expression libraries. Another approach described in this example is based on designing synonymous oligonucleotides for use in PCR amplification using N-terminal and internal amino acid sequence data. Either method can be used to identify DNA clones containing peptide-encoding genes to allow isolation of each gene and determination of their DNA base sequence.

Genomic DNA Isolation: Photorabdus luminescence strain W-14 (ATCC Accession No. 55397) was grown on 2% Proteose peptone # 3 agar (Difco Laboratories, Detroit, Mich.) And described in Example 1 above. One method was used to maintain post repeated passage biological testing. 50 ml shake cultures were produced in 175 ml baffle flasks in 2% Proteose Peptone # 3 medium and grown for approximately 24 hours at 28 ° C. and 150 rpm. 15 ml of this culture was pelleted and frozen at −20 ° C. in its medium until it was thawed for DNA isolation. Thawed cultures were centrifuged (700 × g, 30 min) and the suspended orange mucopolysaccharide material was removed. Residual cell material was centrifuged (25,000 × g, 15 min) to pellet bacterial cells and the media removed and discarded.

Genomic DNA is described in Current Protocols in Moleculat Biology, Ausubel et al. eds, John Wiley & Sons, 1994, section 2.4.1] was isolated by employing the CTAB method (including salt shock and modifying to increase all volumes 10-fold). Pelletized bacterial cells were resuspended in 10 ml final volume in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), followed by addition of 12 ml 5 M NaCl; This mixture was centrifuged at 15,000 xg for 20 minutes. The pellet was resuspended in 5.7 ml TE and 300 ml 10% SDS and 60 ml 20 mg / ml proteinase K (Gibco BRL Products, Grand Island, NY; in sterile distilled water) were added to the suspension. The mixture is incubated at 37 ° C. for 1 hour; Approximately 10 mg lysozyme (Worlington Biochemical Corp., Freehold, NJ) was then added. After an additional 45 minutes, 1 ml of 5 M NaCl and 800 ml of CTAB / NaCl solution (10% w / v CTAB, 0.7 M NaCl) were added. This preparation was incubated at 65 ° C. for 10 minutes, then gently disturbed, then further incubated and disturbed for approximately 20 minutes to aid in the purification of cellular material. Equal volume of chloroform / isoamyl alcohol solution (24: 1 v / v) was added, mixed gently and centrifuged. Equilibrate with equal volume of PCI (phenol / chloroform / isoamyl alcohol solution (50: 49: 1 v / v, 1 M Tris-HCl, pH 8.0; Intermountain Scientific Corporation, Kaysville, UT), then extract DNA Precipitated with 0.6 volume of isopropanol DNA precipitate was gently removed with a glass rod, washed twice with 70% ethanol, dried and STE buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA) 2 Dissolved in ml This formulation contained 2.5 mg / ml DNA as measured by an optical density of 260 nm (ie OD ₂₆₀ ).

The molecular weight size range of the isolated genomic DNA was evaluated for suitability for library construction. CHEF gel analysis was performed with 1.5% agarose (Seakem® LE, FMC BioProducts, Rockland, ME) gel made with 0.5 x TBE buffer (44.5 mM Tris-HCl pH 8.0, 44.5 mM H ₃ BO ₃ , 1 mM EDTA). On a BioRad CHEF-DR II device (Bio-Rad Laboratories, Inc., Richmond, CA) equipped with Pulsewave 760 Switcher. Progression variables were initial A time, 3 seconds; Final A time, 12 seconds; 200 volts; Running temperature 4-18 ° C .; Run time, 16.5 hours. Ethidium bromide staining and examination of the gel under ultraviolet light revealed that the DNA spans 30-250 kbp in length.

Construction of the Library: Partial Sau3A 1 digests were prepared with this Photorabdus genomic DNA preparation. This method is based on section 3.1.3 of Ausbel above. Small scale reactions were carried out under various conditions and modified until near optimum results were obtained. Several scale-up reactions were run using this retrofit condition and the results were analyzed on a CHEF gel and only the best scale-up formulation continued to run. In the best case, 200 μg of photolapis genomic DNA was added to Sau3A 1 (New England Biolabs, “NEB”, Beverly, MA) in 2 ml total volume of 1 × NEB 4 buffer (supplied by manufacturer 10 ×). Incubate at 37 ° C. for 15 minutes using 1.5 units. The reaction was stopped by addition of 2 ml of PCI and centrifuged at 8000 x g for 10 minutes. To the supernatant was added 200 [mu] l of 5 M NaCl and 6 ml of ice cold ethanol. This formulation was quenched at −20 ° C. for 30 minutes and then centrifuged at 12,000 × g for 15 minutes. The supernatant was removed and the precipitate was dried at 40 ° C. in vacuo and then resuspended in 400 μl STE. Spectrophotometric analysis showed about 40% recovery of the input DNA. The digested DNA was size fractionated on a sucrose gradient according to section 5.3.2 of CPMB (see Cited Reference). 10% to 40% (w / v) linear sucrose gradients were prepared using gradient markers in Ultra-Clear® tubes (Beckman Instruments, Inc., Palo Alto, Calif.) And DNA samples were placed on top. After centrifugation (26,000 rpm, 17 hours, Beckman SW41 Rota, 20 ° C.), fractions (about 750 μl) were removed from the top of the gradient and analyzed by CHEF gel electrophoresis as described above. Fractions containing Sau3A 1 in the size range 20-40 kbp were selected and DNA was precipitated by modifying the method of section 5.3.3 of the Ausbel above (increasing the amount of all solutions approximately 6.3-fold). After overnight precipitation, the DNA was recovered by centrifugation (17,000 xg, 15 minutes), dried, redissolved in TE, collected to a final volume of 80 μl, then 8 μl of 3 M sodium acetate and 220 μl of ethanol. And reprecipitated. The pellet recovered by centrifugation as described above was resuspended in 12 μl of TE. Concentrates of DNA were determined by a Hoechst 33258 dye (Polyscience, Inc., Warrington, PA) fluorometer on a Hoefer TKO100 fluorometer (Hoefer Scientific Instruments, San Francisco, CA). Approximately 2.5 μg of size fractionated DNA was recovered.

Thirty μg of cosmid pWE15 DNA (Stratagene, La Jolla, Calif.) Was completed using 100 units of restriction enzyme BamH 1 (NEB) in manufacturer's buffer (final volume 200 μl, 37 ° C., 1 hour). The reaction was extracted with 100 μl PCI and the DNA was precipitated from the aqueous layer by addition of 3M sodium acetate and 550 μl anhydrous ethanol at −20 ° C. After 20 minutes at −70 ° C., the DNA was recovered by centrifugation (17,000 × g, 15 minutes), dried under vacuum and dissolved in 180 μl of 10 mM Tris-HCl, pH 8.0. 20 μl of 10 × CIP buffer (10 mM Tris-HCl, pH 8.3; 10 mM ZnCl ₂ ; 10 mM MgCl ₂ ) and 1: 4 dilution calf enteric alkaline phosphatase (Boehringer Mannheim Corporation, Indianapolis, IN) 1 Μl (0.25 units) were added. After 30 minutes at 37 ° C., the following was added: 2 μl 0.5M EDTA pH 8.0, 10 μl 10% SDS; 0.5 μl 20 mg / ml proteinase K (supplied). It was then incubated at 55 ° C. for 30 minutes. After extraction using 100 μl of PCI and 100 μl of phenol (equilibrated to 1M Tris-Hcl, pH 8.0), dephosphorylated DNA was added by adding 72 μl of 7.5 M ammonium acetate and 550 μl of -20 ° C. ethanol. Precipitate, incubate on ice for 30 minutes and centrifuge as described above. The pelleted DNA was washed once with 500 μl of −20 ° C. 70% ethanol, dried in vacuo and dissolved in 20 μl TE buffer.

Lamation of BamH1-digested and phosphorylated pWE15 vectors of size fractionated Sau3A 1 fragments was modified by modifying the protocol of Ausbel section 3.33 (i.e. using premixed 10 X ligation buffer supplied by the manufacturer). It was carried out using an aze. Ligation was performed overnight at 15 ° C. in a total volume of 20 μl and then stored at −20 ° C.

4 μl of cosmid DNA ligation reaction containing about 1 μl of DNA was packaged into bacteriophage lambda using a commercial packaging extract (Gigapack® III Gold Packaging Ectract, Stratagene) according to the manufacturer's instructions. Packaged preparations were stored at 4 ° C. until use. Packaged cosmid preparations were used to infect Escherichia coli XL1 Blue MR cells (Stratagene) according to the Gigapack® III Gold protocol ("Titering the cosmid Library") as follows: XL1 Blue MR cells were 0.2% ( w / v) LB medium containing maltose and 10 mM MgSO ₄ (g / L: bacto-tryptone, 10; bacto-east extract, 5; bacto-aca, 15, NaCl, 5 (Difco Laboratories, Detroit, MI) ) it was grown in 37 ℃ in. after 5 hours growth, pellet the cells with 700 xg (15 min.) screen, and 10 mM MgSO ₄ 6 ㎖ the resuspended. culture density was OD ₆₀₀ = 0.5 by using 10 mM MgSO ₄ The packaged cosmid library was diluted 1:10 or 1:20 using sterile SM medium (0.1 M NaCl, 10 mM MgSO ₄ , 50 mM Tris-HCl pH 7.5, 0.01% w / v gelatin). 25 μl of diluted formulation was mixed with 25 μl of diluted XL1 Blue MR cells This mixture was shaken at 25 ° C. for 30 minutes without shaking. Amount and then was continued for about one hour, while the addition of 200 ㎕ used LB bromo and occasionally gently shaking the culture the culture aliquots (20 - 40 ㎕) of water. The 100 ㎎ / ℓ ampicillin (i.e., LB-Amp ₁₀₀₎ Sprinkle onto LB agar plates containing and incubate overnight at 37 ° C. To store libraries without amplification, single colonies were drawn and seeded into individual wells of sterile 96-well microwell plates, each well having 75 μl. Terrific Broth (TB medium: 12 g / l bacto-tryptone, 24 g / l bacto-east x, 0.4% v / v glycerol, 17 mM KH ₂ PO ₄ , 72 mM K ₂ HPO ₄ ) and 100 mg / 1 ampicillin (ie TB-Amp ₁₀₀ ) was contained and incubated overnight at 37 ° C. without shaking. After replicating the 96-well plate into the replicate plate, 75 μl / well of filtered sterilized TB: glycerol (1: 1, v / v: with or without 100 mg / L ampicillin) was added to the plate It was then quickly shaken at 100 rpm, 37 ° C., then sealed with Parafilm® (American National Can, Greenwich, CT) and placed and stored in a -70 ° C. freezer. Replication plates were grown and treated identically on master plates. A total of 40 such master plates (and replicas thereof) were made.

Screening of Libraries Using Radiolabeled DNA Probes: To prepare colony filters for probing using radioisotope labeled probes, 96-well plates of the library were thawed at 25 ° C. (bench top at room temperature). A replication plating tool with 96 prongs was used to inoculate a new 96-well replicate plate containing 75 μl / well of TB-Amp ₁₀₀ . Replication plates were grown overnight (fixed) at 37 ° C. and then shaken at 100 rpm for about 30 minutes at 37 ° C. A total of 800 colonies appeared on these replicate plates due to the non-growth of some isolates. Magna NT (100 ml / dish) placed on a solid LB-Amp ₁₀₀ (100 ml / dish) in a bioanalytical plastic dish (Nunc, 243 × 243 × 18 mm; Curtin Mathison Scientific, Inc., Wood Dale, IL) using a replication tool. MSI, Westboro, Mass.) A 96-well array of replication impregnations was inoculated onto nylon membrane (0.45 micron, 220 × 250 mm). These colonies were grown on the membrane at 37 ° C. for about 3 hours.

Positive control colonies (bacterial colonies containing GZ4 sequence inserts, see below) are individual Magna NT membranes (Nunc, 0.45 micron, 82 mm circles) on LB medium supplemented with 35 mg / l chloramphenicol (ie, LB-Cam ₃₅ ). The phases were grown and processed side by side on the library colony membrane side. Bacterial colonies on the membranes were lysed and denatured and neutralized according to a protocol taken from Genius® System User Guide Version 2.0 (Boehringer Mannheim Corporation, Indianapolis, IN). The membranes were denatured by placing on the colony side on filter paper soaked with 0.5 N NaOH and 1.5 M NaCl for 15 minutes and neutralized on filter paper soaked with 1M Tris-HCl pH 8.0, 1.5 M NaCl for 15 minutes. After UV crosslinking using a Stratagene UV Stratalinker set to automatic crosslinking, the membranes were stored at 25 ° C. until use. The membranes were trimmed into strips containing a replica impregnation of a single 96-well plate, then washed extensively by the method of section 6.4.1 of CPMB (see literature) and in 3 × SSC, 0.1% (w / v) SDS. 3 hours at 25 ° C., then 1 hour at 65 ° C. in the same solution, followed by 2 × SSC in the formulation for hybridization step (20 × SSC = 3M NaCl, 0.3M sodium citrate, pH 7.0).

Amplification of Specific Genomic Fragments of the TcaC Gene: Based on the N-terminal amino acid sequence determined for the purified Tcac peptide fragment (described herein as SEQ ID NO: 2), a pool of synonymous oligonucleotides (S4Psh) was applied to Applied Biosystem ABI394. Synthesis was performed by strand β-cyanoethyl chemistry on a DNA / RNA synthesizer (Perkin Elmer, Foster City, Calif.). These oligonucleotides were deprotected at 55 ° C. for 8 hours, dissolved in water, quantified by spectrophotometric measurement and used in dilution. This pool corresponds to the determined N-terminal amino acid sequence of the TcaC peptide. The amino acid sequences determined and the corresponding synonymous DNA sequences are shown below, where A, C, G and T are standard DNA bases and I represents inosine.

Amino Acid Met Gln Asp Ser Pro Glu Val S4Psh 5 'ATG CA (A / G) GA (T / C) (T / A) (C / G) (T / A) CCI GA (A / G) GT 3'

Another set of synonymous oligonucleotides was synthesized (pool P2.3.5R), showing the complement of the coding strand for the determined amino acid sequence of SEQ ID NO: 17:

Amino Acids Ala Phe Asn Ile Asp Asp Val Codon 5 'GCN TT (T / C) AA (T / C) AT (A / T / C) GA (T / C) GA (T / C) GT 3' P2.3.5R 3 'CG (A / C / G / T) AA (A / G) TT (A / G) TA (T / A / G) CT (A / G) CT (A / G) CA 5 '

These nucleotides were used for polymerase chain reaction (PCT®, Roche Molecular System, Branchburg, NJ) to amplify specific DNA fragments (described above) from genomic DNA prepared from Photorabdus strain W-14. Reactions (50 μl) were prepared for each primer pool P2Psh and 125 pmol of P2.3.5R, 253 ng of genomic template DNA, 10 pmol of dCTP, dGTP and dTTP respectively, 1 × GeneAmp® PCR buffer and AmpTaq® 2.5 Units (all of which are commercially available from Roche Molecular Systems; 10X GeneAmp® buffer contained 100 mM Tris-HCl pH 8.3, 500 mM KCl, 0.01% w / v gelatin) Amplification was Perkin Elmer Cetus DNA Thermal 35 cycles of 94 ° C. (1.0 min), 55 ° C. (2.0 min), 72 ° C. (3.0 min) in Cycler (Perkin Elmer, Foster City, Calif.) Followed by an extended period of 7.0 min at 72 ° C. Amplification procedures were performed using 2% w / v NuSieve (etc.) in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.0). Rox) Analysis by electrophoresis through 3: 1 agarose (FMC BioProducts) A specific product of an estimated size of 250 bp was found in other amplification products by ethidium bromide (0.5 μg / ml) staining and examination under ultraviolet light. Was observed.

The area of the gel containing approximately 250 bp product was cut and a small plug (0.5 mm diameter) was removed and used to feed the template for PCR amplification (40 cycles). The reaction (50 μl) contained the same components as above except for genomic template DNA. Following amplification, the ends of the fragments were blunt-ended and phosphorylated by incubation with T15 DNA ligase (NEB), 1 nmol ATP and T4 kinase (Pharmacia Biotec Inc., Piscataway, NJ) 2.15 units for 20 minutes at 25 ° C. I was.

DNA fragments were isolated from the remaining primers by electrophoresis through 1% GTG® agarose (FMC) in TEA. Gel slices containing fragments of apparent size 250 bp were cut and DNA extracted using a Qiaex kit (Qiaex Inc., Chatsworth, Calif.).

The extracted DNA fragments were ligated to the plasmid vector pBC KS (+) (Stratagene) extracted in a similar manner as described for pWE15 DNA after completion of digestion with restriction enzyme Sma 1. Representative ligation reactions (16.3 μl) include 1 × ligation buffer (50 mM Tris-HCl, pH 7.4; 10 mM MgCl ₂ ; 10 mM dithiothreitol; 1 mM spermidine, 1 mM ATP; 100 mg / ml bovine serum albumin) PBC KS (+) DNA 100 ng, 250 bp fragment DNA 70 ng, 1 nmol [Co (NH ₃ ) ₆ ] Cl, and T4 DNA ligase (Collaborative Biomedical Products, Bedford, Mass.) 3.9 Weiss units It was. After overnight incubation at 14 ° C., the ligated product is transformed into frozen E. coli DH5α cells (Gibco BRL), frozen according to the supplier's instructions, containing 119 μg / ml IPTG and 50 μg / ml X-gal. Placed on LB-Cam ₃₅ plate. Independent white colonies were extracted to transform plasmid DNA into modified alkaline-soluble / PEG precipitation methods [PRISM® Ready Reaction DyeDeoxy® Terminator Cycle Sequencing Kit Protocols; ABI / Perkin Elmer]. Two strands of the nucleotide sequence of the insert DNA were sequenced with the T7 primer [pBC KS (+) base 601-623: TAAAACGACGGCCAGTGAGCGCG] and LacZ primer [pBC KS (+) base 792-816: ATGACCATGATTACGCCAAGCGC GC) and PRISM (R) The determination was made using the protocol provided with the kit [ABI / Perkin Elmer]. Unincorporated dye terminator dideoxyribonucleotides were removed by passing through a Centri-Sep 100 column (Princeton Separation, Inc., Adelphia, NJ) according to the manufacturer's instructions. DNA sequences were obtained by analyzing samples on an ABI Model 373 A DNA Sequencer (ABI / Perkin Elmer). The DNA sequences of both isolates, GZ4 and HB14, were found as described in FIG.

This sequence exhibited the following properties: i) Bases 1-20 represented one of the 64 possible sequences of S4Psh synonymous oligonucleotides, and ii) the sequences of amino acid sequences 1-3 and 6-12 were TcaC (SEQ ID NO: The fourth amino acid encoded was cysteine rather than serine. This difference is encoded within the synonym for the serine codon (see above), iv) the fifth amino acid is proline, corresponding to the TcaC N-terminal sequence given in SEQ ID NO: 2, and v) bases 257-276 are synonymous pools. A shortened translation frame for the corresponding gene, which encodes one of the 192 possible sequences designed into it, vi) the TGA termination codon introduced at bases 268-270 is the result of complementarity to synonym assembled into the nucleotide pool at the corresponding position Does not provide.

<Labeling of TcaC peptide gene-specific probes>

DNA fragments corresponding to the 276 bases were 100 pmol each of P2Psh and P2.3.5R primers, 10 ng of plasmid GZ4 or HB104 as template, 20 nmol of dATP, dCTP, dGTP and dTTP, respectively, AmpliTaq® DNA polymerase 5 units , And 1 × Concentration of GeneAmp® buffer was amplified (100 cycles) to 100 μl reaction volume by PCR (registered trademark) under the same temperature control as above. Amplification products were extracted from 1% GTG® agarose gel by Qiaex kit and quantified by fluorimeter.

Amplification products extracted from plasmid HB14 template (approximately 400 ng) were divided into 5 aliquots and labeled with ³² P-dCTP using the High Prime Labeling Mix (Boehringer Mannheim) according to the manufacturer's instructions. Unincorporated radioisotopes were removed by passing through NucTrap® probe purification column (Stratagene) according to the manufacturer's instructions. Specific activity of the labeled DNA product was determined to be 3.11 × 10 ⁸ dpm / μg by liquid scintillation counting. This labeled DNA was used to probe membranes prepared from 800 members of the genomic library.

<Screening using TcaC-peptide gene specificity probes>

Radiolabeled HB14 probe was boiled for approximately 10 minutes and then added to a “minimal hyb” solution. [Note: The "least hybrid" method is the CERES protocol; "Restriction Fragment Length Polymorphism Laboratory Manual Version 4.0", sections 4-40 and 4-47; CERES / NPI, obtained from Salt Lake City, Utah. NPI is nonexistent and his successor runs as Linkage Genetics. “Minimal hybrid” solutions include 10% w / v PEG (polyethylene glycol), molecular weight approximately 8000), 7% w / v SDS; 0.6X SSC, 10 mM sodium phosphate buffer (prepared from 1 M stock containing 95 g / l NaH ₂ PO ₄ .1H ₂ O and 84.5 g / l Na ₂ HPO ₄ .7H ₂ O), 5 mM EDTA and 100 Mg / ml contains denatured salmon sperm DNA. The membranes were rapidly pressure dried, and then 5 strips of membranes were placed in each of two plastic boxes containing 75 ml of "minimum hybrid" and 2.6 ng / ml of radiolabeled HB14 probes without preliminary hybridization. They were incubated overnight with gentle shaking at 60 ° C. (50 rpm). The filters were washed three times for approximately 10 minutes each at 25 ° C. in a “minimum hybrid wash solution” (0.25 × SSC, 0.2% SDS) and then twice for 30 minutes with gentle shaking at 60 ° C. in the same solution. The filters are placed on a paper covered with Saran Wrap (registered trademark) (Dow Brands, Indianapolis, Indiana) in a shaded radiographic cassette, and two DuPont Cronex Lightening-Plus C1 enhancers (Sigma Chemical Co., Missouri) X-Omat X-ray film (St. Louis) (Rokster, Kodak) was exposed for 4 hours at -70 ° C. At development (standard photographic process), the important signals were noticeable in both replicates among the high peaks of weaker, more irregular signals. The filters were again washed for about 4 hours at 68 ° C. in “minimal hybrid wash solution”, then placed back in the cassette and the film exposed at −70 ° C. overnight. Twelve possible positives were identified due to strong signals in all replicate 96-well colony impregnations. No signal was seen in the negative control membrane (colonies of XL1 Blue MR cells containing pWE15), but at the same time very strong signal in the positive control membrane (DH5α cells containing GZ4 isolate of PCR product) treated with experimental samples. Appeared.

Twelve putative hybridization-positive colonies were recovered from frozen 96-well library plates and grown overnight at 37 ° C. in LB-Amp ₁₀₀ medium. These were then patched onto solid LB-Amp ₁₀₀ (3 / plate, +3 negative control: XL Blue MR cells containing pWE15 vector). Two sets of membranes (Magna NT nylon, 0.45 micron) were prepared for hybridization. The first set was prepared by placing the filter directly on the colonies on the patch plate, then removing it with adherent bacterial cells and treating as follows. The second set of filters was placed on a plate containing LB-Amp ₁₀₀ medium and then inoculated by moving the cells over the filter from the patch plate. After growing overnight at 37 ° C., the filters were removed from the plate and treated.

Bacteria cells on the filter were lysed and DNA was denatured by placing the colony side of each filter for 3 minutes on a pool of 0.5 N NaOH in a plastic plate (1.0 mL). The filters were dried under pressure on a paper towel and the process repeated with fresh 0.5 N NaOH. After acupressure drying, the filters were neutralized by placing on a 1.0 ml pool of 1 M Tris-HCl pH 7.5 for 3 minutes each, acupressure dried and neutralized again with fresh buffer. Subsequently, two similar immersions (5 min each) were performed on a pool of 0.5 M Tris-HCl pH 7.5 and 1.5 M NaCl. After acupressure drying, the DNA was UV crosslinked to the filter as described above, and the filters were washed in about 100 ml of 3 × SSC and 0.1% (w / v) SDS (25 ° C., 100 rpm) (4 times, each wash). 30 minutes each with fresh solution). These were then subjected to preliminary hybridization solutions [6X SSC and Ficoll 400 (from Pharmacia), polyvinylpyrrolidone (average molecular weight 360,000; from Sigma) and bovine serum albumin fragment V; (Sigma) each was placed at 65 ° C., 50 rpm for 2 hours in a minimum volume of 1% w / v]. The preliminary hybridization solution was removed and replaced with an HB14 ³² P-labeled probe that was stored from previous hybridization of the library membrane and denatured at 95 ° C. for 5 minutes. Hybridization was performed while shaking at 60 rpm at 50 rpm for 16 hours.

Following removal of the labeled probe solution, the membranes were washed three times at 25 ° C. (50 rpm, 15 minutes) in 3 × SSC (about 150 mL each wash). They were then washed in 0.25X SSC and 0.2% SDS (minimum hybrid wash solution) at 68 ° C. for 3 hours (50 rpm) and exposed to X-ray film at 25 ° C. for 1.5 hours as described above (enhancers). No screen). This exposure showed a very strong hybridization signal for cosmid isolates 22G12, 25A10, 26A5 and 26B10 and a very weak signal for cosmid isolate 8B10. No signal was seen in the negative control (pWE15) colonies, while at the same time a very strong signal was seen in the positive control membrane (DH5α cells containing GZ4 isolate of PCR product) treated with experimental samples.

<Amplification of specific genomic fragments of the TcaB gene>

Based on the N-terminal amino acid sequence determined for the purified TcaB _i peptide fragment (described herein as SEQ ID NO: 3), a pool of synonymous oligonucleotides (pool P8F) was synthesized as described for peptide TcaC. The amino acid sequences determined and the corresponding synonymous DNA sequences are shown below, where A, C, G and T are standard DNA bases and I represents inosine.

Another set of synonymous oligonucleotides was synthesized (pool P8.108.3R), which represents the complement of the coding strand to the determined amino acid sequence of the TcaB _i -PT108 internal peptide (SEQ ID NO: 20):

These oligonucleotides were used as primers for PCR (registered trademark) using HotStart 50 Tubes (registered trademark, Molecular Bio-Products, Inc., San Diego, Calif.) And photolabose strain W-14 (described above). Specific DNA fragments were amplified from genomic DNA prepared from. Representative reactions (50 μl) contained 2 nmol of each primer pool P8F and P8.108.3R 25 pmol, dATP, dCTP, dGTP and dTTP each in the 1X GeneAmp® PCR buffer (bottom layer), 1X GeneAmp® ) PCR buffer contained 230 ng of genomic template DNA, 8 nmol of dATP, dCTP, dGTP and dTTP, respectively, and 2.5 units of AmpTaq® DNA polymerase (upper layer). Amplification was performed up to 35 cycles as described for the TCaC peptide. Amplification products were analyzed by electrophoresis through 0.7% w / v SeaKem® LE agarose (FMC) in TEA buffer. A specific product of estimated size 1600 bp was observed.

Four such reactions were pooled and amplified DNA was extracted from 1.0% SeaKem® LE Agarose (FMC) by Qiaex kit as described for the TcaC peptide above. The extracted DNA was used directly as a template for sequencing [PRISM® sequencing kit] using P8F and P8.108.3R primer pools. Each reaction contained about 100 ng template DNA and 25 pmol of one primer pool and treated according to standard protocols as described for the TcaC peptide. Sequence analysis derived from extension of the P8F primer showed a short DNA sequence (and encoded amino acid sequence) corresponding to a portion of the N-terminal peptide sequence of SEQ ID NO: 3 (TcaB _i ):

GAT GCA TTG NTT GCT

Asp Ala Leu (Val) Ala

<Labeling of TcaB _i peptide gene-specific probes>

Approximately 50 ng of gel-purified TcaB _i DNA fragments were labeled with ³² P-dCTP as described above and unincorporated radioisotopes were removed by passing through a NICK column (registered trademark) (Pharmacia). The specific activity of the labeled DNA was determined to be 6 × 10 ⁹ dpm / μg. This labeled DNA was used to probe colony membranes prepared from components of genomic libraries hybridized to TcaC-peptide specificity probes.

The radioactive TcaC-specific label was removed by boiling the membrane containing 12 colonies identified in the TcaC-probe library screen (see above) twice for approximately 30 minutes each time in 1 L of 0.1 × SSC and 0.1% SDS. Removal of the radiolabel was confirmed by film exposure for 6 hours. The removed membrane was then incubated with the TcaB _i peptide specificity probe prepared above. Labeled DNA was denatured by boiling for 10 minutes and then added to a filter incubated for 1 hour at 60 ° C. in 100 ml of a “minimum hybrid” solution. After hybridization at this temperature overnight, the probe solution was removed and the filtrate was washed as follows (both in 0.3X SSC and 0.1% SDS): once at 25 ° C. for 5 minutes, at 60 ° C. in fresh solution. Once for 1 hour, and once for 1 hour at 63 ° C. in fresh solution. After exposure to the X-ray film for 1.5 hours by the standard method, four strongly hybridized colonies were observed. These were isolates, 22G12, 25A10, 26A5 and 26B10 as well as TcaC-specific probes.

The TcaBi probe solution was diluted with an equal volume (about 100 mL) of "minimal hybrid" solution and then used to screen membranes containing 800 members of the genomic library. After exposure to hybridization, washing and X-ray film as described above, only four cosmid clones 22G12, 25A10, 26A5 and 26B10 were found to hybridize strongly to this probe.

<Isolation of subclones containing genes encoding TcaC and YcaBi peptides and determination of their DNA base sequences>

Three hybridization-positive cosmids in strain XL1 Blue MR were grown overnight with shaking (200 rpm) at 30 ° C. in TB-Amp ₁₀₀ . After harvesting the cells by centrifugation, cosmid DNA was prepared using a commercially available kit (BIGprep®, 5 Prime 3 Prime, Inc., Boulder, Colorado) according to manufacturer's protocol. Only one cosmid, 26A5, was successfully isolated by this procedure. When digested with restriction enzyme EcoR 1 (NEB) and analyzed by electrophoresis, fragments of approximate sizes 14, 10, 8 (vector), 5, 3.3, 2.9 and 1.5 kbp were detected. A second attempt to isolate cosmid DNA from the same three strains (8 mL culture; TB-Amp ₁₀₀ , 30 ° C.) was carried out using the boiling miniprep method [Evans G. and G. Wahl., 1987, “Cosmid vectors for genomic walking and rapid restriction mapping. " in Guide to Molecular Cloning Techniques. Meth. Enzymology, Vol. 152, S. Breger and A. Kimmel, eds., Pgs. 604-610] was used. Only one cosmid, 25A10, was successfully isolated by this method. When digested with restriction enzyme EcoR 1 (NEB) and analyzed by gel electrophoresis, this cosmid exhibited the same fragmentation pattern as previously seen in cosmid 26A5.

0.15 μg of a sample of 26A5 cosmid DNA was used to transform 50 ml of E. coli DH5α cells (Gibco BRL) according to the supplier's protocol. A single colony isolate of the strain was inoculated in 4 ml of TB-Amp ₁₀₀ and grown at 37 ° C. for 8 hours. Chloramphenicol was added to a final concentration of 225 μg / ml and the culture continued for 24 hours, after which the cells were harvested by centrifugation and frozen at -20 ° C. Isolation of 26A5 cosmid DNA was carried out by standard alkaline lysis miniprep (Maniatis et al., See literature, p. 382) and increased all volumes to 50% with stirring or light mixing, rather than vortexing at all stages. Modified. After washing the DNA pellet in 70% ethanol, it was dissolved in TE with 25 μg / ml ribonuclease (from Boehringer Mannheim).

<Identification of EcoR I Fragments Hybridized to GZ4-Induced and TcaBi-Probes>

Approximately 0.4 μg of cosmid 25A10 (extracted from XL1 Blue MR cells) and about 0.5 μg cosmid 26A5 (extracted from chloramphenicol-amplified DH5α cells) were digested with EcoR I (NEB) about 15 units for 85 minutes and frozen overnight. And then heated at 65 ° C. for 5 min and electrophoresed in 0.7% agarose gel [Seakem® LE, 1 × TEA, 80 volts, 90 min]. DNA was stained with ethidium bromide as described above and photographed under ultraviolet light. The EcoR I digest of cosmid 25A10 was completely degraded, but the sample of cosmid 26A5 only partially degraded under these conditions. Agarose gels containing DNA fragments are depurinated, denatured, neutralized and then Magna NT nylon membranes using a high salt (20X SSC) protocol as described in section 2.9 of Ausubel et al (CPMB, see literature). Southern blotting was performed on the image. The transferred DNAs were then UV-crosslinked to the nylon membrane as described above.

TcaC-peptide specific DNA fragments corresponding to inserts of plasmid isolate GZ4 were amplified by PCR (registered trademark) in 100 ml reaction volume as described above. The amplification products from these three reactions were pooled, extracted from 1% GTG (registered trademark) agarose gel by Qiaex kit as described above, and quantified by fluorimeter. 100 ng of gel purified DNA was labeled with a specific activity of 6.34 × 10 ⁸ dpm / μg with ³² P-dCTP using High Prime Labeling Mix (Boehringer Mannheim) as described above.

³² P-labeled GZ4 probe was boiled for 10 minutes, then added to 1 ng / ml of "minimal hybrid" buffer, Southern blot membrane containing digested cosmid DNA fragment was added and gently shaken at 50 rpm. Incubated at 60 ° C. for 4 hours. The membranes were then washed three times at 25 ° C. for about 5 minutes each (minimum hybrid wash solution) and then twice at 60 ° C. for 30 minutes each. The blots were exposed to the film (along with the strengthening screen) at −70 ° C. for about 30 minutes. GZ4 probes were strongly hybridized to the 5.0 kbp (apparent size) EcoR I fragments of two cosmids 26A5 and 25A10.

The membrane was boiled in 0.1 × SSC + 0.1% SDS for about 30 minutes to remove radioactivity and exposed to film to confirm the absence of radiolabel. This was then hybridized at 60 ° C. for 3.5 hours using a modified TcaB _i probe in “minimal hybrid” buffer previously used for screening of the colony membrane, washed as described above, and with two strengthening screens − The film was exposed to the film at 70 ° C. for 40 minutes. When both cosmids were used, the TcaB _i probe was weakly hybridized with about 5.0 kbp EcoR 1 fragment and strongly hybridized with about 2.9 kbp fragment.

Samples of cosmid 26A5 DNA (from DH5α cells) were used as DNA sources for subcloning the desired bands. 2.5 μg of this DNA was digested with about 3 units of EcoR 1 (NEB) in a total volume of 30 μl for 1.5 hours to obtain a partial digest and confirmed by gel electrophoresis. 10 μg of pBC KS (+) DNA (manufactured by Stratagene) was digested with 20 units of EcoR 1 in a total volume of 20 μl for 1.5 hours and completely digested and confirmed by electrophoresis. Both EcoR 1 and cleaved DNA preparations were diluted to 50 μl with water, equal volume of PCI was added to each, the suspension was gently mixed, spun with a microcentrifuge and the aqueous supernatant was collected. DNA was precipitated with 150 μl of ethanol and the mixture was left overnight at -20 ° C. After centrifugation and drying, EcoR 1 and digested pBC KS (+) were dissolved in 100 μl of TE and partially digested 26A5 was dissolved in 20 μl of TE. DNA recovery was confirmed by fluorimeter.

In a separate reaction, about 60 ng of EcoR I and digested pBC KS (+) DNA were ligated with about 180 ng or 270 ng of partially digested cosmid 26A5 DNA. Ligation was performed for 5 hours in 20 μl volume at 15 ° C. using T4 ligase and buffer from New England BioLabs. Ligation mixtures diluted to 100 μl with sterile TE were used to transform frozen qualified DH5α cells (Gibco BRL) according to the supplier's instructions. Different amounts (25-200 μl) of transformed cells were plated on freshly prepared solid LB-Cam ₃₅ medium with 1 mM IPTG and 50 mg / 1 X-gal. The plate was incubated at 37 ° C. for about 20 hours and then cooled in the dark for about 3 hours to enhance color for insert selection. White colonies were plated on patch plates of the same composition and incubated overnight at 37 ° C.

Two colony lifts of each selected patch plate were prepared as follows. After white colonies were selected for a new plate, the spherical Magna NT nylon membrane was pressed onto the patch plate, the membrane was lifted and allowed to denature, neutralize and UV crosslink as described above for the library colony membrane. The crosslinked colony lift was washed vigorously and gently wiped off excess cell debris with a tissue. One set was hybridized with the GZ4 (TcaC) probe solution described above according to the "least hybrid" protocol, and the other set was hybridized with the TcaB _i probe solution described above, then washed and described for the library colony membrane. As exposed to the film.

Colonies showing hybridization signals with only GZ4 probes, with both GZ4 and TcaB _i , or only with TcaB _i probes were selected for further study, with IPTG and X-gal for isolation of single colonies as above. Cells were streaked on LB-Cam ₃₅ medium. About 35 single colonies from 16 different isolates are placed in liquid LB-Cam ₃₅ medium, grown overnight at 37 ° C., cells are harvested by centrifugation, and plasmid DNA is described in Maniatis et al. , p. 368] by standard alkaline lysis miniprep. DNA pellets were dissolved in TE + 25 μg / ml ribonuclease A and the DNA concentration was measured with a fluorometer. EcoR I digestion patterns were analyzed by gel electrophoresis. The following isolates were usefully used. Isolate A17.2 contains only the religated pBC KS (+) and was used as a (negative) control. Isolates D38.3 and C44.1 contain only 2.9 kbp TcaB _i -hybridization EcoR I fragments inserted in pBC KS (+), respectively. These plasmids pDAB2000 and pDAB2001 are shown in FIG. 2, respectively.

Isolate A35.3 contains only about 5 kbp of GZ4-hybridization EcoR 1 fragment inserted into pBC KS (+). This plasmid was named pDAB2002 (see FIG. 2). These isolates provided a template for DNA sequencing.

Plasmids pDAB2000 and pDAB2001 were prepared using the BIGprep® kit. 30 ml of culture was grown overnight in TB-Cam ₃₅ until OD ₆₀₀ became 2, and then the plasmid was isolated according to the manufacturer's instructions. DNA pellets were redissolved in 100 μl of TE, respectively, and the integrity of the samples was confirmed by EcoR I digestion and gel electrophoresis analysis.

The sequencing reaction was performed in duplicate using one copy using pDAB2000 DNA as a template and the other copy using pDAB2001 DNA as a template. The reaction was performed using dideoxy dye terminator cycle sequencing as described above for sequencing of GZ4 / HB14 DNA. Initial sequencing used primers based on the LacZ and T7 primers as well as the determined sequences of the TcaB _i PCR amplification product (TH1 = ATTGCAGACTGCCAATCGCTTCGG, TH12 = GAGAGTATCCAGACCGCGGATGATCTG).

After arrangement and editing of each sequencing result, each sequence was cut to 250 to 350 bases according to the integrity of the chromosomal data translated by the Perkin Elmer Applied Biosynthesis Division SeqEd 675 software. Subsequent sequencing “steps” were performed by selecting the appropriate sequence for the new primer. With a few exceptions, the primer (synthesized as above) had a length of 24 bases with a 50% G + C composition. Sequencing by this method was performed for all strands of the about 2.9 kbp EcoR I fragment.

For use as template for DNA sequencing, plasmid DNA from isolate pDAB2002 was prepared by BIGprep® kit. Sequencing reactions were performed and analyzed as described above. Initially, sequencing reactions from adjacent vector sequences were primed with insert DNA using T3 primer (pBS KS (+) bases 774-796: CGCGCAATTAACCCTCACTAAAG) and T7 primer (pBS KS (+) bases 621-643: GCGCGTAATACGACTCACTATAG). Read. Subcloned oligonucleotides of the primer-sequenced oligonucleotide sequence were prepared by different primer sets (GZ4F: GTATCGATTACAACGCTGTCACTTCCC; TH13: GGGAAGTGACAGCGTTGTAATCGATAC; TH14: ATGTTGGGTGCGTCGGCTAATGGACATAAC; and LW1-204: GGGAAGTGACAGCGTTGTAATCGATAC) to determine the sequence of the cloned oligonucleotide-PCR It was. A new set of primers was designed from the data generated during the initial cycle of sequencing and used to prime the total length of about 5 kbp fragments. A total of 55 oligo primers were used to confirm the total sequence of 4832 bp.

When the DNA sequence of the EcoR I fragment insert of pDAB2002 was combined with a portion of the crystal sequence of the pDAB2000 / pDAB2001 isolate, a total of 6005 bp of contiguous sequence was generated (expressed as SEQ ID NO: 25). When translating the long open reading frame to the corresponding amino acid, the sequence clearly showed the TcaB _i N-terminal peptide (shown in SEQ ID NO: 3) encoded by bases 68-124 immediately before the methionine residue (translation start). Upstream is a potential ribosomal binding site (bases 51-58) and downstream encodes the TcaB _i -PT158 internal peptide (as represented by SEQ ID NO: 19) at bases 215-277. Furthermore, in base 1787-1822 downstream of the same translation frame, there is a sequence encoding the TcaB _i -PT108 internal peptide (as represented by SEQ ID NO: 20). In addition, TcaB _ii N-terminal peptide (SEQ ID NO: 5) is encoded at bases 1946-1972 of the same translation frame, and the translation frame continues uninterrupted until the translation termination codon at nucleotides 3632-3634.

TcaB _i of the sequence ends and encoding the -PT108 TcaB _ii coding start part-frame (in-frame) the lack of a stop codon between the regions, and the lack of TcaB _ii coding region a ribosome binding site that can be identified immediately upstream of the Peptides TcaB _ii and TcaB _i are encoded by a single open translation frame of 3567 bp starting at base pair 65 of SEQ ID NO: 25, and by post-translational degradation a single primary gene of 1189 amino acids (131,586 daltons; represented by SEQ ID NO: 26) Most likely from the product TcaB. If the amino acid immediately preceding the TcaB _ii N-terminal peptide represents the C-terminal amino acid of the peptide TcaB _i , the expected mass of TcaB _ii (627 amino acids) is 70,814 Daltons (denoted as SEQ ID NO: 28) and is observed by SDS-PAGE Is somewhat larger than the size (68 kDa). This peptide will be encoded by a continuous stretch of 1881 base pairs (as represented by SEQ ID NO: 27). Native C- terminus of the TcaB _i is considered to be in close proximity than in the C- terminal of a TcaB _i -PT108. The molecular weight of PT108 (3.438 kDa; measured during N-terminal amino acid sequencing of this peptide) can predict the size of 30 amino acids. Using the size of this peptide to represent the C-terminus of the TcaB _i coding region (Glu at position 604 of SEQ ID NO: 28), the derived size of TcaB _i was determined to be 604 amino acids or 68,463 daltons, resulting in experimental observations. More consistent with

Translation of the TcaB _ii peptide coding region of 1686 base pairs (as represented by SEQ ID NO: 29) yields a protein of 562 amino acids (as represented by SEQ ID NO: 30) with an expected mass of 60,789 Daltons, which is in good agreement with the observed 61 kDa.

Potential ribosomal binding sites (bases 3682-3687) are found 48 bp downstream of the stop codon of the tcaB open reading frame. A sequence encoding the N-terminus of peptide TcaC (SEQ ID NO: 2) at bases 3694-3726 is found. The open reading frame initiated by this N-terminal peptide continues undisturbed up to base 6005 (2361 base pairs, represented by the first 2361 base pairs of SEQ ID NO: 31). The gene encoding the total TcaC peptide (apparent size about 165 bp; about 1500 amino acids) (tcaC) will comprise about 4500 bp.

In addition, another isolate E20.6, comprising a cloned EcoR I fragment of cosmid 26A5, was identified by homology with the aforementioned GZ4 and TcaB _i probes. Agarose gel analysis of the EcoR 1 digest of the DNA of the plasmids (pDAB2004, FIG. 2) contained in this strain showed insert fragments of estimated sizes 2.9, 5 and 3.3 kbp. DNA sequence analysis disclosed from primers designed from the sequence of plasmid pDAB2002 showed that a 3.3 kbp EcoR 1 fragment of pDAB2004 was present adjacent to the 5 kbp EcoR 1 fragment shown in pDAB2002. The 2361 base pair open reading frame found in pDAB2002 continues undisturbed with the other 2094 bases in pDAB2004 (denoted 2362 to 4458 of base pair 31). DNA sequence analysis using parent cosmid 26A5 DNA as a template confirmed the continuity of the open reading frame. In addition, the open reading frame (TcaC SEQ ID NO: 31) comprises 4455 base pairs and encodes a protein (TcaC) of 1485 amino acids (denoted as SEQ ID NO: 32). The calculated molecular size 166,214 Daltons matched the estimated size of the TcaC peptide (165 kDa) and the derived amino acid sequence exactly matched the sequence disclosed in the TcaC N-terminal sequence (SEQ ID NO: 2).

The absence of an amino acid sequence corresponding to SEQ ID NO: 17 used to design a synonymous oligonucleotide primer pool in the found sequence indicates that the PCR (registered trademark) product found in isolates GZ4 and HB14 used as probes in the initial library screen. It is shown that the formation of by chance occurs by reverse-strand priming by one of the primers of the synonym pool. In addition, the derived protein sequence does not comprise an internal fragment represented as SEQ ID NO: 18. These sequences indicate that plasmid pDAB2004 contains the complement coding region of the TcaC peptide.

Further analysis of SEQ ID NO: 25 shows the end of the open reading frame (bases 1-43) encoding the last 13 amino acids of TcaA _iii disclosed herein as SEQ ID NO: 35. Only 24 bases separate the start and end of the coding region of the TcaB _i TcaA _iii coding region. Sequences that can act as ribosomal binding sites are included within 24 bases. Although possible, it is unlikely that the photolabduose gene promoter will be encoded within this short region. A genomic region tca comprising three long open reading frames (tcaA (SEQ ID NO: 33), tcaB (SEQ ID NO: 25, bases 65-36334) and tcaC (SEQ ID NO: 31), separated from the tcaB termini by only 59 bases) is defined by The initial upstream of the beginning of SEQ ID NO: 33) is transcribed and regulated as an operon while producing polycistronic messenger RNA.

<Example 9>

Screening of TcbA _ii Peptide Coding Genes in the Photorabdus Genome Library

This example describes the method used to identify DNA clones comprising the TcbA _ii peptide coding gene, isolation of the gene and determination of partial DNA base sequences thereof.

Primer and PCR Reactions

The TcbA _ii polypeptide of the insect active agent is about 206 kDa. The N-terminal amino acid sequence of this peptide is shown as SEQ ID NO: 1. As described in Example 8, four pools of synonymous oligonucleotide primers (“forward primers”: TH-4, TH-5, TH-6 and TH-7) were encoded to encode a portion of this amino acid sequence. It was synthesized and shown below.

In addition, the primary ("a") and secondary ("b") sequences of the internal peptide preparation (TcbA _ii -PT81) were determined and denoted as SEQ ID NO: 23 and SEQ ID NO: 24, respectively. Four pools of synonymous oligonucleotides (“reverse primers”: TH-8, TH-9, TH-10 and TH-11) are similarly designed and the reverse complement of the sequence encoding a portion of the peptide of SEQ ID NO: 23 It was synthesized as follows to code.

This set of primers was used in a PCR reaction to amplify the TcbA _ii coding gene fragment from the genomic photolabduum luminescence W-14 DNA prepared in Example 6. All PCR reactions were performed with the "Hot Start" technique using AmpliWax® gems and other Perkin Elmer reagents and protocols. In general, a total volume of 11 μl of MgCl ₂ , dNTP's, 10X GeneAmp® PCR Buffer II and a mixture of primers were added to a tube containing a single wax bead [10X GeneAmp® PCR Buffer II was 100 mM Tris-HCl, pH 8.3; And 500 mM KCl. The tube was heated to 80 ° C. for 2 minutes and cooled. On top of the wax seal was added a solution containing 10 × GeneAmp® PCR Buffer II, DNA template and AmpliTaq® DNA polymerase. Final reaction conditions (50 μl in volume) after melting the wax seal and heat cycling to mix the components were 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl ₂ ; 200 μM of dATP, dCTP, dGTP, dTTP; 1.25 mM single prospective primer pool; 1.25 μΜ single reverse primer pool, 1.25 units of AmpliTaq® DNA polymerase and 170 ng of template DNA.

The reaction was placed in a thermal cycler as in Example 8 and run with the following program.

Temperature time Cycle repeat 94 ℃ 2 mins 1X 94 ℃ 15 seconds 30X 55-65 ℃ 30 sec 30X 72 ℃ 1 minute 30X 72 ℃ 7 mins 1X 15 ℃ calendar

A series of amplifications were performed at three different annealing temperatures (55, 60, 65 ° C.) using synonymous primer pools. The reaction annealed at 65 ° C. had no visible amplification product after agarose gel electrophoresis. The reaction annealed at 60 ° C. and comprising primer TH-5 + TH-10 produced an amplification product with mobility corresponding to 2.9 kbp. Use of primer TH-7 + TH-10 under these conditions yielded a smaller amount of 2.9 kbp product. When the reaction was annealed at 55 ° C., the primer pairs produced more 2.9 kbp product, which was also produced by primer pairs TH-5 + TH-8 and TH-5 + TH-11. Additional very faint 2.9 kbp bands were found in the lanes containing amplification products from primer pairs TH-7 + TH-8, TH-9, TH-10 or TH-11.

Ten separate PCR reactions were prepared using primers TH-5 + TH-10 to obtain sufficient PCR amplification products for cloning and DNA sequencing and run using the above conditions at annealing temperatures of 55 ° C. All reactions were pooled and the 2.9 kbp product was purified by Qiaex extraction from agarose gel as described above.

Another sequence determined for the TcbA _ii internal peptide is shown in SEQ ID NO: 21 and SEQ ID NO: 22. As described above, synonymous oligonucleotides (reverse primers TH-17 and TH-18) were prepared to correspond to the reverse complement of the sequence encoding a portion of the amino acid sequence of this peptide.

Oligonucleotides TH- synonymous for amplification experiments using photolapux luminescence W-14 DNA as template and primers TH-4, TH-5, TH-6 or TH-7 as 5'- (forward) primers 18 and TH-17 were used. These reactions amplified products of about 4 kbp and 4.5 kbp, respectively. These DNAs were transcribed from agarose gels to nylon membranes and hybridized with ³² P-labeled probes (as described above) prepared from a 2.9 kbp product amplified by TH-5 + TH-10 primer pairs. Both 4 kbp and 4.5 kbp amplification products were strongly hybridized with the 2.9 kbp probe. Using these results, a map of the TcbA _ii internal peptide sequences shown in FIG. 3 was prepared. Approximate distances between the primers are shown in nucleotides of FIG. 3.

DNA sequence of a 2.9 kbp TcbA _ii coding fragment

About 200 ng of the purified 2.9 kbp fragment prepared above was precipitated using ethanol and dissolved in 17 ml of water. Half of this solution was used as sequencing template with 25 pmol of TH-5 pool as primer and the other half as template for TH-10 priming. Sequencing reactions were carried out as shown in Example 8. When the TH-10 primer pool was used, no reliable sequence was generated, but the reaction using the TH-5 primer pool produced the following sequence.

Based on this sequence, the sequencing primer (TH-21, 5'-CCGGGCGACGTTTATCTAGG-3 ') was designed with reverse complement bases 120-139 of the 5' end of the gel-purified 2.9 kbp TcbA _ii coding PCR fragment (ie Polymerization was initiated towards the TH-5 end). The determined sequence is as follows and compared with the biochemically determined N-terminal peptide sequence of TcbA _ii (SEQ ID NO: 1).

TcbA _ii 2.9 kbp PCR Fragment Sequence Identification

(Underlined amino acid = encoded by synonymous oligonucleotide)

It is clear from the homology of the derived amino acid sequence with the biochemically determined sequence that the 2.9 kbp PCR fragment represents the TcbA coding region. This 2.9 kbp fragment was then used to screen the Photorabdus W-14 genomic library prepared in Example 8 for cosmids containing the TcbA _ii coding gene.

Screening of Photorapdus Cosmid Library

2.9 kb gel Purified PCR fragments were labeled with ³² P using the Boehringer Mannheim High Prime labeling kit as described in Example 8. A filter comprising residues of about 800 colonies from the cosmid library was screened as described above in Example 8, and positive clones were streaked and rescreened for isolation colonies. Three clones (8A11, 25G8 and 26D1) showed positive results through several screening and characterization steps. The TcbA _ii specific probe did not hybridize with any of the four cosmids containing the tcaB and tcaC genes identified in Example 8. DNA from cosmids 8A11, 25G8 and 26D1 was digested with restriction enzymes Bgl II, EcoR I or Hind III (alone or mixed with each other), fragments were separated on agarose gels and as described in Example 8 Transferred to nylon membrane. Membranes were hybridized with ³² P-labeled probes prepared from 4.5 kbp fragments (generated by amplifying photolapux genomic DNA with primer TH-5 + TH-17). The pattern generated from cosmid DNA 8A11 and 26D1 was identical to that generated from genomic DNA similarly cleaved on the same membrane. It was concluded that cosmids 8A11 and 26D1 accurately represent the genomic TcbA _ii coding locus. However, cosmid 25G8 has one Bgl II fragment that is slightly larger than genomic DNA. This may be due to the insert present in the vector.

DNA sequence of the tcbA coding gene

Membrane hybridization analysis of cosmid 26D1 showed that the 4.5 kbp probe hybridized to one large EcoR I fragment (greater than 9 kbp). This fragment was gel purified and ligated to the EcoR I site of pBC KS (+) as described in Example 8 to generate plasmid pBC-S1 / R1. The partial DNA sequence of the insert DNA of this plasmid was determined by "primer walking" from adjacent vector sequences using the procedure described in Example 8. New oligonucleotides designed from predetermined sequences were stretched to generate other sequences. Complete homology when compared to the determined DNA sequence of the tcbA gene (designated as SEQ ID NO: 11 as will be described in Example 12) identified by other methods is shown in nucleotides 1-272, 319-826, 2578-3036 and 3068-3540 ( Total base 1712). Two methods were used to determine the DNA fragment encoding the TcbA _ii peptide.

Analysis of Derived Amino Acid Sequences of the tcbA Gene

The sequence of the DNA fragment identified in SEQ ID NO: 11 encodes the protein in which the derived amino acid sequence is represented by SEQ ID NO: 12. Several features confirm the identity of the gene encoding the TcbA _ii protein. TcbA _ii N-terminal peptide (SEQ ID NO: 1; Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala) is encoded as amino acids 88-100. TcbA _ii internal peptide TcbA _ii -PT81 (a) (SEQ ID NO: 23) is encoded as amino acids 1065-1077 and TcbA _ii -PT81 (b) (SEQ ID NO: 24) is encoded as amino acids 1571-1592. In addition, the internal peptide TcbA _ii- PT56 (SEQ ID NO: 22) is encoded as amino acids 1474-1488 and the internal peptide TcbA _ii- 103 (SEQ ID NO: 21) is encoded as amino acids 1614-1639. It is evident that this gene is a true clone that encodes a TcbA _ii peptide isolated from the pesticidal protein preparation of photolapux luminescence strain W-14.

Proteins isolated as peptide TcbA _ii are derived from degradation of longer peptides. Evidence for this is due to the fact that nucleotides encoding the TcbA _ii N-terminal peptide (SEQ ID NO: 1) are followed by 261 bases (coding 87 N-terminal-near amino acids) in a longer open reading frame (SEQ ID NO: 11). Is provided. This translation frame begins with the nucleotides corresponding to the N-terminal sequence of the large peptide TcbA and encoding the amino acid sequence Met Gln Asn Ser Leu represented by SEQ ID NO: 16. TcbA is thought to be the precursor protein of TcbA _ii .

Relationship between the tcbA, tcaB and tcaC genes

tcbB and tcaC genes are closely related and can be transcribed as one mRNA (Example 8). The tcbA gene is present on cosmids that do not clearly overlap with those containing tcaB and tcaC clusters because each genomic library screens for identified different cosmids. However, comparing the amino acid sequences encoded by the tcaB and tcaC genes with the tcbA gene, it can be seen that there is a substantial degree of homology. Amino acid conservation (Protein Alignment Mode of MacVector® sequencing software, scoring matrix pam250, hash value = 2; Oxford Molecular Group, Campbell, Calif.) Is shown in FIG. 4. In FIG. 4 the upper caret (＾) in the score line of each panel represents homologous or conservative amino acid changes, and the lower caret (v) represents nonhomologous.

This analysis shows that the amino acid sequences of TcbA peptide residues 1739-1894 are highly homologous to amino acids 441-603 of the TcaB _i peptide (162 of 627 amino acids in total of TcaB; SEQ ID NO: 28). In addition, the sequences of TcbA amino acids 1932 to 2459 are highly homologous to amino acids 12 to 531 (520 of a total of 562 amino acids; SEQ ID NO: 30) of peptide TcaB _ii . Considering that the TcbA peptide (SEQ ID NO: 12) contains 2505 amino acids, near its C-terminus, a total of 684 amino acids (27%) are homologous to the TcaB _i or TcaB _ii peptide, and this homology is the putative TcaB proprotein. (Linear 26) is arranged in a straight line. A significant gap in the TcbA homolog corresponds to the junction between the TcaB _i and TcaB _ii portions of the TcaB proprotein. Obviously, the TcbA and TcaB gene products are evolutionarily related, suggesting that they share some common function (s) in Photorapdus.

<Example 10>

Characterization of zinc-metalloprotease in photolapus broth; Protease Inhibition, Classification and Purification

Protease Inhibition and Classification Assays: Protease assays were performed using FITC-casein dissolved in water as substrate (final assay concentration 0.08%). Proteolytic reactions were carried out at 25 ° C. for 1 hour with 25 μl of Photorabdus broth (150 μl total reaction volume) in a suitable buffer. Samples were also analyzed in the presence or absence of dithiothreitol. After incubation, an equivalent amount of 12% trichloroacetic acid was added to precipitate undigested protein. After precipitation for 0.5 h and centrifugation, 100 μl of the supernatant was placed in a 96-well microtiter plate and the same amount of 4N NaOH was added to adjust the pH of the solution. Proteolysis was then quantified using a Fluoroskan II fluorescent plate reader at excitation and emission wavelengths of 485 and 538 nm, respectively. Protease activity was tested in 0.5 units over pH 5.0-10.0. The following buffer was used at a final concentration of 50 mM: sodium acetate (pH 5.0-6.5), Tris-HCl (pH 7.0-8.0), and bis-Tris propane (pH 8.5-10.0). To confirm the type of protease (s) observed, the crude broth was treated with different protease inhibitors (final concentration 0.5 μg / μl) followed by protease activity at pH 8.0 using the substrate. Protease inhibitors used include E-64 (L-trans-epoxysakinylsilamido [4-,-guanidino] -butane), 3,4-dichloroisocoumarin, leupeptin, pepstatin, amastatin, Ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline.

Protease analysis conducted over a specific pH range showed that there was a protease (s) showing maximum activity at pH about 8.0 (Table 17). The addition of DTT had no effect on protease activity. The crude broth was then treated with different protease inhibitors (Table 18). Treatment of crude broth with the above inhibitors completely inhibited all protease activity as IC ₅₀ = 5 μg in 100 μl of 2 mg / ml crude broth solution when 50 μg final concentration was added. I could see that. These data indicate that most of the abundant protease (s) found in photolapus broth are enzymes of the zinc-metalloprotease class.

Effect of pH on the Protease Activity Found in the Daily Production of Photolapux Luminesense (Strain W-14) pH Fluorescent unit ^a Percentage active ^b 5.0 3013 ± 78 17 5.5 7994 ± 448 45 6.0 12965 ± 483 74 6.5 14390 ± 1291 82 7.0 14386 ± 1287 82 7.5 14135 ± 198 80 8.0 17582 ± 831 100 8.5 16183 ± 953 92 9.0 16795 ± 760 96 9.5 16279 ± 1022 93 10.0 15225 ± 210 87 a fluorescence unit = fluorescence unit (maximum = about 28,000; background = about 2200) b percentage activity against the maximum at pH 8.0

Effect of Different Protease Inhibitors on Protease Activity at pH 8 Found in the Daily Production of Photolapux Luminesense (Strain W-14) Inhibitor Calibrated fluorescence unit ^a Percentage suppression ^b Control 13053 0 E-64 14259 0 1,10 phenanthroline ^c 15 99 3,4 dichloroisocoumarin ^d 7956 39 Leupeptin 13074 0 Pepstatin ^c 13441 0 Amastatin 12474 4 DMSO control 12005 8 Methanol control 12125 7 a Corrected fluorescence unit = fluorescence unit-background (2200 fluorescence units) b Percent inhibition of protease activity at pH 8.0 c Dissolve the inhibitor in methanol. d Dissolve the inhibitor in DMSO.

The dialyzed 10-80% ammonium sulphate pellets were added to a Q Sepharose column equilibrated at 50 mM Na ₂ PO ₄ , pH 7.0 to describe zinc-metalloprotease as described in Example 5 for Photolabduus toxin. Isolated. After a strong wash, the toxin protein was eluted using a 0-0.5 M NaCl gradient. Most biological activity and proteins were eluted from 0.15 to 0.45 M NaCl. However, it was observed that most proteolytic activity is present in the 0.25-0.35 M NaCl fraction and a small amount of activity is present in the 0.15-0.25 M NaCl fraction. SDS-PAGE analysis of the 0.25-0.35 M NaCl fraction showed a peptide main band of about 60 kDa. The 0.15-0.25 M NaCl fraction included a similar 60 kDa band but had a lower relative protein concentration. This fraction was then subjected to gel filtration using a SuperRoose 12 HR 16/50 column, with the main peak moving at 57.5 kDa, including the 58.5 kDa band predominantly by SDS-PAGE analysis (greater than 90% of total stained protein). Got. Further analysis of the fraction using various protease inhibitors as described above determined that the protease was zinc-metalloprotease. Almost all protease activity present in photolapus broth at day 1 of fermentation corresponded to about 58 kDa zinc-metalloprotease.

Using W-14 photolabdus broth grown for 3 days in the second isolation of zinc-metalloprotease (s), see Schmidt, T.M., Bleakley, B. and Nealson, K.M. Gelatin-bound sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to visualize protease activity as described in 1988]. SDS developing gel (5.5 × 8 cm) is a 12.5% polyacrylamide (acrylamide / bis-acrylamide) that incorporates a final concentration of 0.1% gelatin (Biorad EIA grade reagent; Richmond, CA) upon dissolution in water. 40% stock solution of Sigma Chemical Co., St. Louis, Missouri, USA. SDS-stacking gels (1.0 × 8 cm) were prepared using 5% polyacrylamide with 0.1% gelatin bound. In general, 2.5 μg of test protein was diluted in 0.03 mL of SDS-PAGE loading buffer without dithiothreitol (DTT) and loaded onto gel. Proteins were electrophoresed in SDS development buffer (Laemmli, U.K. 1970. Nature 227, 680) at 0 ° C. and 8 Hz. After the electrophoresis was terminated, the gel was washed for 2 hours in 2.5% (v / v) Triton X-100. The gel was then incubated for 1 hour at 37 ° C. in 0.1 M glycine, pH 8.0. After incubation, the gels were fixed and stained overnight with 0.1% amido black in methanol-acetic acid-water (30:10:60, v / v / v; Sigma Chemical Co.). Proteolytic activity was visualized as a bright area against dark background stained with Amido black by proteolysis and subsequent diffusion of bound gelatin. At least three specific bands produced by proteolytic activity at 58, 41 and 38 kDa were observed.

The activity of different proteases in the W-14 3 day culture broth was analyzed using FITC-casein dissolved in water as substrate (final assay concentration 0.02%). Proteolysis experiments were performed at 37 ° C. for 0-0.5 h in 0.1 M Tris-HCl (pH 8.0) using different protein fractions with a total volume of 0.15 ml. An equal volume of 12% trichloroacetic acid (TCA) dissolved in water was added to terminate the reaction. After incubation at room temperature for 0.25 hours, the samples were centrifuged at 10,000 x g for 0.25 hours and 0.10 ml aliquots were placed in 96-well microtiter plates. An equal volume of 2 N sodium hydroxide was then added to neutralize the solution, followed by quantification with excitation and emission wavelengths of 485 and 538 nm, respectively, using a Fluoroskan II fluorescent plate reader. Activity was measured at 37 ° C. for 0-10 minutes using FITC-casein at different protease concentrations. Units of activity were optionally defined as the amount of enzyme needed to produce 1000 fluorescent units / min, and specific activity was defined as units / protease mg.

Two zinc-metalloprotease inhibitors, 1,10-phenanthroline and N- (a-ramnopyranosyloxyhydroxyphosphinyl) -Leu-Trp, with stock solutions of inhibitors dissolved in 100% ethanol and water, respectively Inhibition studies were conducted using (phosphoramidon). Stock concentrations were generally 10 mg / ml and 5 mg / ml for 1,10-phenanthroline and phosphoramidone, respectively, and the final concentration of inhibitor was 0.5-1.0 mg / ml per reaction. Treatment of W-14 crude broth on day 3 with 1,10-phenanthroline, an inhibitor of all zinc-metalloproteases, completely eliminated all protease activity and inhibited thermosine-like proteases [Weaver, LH , Kester, WR, and Matthews, BW 1977. J. Mol. Biol. 114, 119-132] treated with phosphoramidone reduced protease activity by about 56%. Residual proteolytic activity will not be able to be further reduced with further phosphoramidon treatment.

The protease of W-14 Photorapdus Broth on Day 3 was purified as follows: Concentration of 4.0 L broth was carried out using Amicon spiral ultrafiltration cartridge Type S1Y100 attached to Amicon M-12 filtration apparatus. Amicon helical ultrafiltration cartridge Type S1Y100 attached to an Amicon M-12 filtration device was used to concentrate 3.8 L of passage material with natural protein less than 100 kDa to 0.375 L. Residual material included proteins of size 10 to 100 kDa. This material was loaded onto a Pharmacia HR16 / 10 column filled with PerSeptive Biosystem (Pramington, MD) Poros® 50 HQ strong anion exchange filler equilibrated in 10 mM sodium phosphate buffer, pH 7.0. Protein was loaded onto the column at a flow rate of 5 ml / min and the unbound protein was washed with buffer until A ₂₈₀ = 0.00. The protein was then eluted using a NaCl gradient of 0-1.0 M NaCl within 40 minutes at a flow rate of 7.5 ml / min. As above, fractions were analyzed for protease activity and the active fractions collected. Fractions showing proteolytic activity were diluted in 50% (v / v) 10 mM sodium phosphate buffer (pH 7.0) and loaded onto a Pharmacia HR 10/10 Mono Q column equilibrated in 10 mM sodium phosphate. The column was washed with buffer until A ₂₈₀ = 0.00, then the protein was eluted using a NaCl gradient of 0-0.5 M NaCl for 1 hour at a flow rate of 2.0 ml / min. Fractions were analyzed for protease activity. Phosphoramidone sensitive protease activity was by 41/38 kDa protease (see below) and fractions with the highest phosphoramidone sensitive protease activity were collected. This fraction was found to elute at 0.15-0.25 M NaCl. Fractions containing predominantly phosphoramidone insensitive protease activity, 58 kDa protease were also collected. This fraction was found to elute at 0.25-0.35 M NaCl. The phosphoramidon sensitive protease fractions were then concentrated to a final volume of 0.75 mL using Millipore Ultrafree®-15 centrifugal filtration apparatus Biomax-5K NMWL membrane. This material was loaded at a flow rate of 0.5 ml / min on a Pharmacia HR10 / 30 column packed with Pharmacia Sephadex G-50 equilibrated in 10 mM sodium phosphate buffer (pH 7.0) /0.1 M NaCl. Fractions with maximum phosphoramidon sensitive protease activity were then collected and centrifuged using Millipore Ultrafree®-15 centrifugal filtration apparatus Biomax-50K NMWL membrane. Such proteolytic activity analysis indicated that this material had only phosphoramidone sensitive protease activity. Phosphoramidon insensitive protease, 58 kDa protein was collected, then concentrated in Millipore Ultrafree ™ -15 centrifugal filtration apparatus Biomax-50K NMWL membrane and further separated on a Pharmacia Superdex-75 column. Fractions containing this protease were collected.

Analysis of purified 58 and 41/38 kDa proteases showed that both proteases were completely inhibited with 1,10-phenanthroline but only 41/38 kDa protease was inhibited with phosphoramidone. Further analysis of crude broth showed that the protease activity of W-14 protease at day 1 was 23% of the total protease activity due to 41/38 kDa protease, and increased to 44% at day W-14 broth. Showed that

Standard SDS-PAGE analysis was performed using a 4-20% gradient MiniPlus SepraGels available from Integrated Separation Systems, Natick, Mass., To investigate protein purity and obtain amino terminal sequences. After purification, the protein to be sequenced at the amino terminus is blotted onto a PVDF membrane (ProBlott® membrane from Applied Biosystems, Foster City, Calif.), Visualized with 0.1% amido black and incised to cut and sequenced for Mass. Sent to Cambridge Prochem, Cambridge, USA.

The estimated amino terminal sequences of the 58 (SEQ ID NO: 45) and 41/38 kDa (SEQ ID NO: 44) proteases from the 3 day old W-14 broth were DV-GSEKANEKLK (SEQ ID NO: 45) and DSGDDDKVTNTDIHR (SEQ ID NO: 44), respectively.

Sequencing of the 41/38 kDa protease showed several amino termini, each with additional amino acids removed by proteolysis. The primary, secondary, tertiary and fourth sequencing of the 38 and 41 kDa polypeptides allowed the estimation of the sequences and showed that these two proteases were homologous.

<Example 11a>

Screening Using Antibodies from the Photorabdus Genome Library Against TcbA Peptide Coding Gene

Concurrent with sequencing described above, appropriate probing and sequencing was performed based on the TcbA _ii peptide (SEQ ID NO: 1). This sequencing was performed by preparing bacterial culture broths and purifying the toxins described in Examples 1 and 2 above.

Genomic DNA was isolated from photolabduum luminescence strain W-14 grown in Grace's insect tissue culture medium. The bacteria were grown for about 24 hours at 28 ° C. and 250 rpm in 5 ml of culture medium in a 250 ml Erlenmeyer flask. Bacteria cells were pelleted at 5000 × g for 10 minutes from 100 ml of culture medium. After discarding the supernatant, cell pellets were used for genomic DNA isolation.

Genomic DNA was isolated using a modified CTAB method described in Ausubel, section 2.4.3, supra. Subsequently, a section entitled “Large Scale CsCl prep of bacterial genomic DNA” was conducted via step 6. At this time, an additional chloroform / isoamyl alcohol (24: 1) extraction is performed followed by a phenol / chloroform / isoamyl (25: 24: 1) extraction step and finally a chloroform / isoamyl / alcohol (24: 1) extraction. Was carried out. DNA was precipitated by adding 0.6 volume of isopropanol. Precipitated DNA was wound around a curved glass rod, briefly soaked in 70% ethanol and finally washed, and dissolved in 3 ml of TE buffer.

The DNA concentration estimated by optical density at 280/260 nm was about 2 mg / nm.

The library was prepared using the genomic DNA. About 50 μg of genomic DNA was partially digested with Sau3 A1. Subsequently, partial digested DNA fragments were size fractionated using NaCl density gradient centrifugation. Fractions containing DNA fragments with an average size of at least 12 kb, as determined by agarose gel electrophoresis, were ligated into plasmid BluScript from Stratagene, La Jolla, Calif., And transformed into E. coli DH5α or DHB10 strains.

Separately, purified aliquots of the protein were sent to the Biotechnology Hybridoma Center of the University of Wisconsin, Madison, USA, for the production of monoclonal antibodies against the protein. The material sent to this center was an HPLC purified fraction containing natural bands 1 and 2 denatured at 65 ° C., of which 20 μg were injected into 4 mice, respectively. Stable monoclonal antibody-producing Hibrooma cell lines were recovered after fusion of splenocytes of non-immunized mice with stable myeloma cell lines. Monoclonal antibodies were recovered from hybridoma cells.

Separately, native agarose gel purification band 1 (see Example 1) protein was used to immunize New Zealand white rabbits to generate polyclonal antibodies. The band was cut from a natural agarose gel and the gel pieces were heated to 65 ° C. to melt the agarose and immediately emulsify with adjuvants to prepare proteins. Freund's complete adjuvant was used for primary immunization and an additional 3 injections at 1 month intervals with Freund's incomplete adjuvant. About 0.2 ml of an emulsified band 1 containing 50-100 μg of protein for each injection was delivered by multiple subcutaneous injections in the rabbit's back. Serum was obtained 10 days after the last injection and blood was collected again after 3 weeks. Serum supplements were inactivated by heating to 56 ° C. for 15 minutes and then stored at −20 ° C.

The monoclonal and polyclonal antibodies were then used to screen genomic libraries for expression of antigens that can be detected by epitopes. Positive clones were detected on nitrocellulose filter colony lift. Immunoblot analysis was performed for positive clones.

Four genomic regions were experimentally identified through analysis of clones identified by immunoblot and Southern analysis.

In the clone of the first region there was a gene encoding a peptide named TcbA _ii . The full DNA sequence of this gene (TcbA) was obtained and shown as SEQ ID NO: 11. Confirmation that this sequence encodes the internal sequence of SEQ ID NO: 1 is evidenced by the presence of SEQ ID NO: 1 at amino acid number 88 from the putative amino acid sequence generated by the open translation frame of SEQ ID NO: 11. This can be confirmed through SEQ ID NO: 12, the putative amino acid sequence produced by SEQ ID NO: 11.

Toxin peptides in the second region include the segments referred to as TcaB _i , TcaB _ii and TcaC. After screening the library using polyclonal antiserum, several clones that produce proteins of different sizes were found to cross-react with polyclonal antibodies on the immunoblot and also share DNA homology on the southern blot. The toxin gene of the said 2nd region was confirmed by this. Sequence comparisons revealed that they belonged to the gene complexes named TcaB and TcaC.

Two different regions of antibody toxin clones were isolated on polyclonal screens. These regions produced proteins that cross-react with polyclonal antibodies and shared DNA homology with regions measured by Southern blotting. Thus, the photolapus luminescence extracellular protein gene appears to represent a group of genetically related genes.

To further confirm the notion that evolutionary related mutations may exist in the toxin peptides contained within these organisms, two methods of testing different strains of photolapus luminescence for the presence of related proteins are carried out. It was. This was done by both PCR amplification of genomic DNA and immunoblot analysis using polyclonal and monoclonal antibodies.

This result indicates that the related proteins are photolabduus luminescence strains WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-11, WX-12, WX Produced by -15 and WX-14.

<Example 11b>

Sequencing and Analysis of tcc Toxin Clones

Other DNA sequencing was performed on the plasmids isolated from the E. coli clone described in Example 11a. The nucleotide sequence from the third region of the E. coli clone was found to be three closely related open translation frames in the genomic locus. This locus was named tcc with three open reading frames named tccA (SEQ ID NO: 56), tccB (SEQ ID NO: 58), and tccC (SEQ ID NO: 60). A similar association between these open reading frames is shown in SEQ ID NO: 56 where 93 bp separates the stop codon of tccA from the initiation codon of tccb (bases 2992-2994, SEQ ID NO: 56), or 131 bases of tccB and tccC (bases 4930-4932). , SEQ ID NO: 58 to isolate the stop codon of SEQ ID NO: 58). The natural map is shown in Figure 6B.

The amino acid sequence estimated from the tccA open reading frame represents a gene encoding a protein of 105,459 Da. This protein was named TccA (SEQ ID NO: 57). The first 12 amino acids of this protein match the N-terminal sequence obtained from the 108 kDa protein (SEQ ID NO: 8) identified above as part of the toxin complex.

The amino acid estimated from the tccB open reading frame represents a gene encoding a protein of 175,716 Da. This protein was named TccB (SEQ ID NO: 59). The first 11 amino acids of this protein match the N-terminal sequence obtained from the protein with an estimated molecular weight of 185 kDa (SEQ ID NO: 7). Similar analysis results, TccB protein identified as TcbA (SEQ ID NOs: 12, 37% similar and 28% identical), TcdA (SEQ ID NOs: 47, 35% similar and 28% identical) and TcaB (SEQ ID NOs: 26, 32% similar and 26% identical) The GAP algorithm Wisconsin package version 9.0 from Genetics Computer Group (GCG) of Madison, Wisconsin.

The putative amino acid sequence of tccC indicates that this open reading frame encodes a protein of 111,694 Da and the resulting protein was named TccC (SEQ ID NO: 61).

<Example 12>

Characterization of Photorapduus Strains

In order to confirm that the collections described herein consisted of photolabose strains, the strains of the present invention are characterized by photolabdues and are distinguished from other Enterobacteriaceae and Xenorhabdus species. In terms of the resulting microbiological characteristics [Farmer, JJ 1984. Bergey's Manual of Systemic Bacteriology, vol 1. pp. 510-511. (ed.Kreig N.R. and Holt, J.G.). Williams & Wilkins, Baltimore .; Akhurst and Boemare, 1988, Boemare et al., 1993). These distinctive features include Gram-negative bacillus strains, organism sizes ranging from 0.5 to 2 μm in width, 2 to 10 μm in length, red / yellow colony staining, presence of crystal inclusions, presence of catalase, nitrate reduction ability, presence of bioluminescence, growth Retention of dye uptake from the medium, formation of positive proteases, growth temperature range below 37 ° C., survival and active motility under anaerobic conditions, and the like (Table 20). Reference Escherichia coli, Genorabdus and Photorapdus strains were included in all tests for comparison. Overall results were consistent with all strains that are part of the genus Enterobacteriaceae and Photorapdus.

A luminometer was used to confirm bioluminescence of each strain, quantify light production and measure relative. For determination of relative luminescence units, broths of each strain (cells and media) were inoculated into liquid culture and measured at three intervals (6, 12 and 24 hours) and compared with background luminescence (non-inoculated media and water). . Prior to measuring luminescence from various broths, absorbance (560 nm) was measured in a Gilford Systems (Oberlin, Ohio) spectrometer using a sipper cell to confirm cell density. A suitable dilution was then prepared (to normalize the optical density to 1.0 unit) before measuring the luminescence. Subsequently, aliquots of dilute broth were placed in cuvettes (300 μl each) and read on a Bio-Orbit 1251 luminometer (Bio-Orbit Oy, Twiku, Finland). The total time for each sample was 45 seconds. Samples were read to provide oxygen availability while continuously mixing (rotating in a baffle cuvette). Positive testing was determined to be at least 5 times the background luminescence (about 5 to 10 units). In addition, colony luminescence was measured visually using a photographic film overlay after being maintained in the dark. Gram staining characteristics of each strain were confirmed with Gram staining kit (BBL, Cockisville, Maryland) used with commercially available Gram staining control slides (fisher Scientific, Pittsburgh, PA). Subsequently, microscopic evaluation was performed using a Zeiss microscope (from Carl Zeiss, Germany) 100X oil immersion objective lens (with 10X eyepiece and 2X sieve magnification). Microscopic examination of each strain for organism size, intracellular and inclusion bodies (after log phase growth) was performed using wet mount slides (10X eyepiece, 2X body magnification, 40X objective) with oil immersion and phase contrast microscopes. Akhurst, RJ and Boemare, N.E. 1990. Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R. and Kaya, H.). pp. 75-90. CRC Press, Boca Raton, USA .; Baghdiguian S., Boyer-Giglio M.H., Thaler, J. O., Bonnot G., Boemare N. 1993. Biol. Cell 79, 177-185. Colony staining was observed after inoculation on Bacto nutritional agar (Difco Laboratories, Detroit, Mich.) Prepared according to the manufacturer's instructions. Incubation was at 28 ° C. and occurred after 5-7 days. To test for the presence of enzyme catalase, colonies of test organisms were removed from the nutrient agar plates on a small plug and placed in the bottom of a glass test tube. 1 ml of household hydrogen peroxide solution was gently added along the side of the test tube. Positive responses were recorded when bubbles of gas (estimated oxygen) occurred immediately or within 5 seconds. Controls of vaccinated nutrient agar and hydrogen peroxide solution were also investigated. To test nitrate reduction, each culture was inoculated in 10 ml of Bacto nitrate broth (Difco Laboratories, Detroit, Mich.). After 24 hours incubation at 28 ° C., 2 drops of sulfanylic acid reagent and 2 drops of alpha-naphthylamine reagent (see Difco Manual, 10th edition, Difco Laboratories, Detroit, Michigan, 1984) were tested for nitrite production. The distinctive pink or red formation indicates the formation of nitrite from nitrates. The ability of each strain to absorb dyes from the growth medium can include Bacto MacConkey agar, including dye neutral red; Bacto Tergitol-7 agar with dye bromothymol blue and Bacto EMB agar with dye eosin-Y (Difco Laboratories, Detroit, Mich., All prepared according to manufacturer's instructions) were tested. After inoculation on the medium, the dye uptake was recorded by incubating at 28 ° C. for 5 days. Growth on these media is characteristic of the Enterobacteriaceae family. The motility of each strain was tested using a solution of Bacto Motility test medium (Difco Laboratories, Detroit, Mich.) Prepared according to the manufacturer's instructions. Butt-stab inoculation was performed using each strain, and motility was determined microscopically by the growth diffusion region spread from the inoculation line. In many cases, motility was observed microscopically from liquid culture under wet mount slides. Biochemical nutritional assessment for each strain was carried out using BBL Enterotube II (Benton, Dickinson, Germany). According to the manufacturer's instructions, the incubation was carried out at 28 ° C. for 5 days. The results were in agreement with the record for Photorabdus quoted above. Protease production was tested by hydrolyzing gelatin by preparing Bacto gelatin (Difco Laboratories, Detroit, Mich.) Plates according to the manufacturer's instructions. Cultures were inoculated and plates were incubated at 28 ° C. for 5 days. To assess growth at different temperatures, agar plates (2% Proteose peptone 3 (2 from Difco, Detroit, Mich.) With 2% Bacto-Agar in deionized water) were streaked with a conventional inoculum source. Plates were sealed with Nesco® film and incubated at 20, 28 and 37 ° C. for up to 3 weeks. Plates showing no growth at 37 ° C. did not show cell viability after being transferred to a 28 ° C. incubator for 1 week. Oxygen demand for the Photorabdus strain was tested as follows. Butt-stop inoculation was performed with fluid thioglycolate broth medium (Difco, Detroit, Mich.). After incubation of the tube for 1 week at room temperature, the cultures were examined for type and extent of growth. Indicator resazurin indicates media oxidation levels or aerobic living areas (Difco Manual, 10th edition, Difco Laboratories, Detroit, MI). The growth area results obtained for the photolabduus strains tested were consistent with those for the anaerobic microorganisms.

*-A = Gram strain, B = Crystal inclusion body, C = bioluminescence, D = cell morphology, E = motility, F = nitrate reduction, G = presence of catalase, H = gelatin hydrolysis, I = dye uptake, J = Coloring, K = growth on EMB agar, L = growth on MacConkey agar, M = growth on Tergitol-7 agar, N = anaerobic bacteria, O = growth at 20 ° C, P = growth at 28 ° C, Q = growth at 37 ° C, †-+/- = positive or negative of features, rd = bacilli, S = size within the genus descriptor range, RO = red-orange, LR = light red, R = red, O = orange , Y = yellow, T = tan, LY = pale yellow, YT = pale yellow, and LO = pale orange.

Cellular fatty acid analysis is an identification tool for characterizing bacteria at the genus and species level (Tornabene, TG 1985. Lipid Analysis and the Relationship to Chemotaxonomy in Methods in Microbiology, Vol 18, 209-234 .; Goodfellow, M. and O'Donnell, AG 1993. Roots of Bacterial Systematics in Handbook of New Bacterial Systematics (ed. Goodfellow, M. & O'Donnell, AG) pp.3-54.London: Academic Press Ltd., the disclosures of these references (Incorporated herein by reference), the collection of the invention was used to confirm that it was relevant at the genus level. The cultures were sent to an external contract laboratory for fatty acid methyl ester analysis (FAME) using the Microbial ID (MIDI, Newark, Delaware, USA) using the Microbial Identification System (MIS). The MIS system consists of Hewlett Packard HP5890A gas chromatography with 25 mm × 0.2 mm 5% methylphenyl silicone fused silica capillary column. Hydrogen is used as the carrier gas and a flame-ionization detector functions with the automated sampler, integrator and computer. The computer compares the fatty acid methyl ester of the sample with the microbial fatty acid library and known fatty acid assay calibration mixture. Strains selected in contract laboratories were grown on tryticase soybean agar for 24 hours at 28 ° C. prior to analysis. Sampling was performed in a contract laboratory by standard FAME methods. There was no direct identification of strains against other groups of luminescent bacteria other than photolabose. When performing a cluster analysis comparing the fatty acid profiles of Group 1 isolates, strain fatty acid profiles were related at the genus level.

The evolutionary diversity of the Photorabdus strains in the collection of the present invention was determined by PCR (polymerase chain reaction) mediated genomic fingerprint analysis using the genomic DNA of each strain. This technique is based on a group of repeat DNA sequences that exist throughout the genome of various bacterial species (Versalovic, J., Schneider, M., DE Bruijn, FJ and Lupski, JR 1994. Methods Mol. Cell. Biol. , 5, 25-40). Of these sequences, the repeat extragenic palindromic sequence (REP), the enteric bacterial repeat gene common sequence (ERIC), and the BOX component are believed to play an important role in the organization of the bacterial genome. Genomic tissue is believed to be shaped by selection, and different dispersions of these components in the genome of closely related bacterial strains can be used for identification of these strains (eg Louws, FJ, Fulbright, DW, Stephens, CT). and DE Bruijn, FJ 1994. Appl. Environ. Micro. 60, 2286-2295). Rep-PCR uses oligonucleotide primers complementary to the repeat sequence to amplify DNA fragments of different sizes that exist between the repeat sequences. The product is separated by electrophoresis to establish the DNA "fingerprint" for each strain.

To isolate genomic DNA from the strain of the present invention, the cell pellet is resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to a final volume of 10 ml and then 12 ml of 5 M NaCl. Was added. This mixture was centrifuged at 15,000 × g for 20 minutes. The resulting pellet was resuspended in 5.7 mL TE and 300 μL 10% SDS and 60 μL 20 mg / mL proteinase K (Gibco BRL, Grand Island, NY) was added. After incubating the mixture at 37 ° C. for 1 hour, about 10 mg of lysozyme was added and the mixture was incubated for an additional 45 minutes. Subsequently, 1 ml of 5 M NaCl and 800 µl of CTAB / NaCl solution (10% w / v CTAB, 0.7 M NaCl) are added, the mixture is incubated at 65 ° C. for 10 minutes, gently shaken and incubated, and an additional 20 Shaking for minutes aided in washing of cellular material. An equal volume of chloroform / isoamyl alcohol solution (24: 1, v / v) was added, mixed gently and centrifuged. Then extracted twice using an equal volume of phenol / chloroform / isoamyl alcohol (50: 49: 1). Genomic DNA was precipitated using 0.6 volume of isopropanol. The precipitated DAN was collected with glass sticks, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantified by optical density at 260 nm. In order to perform rep-PCR analysis of photolabdus genomic DNA, the following primers were used: REP1R-I; 5'-IIIICGICGICATCIGGC-3 'and REP2-I; 5'-ICGICTTATCIGGCCTAC-3 '. PCR was performed using the following 25 μl reactions: 7.75 μl H ₂ O, 2.5 μl 10X LA buffer (PanVera Corp., Madison, WI), 16 μl dNTP mixture (2.5 mM each), each primer 1 μl (50 pM / μl), 1 μl DMSO, 1.5 μl genomic DNA (concentration 0.075 to 0.480 μg / μl) and 0.25 μl TaKaRa EX Taq (PanVera Corp., Madison, Wisconsin). PCR amplification was carried out in a Perkin Elmer DNA thermal cycler (Norwalk, CT) using the following conditions: 95 ° C./7 min, followed by 94 ° C./1 min, 44 ° C./1 min, 65 ° C./8 min. Repeat 35 times, followed by 15 minutes at 65 ° C. After cycling, 25 μl of the reaction was added to 5 μl of 6 × gel loading buffer (0.25% bromophenol blue, 40% w / v sucrose). A 15 × 20 cm 1% agarose gel was then developed in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) using 8 μl of each reaction. The gel was run at 45 V for about 16 hours. The gel was then stained for 1 hour in 20 μg / ml ethidium bromide and destained for about 3 hours in TBE buffer. Polaroid pictures of the gel were printed under UV light.

The presence or absence of a band at a size specific to each stain was recorded from the photographs and entered as a similar matrix in the numerical classification software program NTSYS-pc (Exeter Software, Setauquet, NY). E. coli strains HB101 and Xanthomonas duck fib. A control of duck (Xanthomonas oryzae pv. Oryzae) produced a "fingerprint" according to published literature (Versalovic, J., Koeuth, T. and Lupski, JR 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, CM, Halda-Alija, L., Louws, F., Skinner, DZ, George, ML, Nelson, RJ, DE Bruijn, FJ, Rice, C. and Leach, JE 1995. Int.Rice Res. Notes, 20, 23-24 .; Vera Cruz, CM, Ardales, EY, Skinner, DZ, Talag, J., Nelson, RJ, Louws, FJ, Leung, H., Mew, TW and Leach, JE 1996. Phytopathology). Then a series of programs in NTSYS-pc; SIMQUAL (similarity to quantitative data) and related strains to generate similarity coefficients (using Jaccard coefficients) of the matrix and SAHNs (continuous, cohesive, high) that can be pooled and represented as phenograms (FIG. 5). Data from Photorabdus strains were analyzed using Rakikal and Nested clustering [using UPGMA (Unweighted Pair-Group method using arithmetic mean)]. (Cophenetic values) and the MXCOMP (matrix comparison) program were used to generate a cohenetic matrix and to compare the relationship between this and the original matrix on which clustering is based. A normalized Mantel statistic (r) was produced which was produced phosphorus (r = 0.8 to 0.9 indicating very good suitability), in the case of the present invention r is 0 Therefore, the collection of the present invention consists of easily identifiable strains from a diverse and representative group of the genus Photorapdus.

<Example 13>

Insecticidal Utility of Toxin (s) Produced by Various Photoractus Strains

In 175 ml of 2% Proteose Peptone # 3 (PP3, Difco Laboratories, Detroit, MI) liquid medium in a 500 ml Tribaffle flask with Kaput Primary strain subclones were inoculated to prepare initial seed cultures of the various photolapse strains. Inoculum for each seed culture was derived from an oil covered agar slope culture or plate culture. After inoculation, the flasks were incubated at 28 ° C. for 16 hours at 150 rpm on a rotary shaker. The seed culture was then used as a uniform inoculum for a given fermentation of each strain. The post-log seed cultures are also covered with sterile mineral oil, a sterile magnetic stir bar is added for future resuspension, and the culture is stored in the dark, at room temperature, thereby inoculating the inoculum toxin production. The state was stored for a long time. Production broth was inoculated by adding 1% of actively growing seed culture (eg, 1.75 ml per 175 ml of fresh culture) to 2% PP3 fresh medium. The broth was produced in a 500 ml tribaffle flask (see above) or a 2800 ml baffle uneven bottom flask (500 ml capacity) sealed with a silicone foam stopper. Production flasks were incubated for 24 to 48 hours under these conditions. After incubation, the broth was dispensed into cells and lysate pellets by dispensing into sterile 1 L polyethylene bottles and spinning at 2600 × g for 1 hour at 10 ° C. The liquid broth was then vacuum filtered through Whatman GF / D (2.7 μm retention) and GF / B (1.0 μm retention) glass filters to remove debris. The broth was further purified using a 0.5 μm open conduit filter with a tangential outflow microfiltration device (Pall Filtron, Northborough, Mass.). If necessary, the broth can be further clarified by cooling (up to 4 ° C.) and centrifuging at 2600 × g for several hours. After this procedure, the broth was filtered and sterilized using a 0.2 μm nitrocellulose membrane filter. Sterile broth was then either used directly for biological, biochemical analysis or a molecular weight 10000 cutoff M12 ultrafiltration apparatus (manufactured by Amicon, Beverly, Mass.) Or a centrifugal concentrator with a pore size of molecular weight 10000 (Massachusetts) Concentrated (up to 15 times) using Millipore, Bedford and Paul Filtron, Notsboro, Mass., USA. For centrifugal concentrators, the broth was centrifuged at 2000 × g for about 2 hours. A molecular weight 10000 permeate was added to the corresponding residue to give the desired concentrate of components with molecular weight greater than 10000. The treated broth sample was heat inactivated by heating the sample at 100 ° C. for 10 minutes in a sand filled heat block.

Broth (s) and toxin complex (s) from different photolabose strains are useful for reducing insect populations, and insects consisting of using the described active substances in insect habitats in an amount effective to inactivate insects. It was used in the population suppression method. Evidence of the range of pesticidal activity observed from broths of the select group of photolabduus strains fermented as above is shown in Table 20. Increasing the broth concentration or using other fermentation methods allows detection of additional pesticidal activity in the strain. Consistent with the activity associated with the protein, the pesticidal activity of all strains tested was heat labile (see above).

Culture broth (s) from various photolabose strains exhibit differential pesticidal activity (decrease in mortality and / or growth inhibition, reduction in adult appearance) with respect to insect numbers. More specifically, activity is shown against corn rootworm larvae and cotton weevil larvae that are members of the Coleoptera insect throat. Other members of the beetle neck include bugs, pollen beetles, jump flea beetles, seed beetles and Colorado potato beetles. Activity is also observed for star cicada and corn extinct, which are members of the same tree. Other members of the same tree include extinction, pear tree, apple tree, beetle, white fly, stingerworm and numerous host specific aphid strains. The broth and purified toxin complex (es) also have activity against gray tobacco moth, horn moth and European corn borer, which are members of the Lepidoptera. Other typical members of the neck include beetroot maggots, cabbage bugs, black beetles, corn ear beetles, apple moths, antagonists, gallery moths, leaf moths, cabbage beetles, cotton beetle moths, salamanders, eastern tent moths and grass. There are insect moths and pole matrix maggots. Activity is also shown for fruit flies and mosquito larvae that are members of the fly. Other members of the fly tree include pea worms, carrot flies, cabbage root flies, turnip root flies, onion flies, school flies and house flies and various mosquito species. The broth (s) and toxin complex (s) are also active against spotted spider mites that are members of mite that include strawberry ant mites, broad mites, citrus red mites, European red mites, pear green mites and tomato reddish brown mites Indicates.

Activity against corn rootworm larvae was tested as follows. Photolapdus culture broth (s) (0-15 fold concentration, filtration sterilization), 2% proteose peptone # 3, purified toxin complex (s), 10 mM sodium phosphate buffer at pH 7.0 40 μl were applied directly to about 1.5 cm ² ) [Rose, RI and McCabe, JM (1973). J. Econ. Entomol. 66, (389-400). Toxin complexes were diluted in 10 mM sodium phosphate buffer at pH 7.0. The food plate was air dried in a sterile flow hood and wells were infested with a single, newborn Diabrotica undecimpunctata howardi (Sudden Corn Root, SCR) hatched from surface sterilized eggs. The plates were sealed and placed in a wet growth chamber and kept at 27 ° C. for a suitable period (3 to 5 days). Mortality and larval weight measurements were then recorded. In general, 16 insects were used per treatment in all studies. Control mortality was generally less than 5%.

Activity against cotton weevil (Anthomonas grandis) was tested as follows. 0.35 g of concentrated (1-10 fold) photolabose broth, control medium (2% proteose peptone # 3), purified toxin complex (s) (0.23 g / ml) or 10 mM sodium phosphate buffer at pH 7.0 60 μl of the surface of artificial food (Stoneville Yellow lepidopteran food) was applied and dried. One 12 to 24 hour old cotton weevil larva was placed in the feed and the wells were sealed and kept at 25 ° C. for 5 days at 50% relative humidity. Thereafter, mortality and larval weight were evaluated. Control mortality was 0-13%.

Activity against mosquito larvae was tested as follows. Assays were done in 96 well microtiter plates. In each well, 200 μl of an aqueous solution (10-fold concentrated photolabose culture broth (s)), control medium (2% proteose peptone # 3), 10 mM sodium phosphate buffer, toxin complex (s) (0.23 mg / ml) or water and about 20, daily larvae (Aedes aegypti). 6 wells were used per treatment. Results were interpreted three to four days after infestation. Control mortality was 0-20%.

The activity against fruit flies was tested as follows. The Drosophila melanogaster medium purchased was 50% dry medium, water, control medium (2% proteose peptone # 3), 10-fold concentrated photolabose culture broth (s), tablets Prepared using 50% liquid of either toxin complex (s) (0.23 mg / ml) or 10 mM sodium phosphate buffer at pH 7.0. This was accomplished by placing 4.0 ml of dry media in each of three rearing vials and adding 4.0 ml of the appropriate liquid. Then 10 late Young Drosophila melanogas Maggots were added to each 25 ml vial. The vial was placed on a laboratory bench at room temperature under fluorescent lamps. After 15 days of exposure, pups or adult numbers were counted. Adult appearance was reduced (0-16% reduction) compared to water and control medium.

Ingestion assays designed to consume actives without other external contact were tested for activity against star larvae (Macrosteles severini) and corn plant beetle larvae (Peregrinus maidis). The reservoir of active / feed solution is made by drilling two holes in the bottom center of a 35 × 10 mm Petri dish. A square 2-inch Parafilm M is placed across the top of the dish and secured with an "O" ring. Thereafter, about 7 grasshoppers are placed in a 1 oz. Plastic cup and the reservoir is placed on top of the cup with the parafilm underneath. The test solution is then added to the reservoir through the hole. In a test using 10-fold concentrated photolabose culture broth (s), the broth and control medium (2% proteose peptone # 3) were dialyzed against 10 mM sodium phosphate buffer at pH 7.0 and as a result Sucrose (up to 5%) was added to the resulting solution to reduce control mortality. Purified toxin complex (es) (0.23 mg / ml) or 10 mM sodium phosphate buffer at pH 7.0 was also tested. The mortality rate is recorded on day 3. The analytes were stored in the incubator at 28 ° C. under 70% relative humidity with a 16/8 photoperiod. Analytes recorded mortality at 72 hours. Control mortality was less than 6%.

Activity against Lepidoptera larvae was tested as follows. Concentrated (10-fold) photolabose culture broth (s), control medium (2% proteose peptone # 3), purified toxin complex (s) (0.23 mg / ml) or 10 mM sodium phosphate buffer at pH 7.0 40 μl was applied directly to the surface of standard artificial lepidoptera food (Stoneville yellow food, about 1.5 cm ² ). The food plate was air dried in a sterile flow hood and each well was ramped up with one newborn larva. European corn borer (Ostrinia nubilalis) and Manuca sexta eggs were purchased and hatched, and gray tobacco moth (Heliothis virescens) larvae were supplied internally. After spreading with the larvae, the food plate was sealed and placed in the wet growth chamber and kept in the dark at 27 ° C. for an appropriate period. Lethality and weight measurements were performed on day 5. In general, 16 insects were used per treatment in all studies. The control lethality of the control medium was generally 4 to about 12.5% and the control lethality of the phosphate buffer was less than 10%.

Activity against spotted spider mite (Tetranychus urticae) was measured as follows. Young pumpkin plants are trimmed to single cotyledons and sprayed to flush 10-fold concentrated broth (s), control medium (2% proteose peptone # 3), purified toxin complex (s), 10 mM sodium phosphate buffer at pH 7.0 I was. After drying, the plants were infested with a mixed population of spider mites and held for 72 hours at laboratory temperature and humidity. The live ticks were then counted to determine the level of the control group.

Insecticidal range observations of broth from different photoractose strains Fotolapus species Susceptibility * Insect Species WX-1 3 **, 4, 5, 6, 7, 8 WX-2 2, 4 WX-3 1, 4 WX-4 1, 4 WX-5 4 WX-6 4 WX-7 3, 4, 5, 6, 7, 8 WX-8 1, 2, 4 WX-9 1, 2, 4 WX-10 4 WX-11 1, 2, 4 WX-12 2, 4, 5, 6, 7, 8 WX-14 1, 2, 4 WX-15 1, 2, 4 W30 3, 4, 5, 8 NC-1 1, 2, 3, 4, 5, 6, 7, 8, 9, WIR 2, 3, 5, 6, 7, 8 HP88 1, 3, 4, 5, 7, 8 Hb 3, 4, 5, 7, 8 Hm 1, 2, 3, 4, 5, 7, 8 H9 1, 2, 3, 4, 5, 6, 7, 8 W-14 1, 2, 3, 4, 5, 6, 7, 8, 10 ATCC 43948 4 ATCC 43949 4 ATCC 43950 4 ATCC 43951 4 ATCC 43952 4 * = At least 25% mortality and / or growth inhibition compared to the control ** = 1; Tobacco caterpillar, 2; European corn borer, 3; Tobacco hornworm, 4; Southern Corn Roots, 5; Cotton weevil, 6; Mosquito, 7; Fruit fly, 8; Astor extinction, 9; Corn plant grasshopper, 10; Spotted Spider Mite

Example 14

Non W-14 Photorapdu Strains: Purification, Characterization, and Scope of Activity

refine

The following experimental method is similar to that developed for the purification of W-14 and purifying only those fractions with the highest activity against Southern Corn Root (SCR) as determined in biological quantification (see Example 13). Was established on the basis of In general, as described in Example 13, filtered 4-20 L broth was received and concentrated using an Amicon helical ultrafiltration cartridge type S1Y100 attached to an Amicon M-12 filtration device. The residue contained natural proteins of molecular size greater than 100 kDa, and the permeate contained natural proteins of size less than 100 kDa. Most of the activity for SCR was contained in 100 kDa residues. The residue was then diafiltered with 10 mM sodium phosphate buffer (pH 7.0) until the A ₂₈₀ of the filtrate was less than 0.100. Unless otherwise noted, all treatments from now on were performed in buffer limited to 10 mM sodium phosphate (pH 7.0). The residue was then concentrated to a final volume of about 0.20 L and filtered using 0.45 mm sterile filtration units from Nalgene Filterware. Pharmacia filled with filtered material with Perceptep Biosystem Poros® 50 HQ strong anion exchange matrix equilibrated with buffer using PerSeptive Biosystem Sprint HPLC system ) Loaded into the HR16 / 10 column at 7.5 ml per minute. After loading, the column was washed with buffer until A ₂₈₀ was 0.100. Proteins were then eluted from the column at 50 ml total volume for 50 minutes at 2.5 ml per minute using a buffer with 0.4 M NaCl. The column was then washed further with buffer with 1.0 M NaCl at the same effluent rate for 20 minutes (final volume 50 ml). Proteins eluted with 0.4 M and 1.0 M NaCl were placed in separate dialysis bags (Spectra / Por membrane MWCO: 2000) and dialyzed overnight at 4 ° C. in 12 L buffer. Most were contained in a 0.4 M fraction, further by loading 20 ml of 0.4 M fraction at 0.75 ml per minute into a Pharmacia XK 26/100 column prefilled with Sepharose CL4B (from Pharmacia). The fractions were further collected on the basis of the A ₂₈₀ peak profile and the final dose 0.75 ml using a Millipore Ultrafree-15 centrifugal filtration device Biomax-50K NMWL membrane. Protein concentration was determined using the Biorad Protein Assay Kit, based on bovine gamma globulin as a standard.

Specialization

The natural molecular weight of the SCR toxin complex was determined using Pharmacia HR 16/50 precharged with Sepharose CL4B in buffer. The column was then calibrated using known molecular size proteins to calculate the approximate natural molecular size of the toxin. As shown in Table 21, the molecular size of the toxin complex ranged from 777 kDa for strain Hb to 1900 kDa for strain WX-14. Yields of the toxin complex also varied, producing 0.8 mg per liter of strain WX-12 and 7.0 mg per liter of strain Hb.

Proteins found in toxin complexes were examined for individual polypeptide sizes using SDS-PAGE analysis. In general, 20 mg of toxin complex protein from each strain was loaded on a 2-15% polyacrylamide gel (Integration Separation Systems) and loaded at 20 mA in Biorad SDS-PAGE buffer. Electrophoresis. After completion of electrophoresis, overnight coloring was performed on Biorad Coomassie blue R-250 (Coomassie blue R-250, methanol: acetic acid: water (2% in a volume ratio of 40: 10: 40) mixed solution). The gel was then decolorized in methanol: acetic acid: water (volume ratio 40:10: 40). The gel was then rinsed with water for 15 minutes and injected using a Personal Molecular Dynamics Personal Laser Densitometer. Lanes were quantified to estimate molecular size compared to Biorad high molecular weight standards ranging from 200 to 45 kDa.

Table 22 shows the size of the individual polypeptides that make up the SCR toxin complex from each strain. The size of the individual polypeptides ranged from 230 kDa for strain WX-1 to 16 kDa for strain WX-7. All strains except strain Hb had a polypeptide comprising a toxin complex of 160-230 kDa, 100-160 kDa and 50-80 kDa. The data indicate that in all cases the toxin's properties consist of large oligomeric protein complexes, although the toxin complex may vary depending on the strain in terms of peptide composition and composition.

Characterization of Toxin Complexes from Non-W-14 Photorapid Strains Strain Natural molecular weight approximation ^a Active fraction yield (mg / L) ^b H9 972,000 1.8 Hb 777,000 7.0 Hm 1,400,000 1.1 HP88 813,000 2.5 NC1 1,092,000 3.3 WIR 979,000 1.0 WX-1 973,000 0.8 WX-2 951,000 2.2 WX-7 1,000,000 1.5 WX-12 898,000 0.4 WX-14 1,900,000 1.9 W-14 860,000 7.5 a Natural molecular weight determined using a Pharmacia HR 16/50 column packed with Sepharose CL4B b amount of toxin complex recovered from the culture broth

Active range

As shown in Table 23, the toxin complexes purified from strains Hm and H9 were compared to the toxin complexes from strain W-14 to test the activity against various insects. The analysis was performed as described in Example 13. Toxin complexes from all three strains showed activity against tobacco bud worms, European corn borer, southern corn root beetle, and star cicada. In addition, toxin complexes from strains Hm and W-14 showed activity against spotted spider mites. In addition, the toxin complex from W-14 also showed activity against mosquito larvae. This data means that while the toxin complex has similar activity among some insect species, it may also exhibit differential activity against other insect species.

Approximate Value of Peptide Size (kDa) in Purified Toxin Complex from Non W-14 Photorapdus H9 Hb Hm HP88 NC-1 WIR WX-1 WX-2 WX-7 WX-12 WX-14 W-14 180 150 170 170 180 170 230 200 200 180 210 190 170 140 140 160 170 160 190 170 180 160 180 180 160 139 100 140 140 120 170 150 110 140 160 170 140 130 81 130 110 110 160 120 87 139 120 160 120 120 72 129 44 89 110 110 75 130 110 150 98 100 68 110 16 79 98 82 43 110 100 130 87 98 49 100 74 76 64 33 92 95 120 84 88 46 86 62 58 37 28 87 80 110 79 81 30 81 51 53 30 26 80 69 93 72 75 22 77 40 41 23 73 49 90 68 69 20 73 39 35 22 59 41 77 60 60 19 60 37 31 21 56 33 69 57 57 58 33 28 19 51 65 52 54 45 30 24 18 37 63 46 49 39 28 22 16 33 60 40 44 35 27 32 51 37 39 25 26 46 37 23 40 35 39 29

Insecticidal Range Observations of Purified Toxin Complexes from Photoractose Strains Photolapux strain Susceptibility * Insect Species Hm toxin complex 1 **, 2, 3, 5, 6, 7, 8 H9 toxin complex 1, 2, 3, 6, 7, 8 W-14 Toxin Complex 1, 2, 3, 4, 5, 6, 7, 8 * = ＞ 25% mortality rate or growth inhibition rate * = ＞ 25% mortality rate or growth inhibition rate ** = 1; Tobacco Bud Worm, 2; European corn borer, 3; Southern Corn Roots, 4; Mosquitoes, 5; Spotted spider mite, 6; Astor extinction, 7; Fruit fly, 8; Cotton weevil

<Example 15>

Subfraction of the Photoractus Protein Toxin Complex

Photolapus protein toxin complex was isolated as described in Example 14. About 10 mg of toxin was then loaded into a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl, pH 7.0, at an effluent rate of 1 ml / min. The column was washed with 20 mM Tris-HCl at pH 7.0 until the concentration of the optical phase at 280 nm returned to baseline absorbance. The protein attached to the column was eluted for 30 minutes at a rate of 1 ml / min using a 0 to 1.0 M linear concentration gradient NaCl dissolved in 20 mM Tris-HCl at pH 7.0. A single corn rootworm (SCR) biological quantification was performed by collecting the 1 ml fraction (see Example 13). Activity peaks were determined by diluting each fraction in turn in SCR biological quantification. Two activity peaks for SCR were observed, named A (eluted in about 0.2-0.3 M NaCl) and B (eluted in 0.3-0.4 M NaCl). The active peaks A and B were collected separately and subjected to the following three steps treatment to further refine both peaks.

Solid (NH ₄ ) ₂ SO ₄ was added to the protein fraction to a final concentration of 1.7 M. The protein was then loaded at 1 ml / min on a phenyl-Suprose 5/5 column equilibrated with 1.7 M (NH ₄ ) ₂ SO ₄ dissolved in 50 mM potassium phosphate buffer at pH 7. The proteins attached to the column are 1.7 M (NH ₄ ) ₂ SO ₄ , 0% ethylene glycol, 50 mM sodium phosphate at pH 7.0 to 25% ethylene glycol, 25 mM sodium phosphate ((NH ₄ ) ₂ SO _{4 at} pH 7.0 Eluted at a rate of 0.5 ml / min. Fractions were dialyzed overnight against 10 mM sodium phosphate at pH 7.0. Activity in each fraction for SCR was determined by biological quantitation.

Fractions with the highest activity were collected and loaded at 1 ml / min on a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl at pH 7.0. Proteins attached to the column were eluted at a rate of 1 ml / min with NaCl in a 0-1 M linear concentration gradient dissolved in 20 mM Tris-HCl at pH 7.0.

For the final step of purification, the highest active fractions were collected and loaded onto a second phenyl-superrose 5/5 column. Solid (NH ₄ ) ₂ SO ₄ was added to a final concentration of 1.7 M. The solution was then loaded at 1 ml / min on a column equilibrated with 1.7 M (NH ₄ ) ₂ SO ₄ dissolved in 50 mM potassium phosphate buffer at pH 7.0. The proteins attached to the column were eluted at 0.5 ml / min with a linear gradient of 1.7 M (NH ₄ ) ₂ SO ₄ , 50 mM potassium phosphate at pH 7.0 to 10 mM potassium phosphate at pH 7.0. Fractions were dialyzed overnight against 10 mM sodium phosphate buffer at pH 7.0. Activity in each fraction for SCR was determined by biological quantitation.

With this three step treatment, the final purified protein from peak A was named toxin A, and the final purified protein from peak B was named toxin B.

Characterization and Amino Acid Sequencing of Toxin A and Toxin B

In SDS-PAGE, toxin A and toxin B both contained two major peptides (> 90% of total Kumaji coloring protein), which were both 192 kDa (named A1 and B1) and 58 kDa (named A2 and B2, respectively). Included. Toxin A and toxin B both showed only one major band on natural PAGE, indicating that A1 and A2 are subunits of one protein complex and B1 and B2 are subunits of one protein complex. In addition, the natural molecular weight of both toxin A and toxin B was determined to be 860 kDa by gel filtration chromatography. Relative molar concentrations of A1 to A2 were determined to be one-to-one equivalent, as determined by densitometry analysis of the SDS-PAGE gel. Similarly, the B1 and B2 peptides were present at equal molar concentrations.

Toxin A and Toxin B were electrophoresed on 10% SDS-PAGE and blotted by transfer to PVDF membrane. For amino acid analysis and N-terminal amino acid sequencing, blots were sent to Harvard MicroChem and Cambridge ProChem, respectively. The N-terminal amino sequence of B1 was found to be identical to the TcbA _ii region (SEQ ID NOs. 12, 87-99) of the tcbA gene, SEQ ID NO: 1. The N-terminal specific sequence of peptide B2 was obtained (SEQ ID NO: 40). The N-terminal amino acid sequence of peptide B2 was identical to the TcbA _iii region (SEQ ID NOs: 12, 1935-1945) of the amino acid sequence estimated from the tcbA gene. Therefore, the B toxin mainly contains two peptides TcbA _ii and TcbA _iii which have been observed to be derived from the same gene product TcbA.

The N-terminal sequence of A2 (SEQ ID NO: 41) is unique when compared to TcbA _iii peptides and other peptides. The A2 peptide is shown as TcdA _iii (see Example 17). SEQ ID NO: 6 was found to be the mixed amino acid sequence of SEQ ID NO: 40 and 41.

Internal amino acid sequencing was also performed for peptides A1 and A2. For internal amino acid sequencing 10 μg of toxin A was electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes for blotting. After blots were stained with amido black, peptides A1 and A2, represented by TcdA _ii and TcdA _iii , respectively, were cut out of the blot and sent to Harvard microchem and Cambridge prochem. Peptides were digested with trypsin followed by HPLC chromatography to separate individual peptides. N-terminal amino acid analysis was performed on selected trypsin treated peptide fragments. The two internal amino acid sequences of peptide A1 (TcdA _ii -PK71, SEQ ID NO: 38 and TcdA _ii -PK44, SEQ ID NO: 39) were found to have significant homology with the putative amino acid sequence of the tcbA gene (SEQ ID NO: 12) TcbAii region. . Similarly, the N-terminal sequence of SEQ ID NO: 41 (SEQ ID NO: 41) and the two internal sequences (TcdA _iii -PK57, SEQ ID NO: 42 and TcdA _iii -PK20, SEQ ID NO: 43) also represent the tcbA gene (SEQ ID NO: 12) TcbA _iii There was marked homology with the putative amino acid sequence of the region.

Summarizing the results, the toxin complex has two or more active protein toxin complexes of toxin A and toxin B for SCR. Toxin A and toxin B are similar in their natural and subunit molecular weights, but the composition of these peptides is different. Toxin A contains peptides TcdA _ii and TcdA _iii as main peptides, and toxin B contains peptides TcbA _ii and TcbA _iii as main peptides.

Purification and Characterization of Toxin C, Tca Peptides

Photolapus protein toxin complex was isolated as described above. About 50 mg of toxin was then added to a MonoQ 10/10 column equilibrated with 20 mM Tris-HCl at pH 7.0 at a flow rate of 2 ml / min. The column was washed with 20 mM Tris-HCl at pH 7.0 until the optical density at 280 nm returned to baseline level. The protein bound to this column was eluted using a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl at pH 7.0 at 2 ml / min for 60 minutes. Fractions of 2 ml were pooled and subjected to Western analysis using pAb TcaB _ii- syn antibody (see Example 21) as the primary antibody. Fractions reacted with the pAb TcaB _ii- syn antibody were collected and solid (NH ₄ ) ₂ SO ₄ was added until the final concentration was 1.7M. The protein was then added to a phenyl-superrose 10/10 column equilibrated with 1.7 M (NH ₄ ) ₂ SO ₄ in 50 mM potassium phosphate buffer at pH 7 at 1 ml / min. 1.7M (NH ₄₎ at 1ml / min for 120 minutes to a protein that is bound to this column and eluted using a linear gradient of ₂ SO _4, 10mM potassium phosphate, 50mM of potassium phosphate of pH 7.0 to pH 7.0. Fractions of 2 ml were combined and dialyzed overnight with 10 mM sodium phosphate buffer at pH 7.0 and analyzed by Western blot using pAb TcaB _ii- syn antibody as the primary antibody.

Fractions cross reacted with the antibody were pooled and added to a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl at pH 7.0 at 1 ml / min. The protein bound to this column was eluted at 1 ml / min by a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl at pH 7.0 for 30 minutes.

Fractions reacted with the pAb TcaB _ii- syn antibody were collected and added to the Phenyl-Superrose 5/5 column. Solid (NH ₄ ) ₂ SO ₄ was added until the final concentration was 1.7M. This solution was then added to a column equilibrated with 1.7 M (NH ₄ ) ₂ SO ₄ in 50 mM potassium phosphate buffer at pH 7.0 at 1 ml / min. The protein bound to this column was then eluted for 60 minutes using a linear gradient of 1.7 M (NH ₄ ) ₂ SO ₄ , 50 mM potassium phosphate at pH 7.0 to 10 mM potassium phosphate at pH 7.0. Fractions were dialyzed overnight with 10 mM sodium phosphate buffer at pH 7.0.

In the final purification step, fractions reacted with the pAb TcaB _ii- syn antibody measured above by Western analysis were collected and added to a MonoQ 5/5 column equilibrated with 20 mM Tris-HCl at pH 7.0 at 1 ml / min. The protein bound to this column was eluted at 1 ml / min by a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl at pH 7.0 for 30 minutes.

The final purified protein fraction contained six major peptides, 165kDa, 90kDa, 64kDa, 62kDa, 58kDa and 22kDa, tested by SDS-PAGE. The LD50 of the pesticidal activity of this purified fraction was determined to be 100ng and 500ng for SCR and ECB, respectively.

The peptides were blotted into PVDF membranes and blots were sent to Harvard MicroChem and Cambridge ProChem for amino acid analysis and five amino acid N-terminal sequencing, respectively. The N-terminal amino acid sequence of the 165 kDa peptide was determined to be the same as peptide TcaC (SEQ ID NOS: 2, 1-5 positions). The N-terminal amino acid sequence of the 90 kDa peptide was determined to be the TcaA _ii region derived from the amino acid sequence of the tcaA gene (SEQ ID NOs: 33, 254-258). The N-terminal amino acid sequence of the 64 kDa peptide was determined to be identical to the peptide TcaB _i (SEQ ID NOS: 3, 1-5 positions). The N-terminal amino acid sequence of the 62 kDa peptide was determined to be the TcaA _ii region derived from the amino acid sequence of the tcaA gene (positions 33, 489-493). The N-terminal amino acid sequence of the 58 kDa peptide was determined to be identical to peptide TcaB _ii (SEQ ID NOs: 5, 1-5 positions). The N-terminal amino acid sequence of the 22 kDa peptide (SEQ ID NO: 62) was determined to be the TcaA _i region designated TcaA _iv derived from the amino acid sequence of the tcaA gene (positions 34, 98-102). It is noted that all tcaA, tcaB and tcaC genes are in the same tca operon (FIG. 6A).

5 μg of purified Tca fraction, purified toxin A, and purified toxin B as the primary antibody, antibodies such as pAb TcaB _ii- syn antibody, mAb CF52 antibody, pAb TcdA _ii- syn antibody, and pAb Tcd _iii- syn antibody Were analyzed by Western blot using individually (Example 21). Only the purified Tca peptide fraction was reacted with the pAb TcaB _ii- syn antibody, but not toxin A and toxin B. Only toxin B is reacted with mAb CF52 antibody but not the Tca peptide fraction or toxin A. Only toxin A is reacted with the pAb TcdA _ii- syn antibody or pAb Tcd _iii- syn antibody, but not with the Tca peptide fraction or toxin B. This demonstrates that the pesticidal activity observed in the purified Tca peptide fraction is independent of toxin A and toxin B. The purified Tca peptide fraction is the third unique protein toxin represented by toxin C.

<Example 16>

Degradation and Activation of TcbA Peptides

Peptides TcbA _ii and TcbA _iii in the toxin B complex are derived from the single gene product TcbA (Example 15). The processing of the TcbA peptides with TcbA _ii and TcbA _iii is probably due to the action of photolabose protease (s), most likely the metalloprotease described in Example 10. In some cases, it was found that TcbA peptides, as well as peptides TcbA _ii and TcbA _iii , were present as major components in the toxin B complex when the Photorabdus W-14 broth was processed. To collect peptide TcbA from the toxin complex fraction of W-14 broth, the same method as described in the purification of toxin B complex (Example 15) was used. The final purified material was analyzed on 4-20% gradient SDS-PAGE and the main peptides were quantified by densitometry. TcbA, TcbA _ii and TcbA _iii were 58%, 36% and 6% of total protein, respectively. The nature of the peptides was confirmed by Western blot analysis using individual molecular sizes and monospecific antibodies in SDS-PAGE. The natural molecular weight of the fraction was determined to be 860 kDa.

The purification material was treated with purified W-14 photolabose metalloprotease, 38 kDa and 58 kDa (Example 10) and digestion of TcbA by treatment with trypsin (Sigma, Missouri) as a control enzyme. Evaluated. Standard reactants included 17.5 μg of the purified fraction, 1.5 unit protease, and 0.1 M Tris buffer at pH 8.0 in 100 μl total volume. There is no protease in the control reaction. The reaction mixture was left at 37 ° C. for 90 minutes. 20 μl was taken at the end of the reaction and immediately boiled in SDS-PAGE sample buffer for electrophoresis analysis at 4-20% gradient SDS-PAGE. In both 38 kDa and 58 kDa protease treatments, it was determined from SDS-PAGE that the amounts of peptides TcbA _ii and TcbA _iii increased about threefold, while the amount of TcbA peptides decreased proportionally. Relative decreases and increases in selected peptides were confirmed by Western blot analysis. In addition, as a result of gel filtration of the degraded material, it was found that the natural molecular size of the complex did not change. Upon trypsin treatment, peptides TcbA and TcbA _ii were nonspecifically degraded into small peptides. This indicated that the 38 kDa and 58 kDa photolabose proteases could specifically process peptide TcbA with peptides TcbA _ii and TcbA _iii . Protease treated and untreated controls of the remaining 80 μl reaction mixture were serially diluted with 10 mM sodium phosphate buffer at pH 7.0 and analyzed by SCR biological quantification. Comparing the activity at different dilutions showed that SCR insecticidal activity was increased by about 3 to 4 fold upon treatment with 38 kDa protease. In addition, in protease treatment, the growth inhibition rate of the remaining insects was more severe than the control (Table 24).

Conversion and activation of peptide TcbA to peptide TcbA _ii and TcbA _iii by protease treatment. Control 38 kDa protease treatment S0 (% of total protein) 58 18 S1 (% of total protein) 36 64 S9 (% of total protein) 6 18 LD50 (μg protein) 2.1 0.52 SCR Weight (mg / insect) * 0.2 0.1 *: In this assay, inhibition of growth is indicated by measurement of the average weight of live insects after 5 days of diet.

Activation and Treatment of Toxin B by SCR Intestinal Protease

In a second demonstration of proteolytic activity, it was tested whether the W-14 toxin was processed by insects. Toxin B purified from Photolab Dux W-14 broth (see Example 15) consisted mainly of complete TcbA peptides determined by SDS-PAGE and Western blot analysis using monoclonal antibodies. The LD 50 of this fraction for SCR was determined to be about 700 ng.

SCR larvae were grown on coleopteran feed until the fourth instar stage (about 100-125 mg total weight of each insect). SCR intestinal components were collected by removing them using incision scissors and tweezers. After removing the large amount of fat surrounding the intestinal lining, about 40 intestines were homogenized in a microcentrifuge tube containing 100 μl of sterile water. The tube was then centrifuged at 14,000 rpm for 10 minutes and the pellet discarded. Supernatants were stored in a -70 ° C. freezer until use.

Treatment of toxin B by the intestine of the insect was evaluated by treating the purified toxin B with the collected SCR intestinal components. This reaction included 40 μg of toxin B (1 mg / ml), 50 μl of SCR intestinal component, and 0.1 M Tris buffer at pH 8.0 in 100 μl total volume. In the control reaction, the SCR field component was subtracted. The reaction mixture was incubated overnight at 37 ° C. At the end of the reaction, 10 μl was removed and boiled with an equal volume of 2 × SDS-PAGE sample buffer for SDS-PAGE analysis. The remaining 90 μl of reaction mixture was serially diluted with 10 mM sodium phosphate buffer at pH 7.0 and analyzed by SCR bioanalysis. SDS-PAGE analysis demonstrated that peptide TcbA was completely digested into smaller peptides in SCR field component treatment. Analysis of the undenatured toxin fraction demonstrated that the original peptide, about 860 kDa, remained intact even though the larger peptide was cleaved. In SCR bioanalysis, the LD50 of SCR intestines treated with toxin B was found to be about 70 ng, a 10-fold increase. In a separate experiment, protease K treatment completely eliminated toxin activity.

<Example 17>

Screening of Gene Libraries Encoding TcdA _ii Peptides

Cloning and characterization of the gene encoding the TcdA _ii peptide, described in SEQ ID NO: 17 (internal peptide TcdA _ii -PT111 N-terminal sequence) and SEQ ID NO: 18 (internal peptide TcdA _ii -PT79 N-terminal sequence), was complete. Two pools of synonymous oligonucleotides designed to encode amino acid sequences and reverse complements of those sequences, SEQ ID NO: 17 (Table 25) and SEQ ID NO: 18 (Table 26), were synthesized as described in Example 8. The DNA sequence of the oligonucleotides is shown below.

Synonym oligonucleotides for SEQ ID NO: 17 P2-PT111 One 2 3 4 5 6 7 8 amino acid Ala Phe Asn Ile Asp Asp Val Ser Codon 5 'GCN TT (T / C) AA (T / C) AT (T / C / A) GA (T / C) GA (T / C) GTN 3 ' P2.3.6.CB 5 'GC (A / C / G / T) TT (T / C) AAT ATT GAT GAT GT 3 ' P2.3.5 5 'GC (A / C / G / T) TT (T / C) AA (T / C) AT (T / C / A) GA (T / C) GA (T / C) GT 3 ' P2.3.5.R 5 'AC (G / A) TC (G / A) TC (T / G / A) AT (G / A) TT (G / A) AA (A / C / G / T) GC 3 ' P2.3.5.RI 5 'ACI TCI TCI ATI TTI AAI GC 3 ' P2.3R.CB 5 'CAG (A / G) CT (A / C) AC ATC ATC AAT ATT AAA 3 '

Synonymous oligonucleotides for SEQ ID NO: 18 P2-PT79 One 2 3 4 5 6 7 8 9 10 11 12 13 amino acid Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Codon * 5 'TTY ATH GTN TAY ACN 6 6 GGN GTN AAY CCN AAY AAY 3 ' P2.79.2 5 'TTY ATY GTK TAT ACY TCI YTR GGY GTK AAT CCR AAT AAT 3 ' P2.79.3 5 'TTT ATT GTK TAT ACY AGY YTR GGY GTK AAT CCR AAT AAT 3 ' P2.79.R.1 5 'ATT ATT YGG ATT MAC RCC YAR RCT RGT ATA MAC AAT AAA 3 ' P2.79R.CB 5 'ATT ATT YGG ATT MAC ACC CAG RCT GGT ATA MAC AAT AAA 3 ' * Y = C or T, H = A, C or T, N = A, C, G or T, K = G or T, R = A or G, and M = A, depending on the IUPAC-IUB code of the nucleotide Or C

In all forward / reverse combinations, use P2.3.6CB or P2.3.5 as the forward primer and P2.79.R.1 or P2.79R.CB as the reverse primer, and photolabdus W-14 genomic DNA as template Use, essentially as described in Example 8, polymerase chain reaction was carried out. In another reaction set, primers P2.79.2 or P2.79.3 were used as forward primers and P2.3.5R, P2.3.5RI, and P2.3R.CB as forward primers in all forward / reverse combinations. Only similar reactions with P2.3.6.CB as forward primers and P2.79.R.1 or P2.79R.CB as reverse primers had an estimated size of 2500 base pairs (mobility on agarose gels) observed It became. Sequence of the primers used in order to obtain the amplification products, peptide fragments TcdA _ii -PT111 a peptide fragment of amino TcdA _ii -PT79 - shows that in the vicinity.

The 2500 bp PCR product was ligated to the plasmid vector pCR ^TM II (manufactured by Invitrogen, San Diego, Calif.) According to the manufacturer's instructions, and the DNA of the insert fragments of the two isolates (HS24 and HS27) Sequences were determined using the primers and sequencing methods suggested by the manufacturer. The sequences of both isolates were identical. New primers were synthesized based on the determined sequences, and the new primers were used to prime additional sequencing reactions to obtain sequences of inserts (SEQ ID NO: 36) of a total 2557 bases. Partial translation of the peptide encoded by SEQ ID NO: 36 results in the 845 amino acid sequence disclosed in SEQ ID NO: 37. Protein homology analysis of this site of the TcdA _ii peptide fragment includes a significant amino acid homology of the protein TcbA (SEQ ID NO: 12) using Wisconsin package version 8.0 of the Genetics Computer Group (GCG) of Madison, WI. 68% similarity and 53% identity to 542-1390 residues, or 60% similarity and 54% identity to 567-1389 residues using Wisconsin Package Version 9.0 of the Genetics Computer Group (GCG) of Madison, Wisconsin) appear. Thus, the genes partially depicted in SEQ ID NO: 36 exhibit amino acid sequences that are similar but not identical to the TcbA protein and are evident in biological activity similar to, but not identical to, the TcbA protein.

In other cases, the peptides TcdA _ii- PK44 and TcdA _iii 58 kDa N-terminal peptides described in SEQ ID NO: 39 (internal peptide TcdA _ii -PK44 sequence) and SEQ ID NO: 41 (TcdA _iii 58 kDa N-terminal peptide sequences) are encoded. Gene was isolated. Two pools of synonymous oligonucleotides and reverse complements of those sequences, designed to encode amino acid sequences as set forth in SEQ ID NO: 39 (Table 28) and SEQ ID NO: 41 (Table 27), are described in Example 8 and their DNA. Synthesis was as described in the sequence.

Synonym oligonucleotides for SEQ ID NO: 41 Codon # One 2 3 4 5 6 7 8 9 10 11 12 13 14 amino acid Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln A2.1 5 'YTR CGY AGY GCI AAT ACY YTR ACY GAT YTR TTT YTR CCR CA 3 ' A2.2 GCI AAT ACI YTR ACI GAY YTR TTY YTR CCI CA 3 ' A2.3.R 5 'TG YGG YAR AAA YAR RTC RGT YAR RGT RTT IGC RCT RCG 3 ' A2.4.R 5 'TG IGG CAG AAA CAG RTC IGT CAG IGT ATT ICG 3 '

Synonym oligonucleotides for SEQ ID NO: 39 amino acid # (8) (9) 10 (11) (12) (13) (14) (15) (16) Codon # One 2 3 4 5 6 7 8 9 amino acid Gly Pro Val Glu Ile Asn Thr Ala Ile A1.44.1 5 'GGY CCR GTK GAA ATT AAT ACC GCI AT 3 ' A1.44.1R 5 'ATI GCG GTA TTA ATT TCM ACY GGR CC 3 ' A1.44.2 5 'GGI CCI GTI GAR ATY AAY ACI GCI AT 3 ' A1.44.2R 5 'ATI GCI GTR TTR ATY TCI ACI GGI CC 3 '

In all forward / reverse combinations, essentially using A1.44.1 or A1.44.2 as the forward primer and A2.3R or A2.4R as the reverse primer, and Photorabdus W-14 genomic DNA as the template, essentially The polymerase chain reaction was performed as described in 8. In another reaction set, in all forward / reverse combinations, primers A2.1 or A2.2 were used as forward primers, and A1.44.1R and A1.44.2R were used as reverse primers. Only similar reactions with an estimated size of 1400 base pairs (mobility on agarose gel) were observed in reactions containing A1.44.1 or A1.44.2 as forward primers and A2.3R as reverse primers. The sequence of the primers used to obtain the amplification product indicates that the peptide fragment TcdA _ii -PK44 is at the amino-contiguous site of the 58 kDa peptide fragment of TcdA _iii .

The 1400 bp PCR product was ligated to the plasmid vector pCR ^™ II according to the manufacturer's instructions. DNA sequences of the insert fragments of the four isolates were determined using primers similar in sequence to the primers suggested by the manufacturer and the above sequencing method. The nucleic acid sequences of all isolates differed at sites corresponding to synonymous primer sequences as expected, but the amino acid sequences estimated from the data were identical to the actual amino acid sequences of the peptides determined above (SEQ ID NOs: 41 and 39).

As described in Example 8, five hybridized cosmids were isolated by screening a W-14 genomic cosmid library using an isotopically labeled probe consisting of the DNA (SEQ ID NO: 36) prepared above. Water, 17D9, 20B10, 21D2, 27B10, and 26D1, were identified. The cosmids were different from the cosmids previously identified with probes corresponding to genes as described in SEQ ID NO: 11 or SEQ ID NO: 25. Restriction enzyme analysis and DNA blot hybridization were performed to identify three EcoRI fragments with sizes of about 3.7, 3.7, and 1.1 kbp, which are the sites containing the DNA of SEQ ID NO: 36. The same 14 cosmids (17D9, 20B10, 21D2, 27B10, and 26D1) were identified by screening the W-14 genomic cosmid library using isotope labeled 1.4 kbp DNA fragments prepared in this example as probes. . DNA blot hybridization to cosmid DNA digested with EcoR1 also showed hybridization to the same subset of EcoRI fragments, as shown in the 2.5 kbp TcdA _ii gene probe, both fragments on genomic DNA. It is coded.

DNA sequencing of the cloned EcoRI fragment revealed an uninterrupted translation frame of 7551 base pairs (SEQ ID NO: 46) encoding a 282.9 kDa protein of 2516 amino acids (SEQ ID NO: 47). The amino acid sequencing of the protein revealed that all expected internal fragments of peptide TcdA _ii (SEQ ID NOs: 17, 18, 37, 38 and 39) and TcdA _iii peptide N-terminus (SEQ ID NO: 41) and all TcdA _iii internal peptides (SEQ ID NO: 42 and 43). Peptides isolated and identified by TcdA _ii and TcdA _iii are the respective products of the open reading frame represented by tcdA, shown in SEQ ID NO: 46. In addition, SEQ ID NO: 47 illustrates the sequence set forth in SEQ ID NO: 13, which is the N-terminal sequence of the peptide of about 201 kDa size starting at position 89, wherein the initial protein generated from SEQ ID NO: 46 is processed in a similar manner as in SEQ ID NO: 12 above. It is displayed. In addition, the protein was further digested to a 209.2 kDa size product encoded by SEQ ID NO: 48 and disclosed as SEQ ID NO: 49 (TcdA _ii peptide), and a 63.6 kDa size product encoded by SEQ ID NO: 50 and disclosed as SEQ ID NO: 51 (TcdA _iii peptide). To produce. Thus, as exemplified by the 282.9 kDa full-length protein disclosed in SEQ ID NO: 47, the pesticidal activity identified as toxin A (Example 15) derived from the product of SEQ ID NO: 46 was processed to produce the peptides disclosed in SEQ ID NOs: 49 and 51. I think. The pesticidal activity identified as toxin B (Example 15) is thought to be derived from the product of SEQ ID NO: 11 exemplified by the 280.6 kDa protein disclosed as SEQ ID NO: 12. The protein was processed by proteolysis to produce a 67.6 kDa peptide having a 207.6 kDa peptide as set out in SEQ ID NO: 53, encoded by SEQ ID NO: 52, and an N-terminal sequence set forth as SEQ ID NO: 40 and SEQ ID NO: 55, encoded by SEQ ID NO: 54 Produces peptides.

Comparing the amino acid sequence of the protein set forth in SEQ ID NO: 12 with the protein set forth in SEQ ID NO: 47, 69% similarity and 54% identity, or of this program, using the Wisconsin Package Version 8.0 of the Genetics Computer Group (GCG) of Madison, Wisconsin Version 9.0 is used to show 60% similarity and 54% identity. This highly evolutionary relationship is not uniform throughout the entire amino acid sequence of the peptides, but becomes greater towards the carboxy-terminal end of the protein, which is SEQ ID NO: 51 (derived from SEQ ID NO: 47) and SEQ ID NO: 55 (SEQ ID NO: 12). Peptides disclosed herein are 76% similarity and 64% identity using Wisconsin package version 8.0 of the Genetics Computer Group (GCG) of Madson, WI, or 71% similarity and 64% using version 9.0 of this program. This is because they have identity.

<Example 18>

Control of Maize Plant Leaf Damage Caused by European Corn Borer by Spray Application of Photorapdus (Strain W-14) Broth

The ability of the photolabduus toxin (s) to reduce plant damage caused by insect larvae, and the leaf damage caused by the European corn periphery (Ostrinia nubilalis), which prevailed on corn plants treated with photolabdus broth Proven by measurement. Fermentation broth from Photolabduus strain W-14 was made and concentrated about 10-fold using ultrafiltration (10000 MW pore size) as described in Example 13. The resulting concentrated broth was filtered sterilized using a 0.2 micron nitrocellulose membrane filter. Similarly prepared, uninoculated 2% proteose peptone # 3 samples were used for control purposes. Corn plants (breeding series) were grown from seed to plant stage 7 or 8 in pots containing mixtures without soil in greenhouses (day 27 ° C., night 22 ° C., relative humidity of about 50%, low length 14). Time, water / fertilizer supply if required). The test plants were randomly cut off design (3 reps / treatment) in daytime temperatures of about 22 ° C, night temperatures of about 18 ° C, artificial lightless and partial shading, relative humidity of about 50% and water / fertilizers as needed , 6 plants / treatment). The treatment (uninoculated media and concentrated photolabose broth) was applied 2.0 ml directly from the spiral (approximately 6 inches) using a syringe sprayer, and further 2.0 ml was applied about 30 cm (approx. 1 step) in a circular motion. In addition, one group of plants did no treatment. After the treatment had dried (approximately 30 minutes), twelve new European corn puncture larvae (eg, eggs purchased from the source of purchase) were applied directly to the helix. After one week, damage to the leaves was recorded using a modified Guthrie Scale [Koziel, MG, Beland, GL, Bowman, C., Carozzi, NB, Crenshaw, R., Crossland, L. , Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K., Lewis, K., Maddox, D., McPherson, K., Meghji, MZ, Merlin, E., Rhodes, R., Warren, GW, Wright, M. and Evola, SV, 1993, Bio / Technology, 11, 194-195], and the records were statistically compared (T-test (LSD) p <0.05 and Tukey's Studentized Range (HSD) test p <0.1 The results are illustrated in Table 29. For reference, score 1 indicates no damage and score 2 on needles that did not open without needle holes. Indicative of fine "window pain" damage and a score of 5 indicates leaf penetration of elongated damage and / or clear central leaf vein feeding (damage <1 inch) to three or more leaves. When other protein containing fractions is or can be delivered to the spray formulation the gene or its derivative encoding a protein or a portion thereof is passed through the plant or bacterium a gene switch, it indicates that it is possible to provide toughness for a specific pest.

Effect of Photolab Dose Culture Broth on Leaf Damage on Corn Caused by European Corn Borer process Average Gutri Score Untreated 5.02 ^a Unvaccinated Badge 5.15 ^a Potorabdus Broth 2.24 ^b Means with different letters are statistically different (p <0.05 or p <0.1).

<Example 19>

Genetic Engineering of Genes for Expression in Escherichia Coli

Summary of the structure

A series of plasmids were constructed to express the tcbA gene of Photolapis W-14 in E. coli. A list of plasmids is shown in Table 30. A brief description of each construct and a summary of the E. coli expression data obtained are also shown.

Plasmids for tcbA Gene Expression Plasmid gene Vector / Selection Cover compartment pDAB2025 tcbA pBC / Chl Intracellular pDAB2026 tcbA pAcGP67B / Amp Baculovirus, secreted pDAB2027 tcbA pET27b / Kan Periplasm pDAB2028 tcbA pET15-tcbA Intracellular Abbreviations: Kan = Kanamycin, Chl = Chloramphenicol, Amp = Ampicillin

Fabrication of pDAB2025

A large EcoRI fragment that hybridizes to the TcbA _ii probe is described in Example 9. The fragment was subcloned into pBC (Stratagene, La Jolla, Calif.) To make pDAB2025. Sequencing revealed that the fragment was 8816 base pairs. The fragment encodes the tcbA gene with the starting ATG at position 571 and the terminating TAA at position 8086. Thus, the fragment has 570 base pairs of photolabose DNA upstream of ATG and 730 base pairs downstream of TAA.

Construction of Plasmid pDAB2026

5 'TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3' with 5 'Primer (S1Ac51) and 5' TTT AAA GCC GCT TAA CGG ATG GTA TAA CGA ATA TG 3 'with 3' Primer (S1Ac31) tcbA gene was amplified by PCR from plasmid pDAB2025. 57.5 ml of water, 10 ml of 10X LA buffer, 16 ml of dNTPs (2.5 mM each), 300 ng of plasmid pDAB2025 containing 20 ml of each primer and W-14 tcbA gene at 10 pmoles / ml, TaKaRa LA Taq PCR was performed using a TaKaRa LA PCR kit purchased from PanVera (Medicine, Wisconsin) at reaction conditions of 1 ml of polymerase. Cycling conditions were 30 cycles of 98 degreeC / 20 second, 68 degreeC / 5 minutes, and 72 degreeC / 10 minutes. The PCR product, expected to be about 7526 bp, was isolated from Qiagen (Qiagen, Chatsworth, CA) by isolating a 0.8% agarose gel using TBE buffer (100 mM Tris, 90 mM Boric acid, 1 mM EDTA). Purification was performed using the purchased Qiaex II kit. The purified tcbA gene was cut into Nco I and Not I and ligated into the baculovirus transfer vector pAcGP67B (manufactured by PharMingen, San Diego, Calif.) And used to transform DH5α Escherichia coli. The resulting recombinant was named pDAB2026. The tcbA gene was then cut out of pDAB2026 and transferred to pET27b to make plasmid pDAB2027. Missense mutations in the tcbA gene were recovered in pDAB2027.

The recovered tcbA gene contains two changes from the sequence shown in SEQ ID NO: 11, where the A> G transformation at 212 changes asparagine 71 to serine 71 and the G> A transformation at 229 changes alanine 77 to threonine 77. . All of these changes occur upstream of the proposed TcbA _ii N-terminus.

Fabrication of pDAB2028

The tcbA coding region of pDAB2027 was transferred to vector pET15b. This was done using shotgun ligation and DNA was digested with restriction enzymes Nco I and Xho I. The resulting recombinant was named pDAB2028.

TcbA Expression from Plasmid pDAB2028 in Escherichia Coli

Expression of tcbA in E. coli was achieved by modifying methods previously described by Studier et al. (Studier, FW, Rosenberg, A., Dunn, J., and Dubendorff, J., (1990) Use of T7 RNA polymerase to direct expression of cloned genes.Methods Enzymol., 185: 60-89). Water soluble E. coli cell strain BL21 (DE3) was transformed with plasmid pDAB2028 and plated on LB agar containing 100 μg / ml of ampicillin and 40 mM glucose. The transformed cells were plated to the density of hundreds of isolated colonies / plates. After standing overnight at 37 ° C., cells were scraped from the plate and suspended in LB broth containing 100 μg / ml of ampicillin. Typical culture doses were 200 to 250 ml. At time zero, the culture density (OD600) was 0.05 to 0.15 depending on the experiment. Cultures were shaken at any one of three temperatures (22 ° C., 30 ° C. or 37 ° C.) until a density of 0.15 to 0.5 was obtained and 1 mM isopropylthio-β- was obtained when the density was obtained. Induced by galactoside (IPTG). The cultures were incubated at the designated temperature for 4-5 hours and then left to move to 4 ° C. until treatment (12-72 hours).

Purification and Characterization of TcbA from Plasmid pDAB2028 Expressed in Escherichia Coli

E. coli cultures expressing TcbA peptides were treated as follows. Cells were harvested by centrifugation at 17000 × g and collected in separate containers along the medium.

The medium was concentrated about 8 × using an M12 (Amicon, Beverley, Mass.) Filtration system and a 100 kD molecular size cut off filter. The concentrated medium was loaded on an anion exchange column and the attached protein was eluted with 1.0 M NaCl. A 1.0 M NaCl elution peak was found to cause pesticidal effects against Southern Corn Root Worm (SCR) larvae (Table 30). The 1.0 M NaCl fractions were concentrated by dialysis against 10 mM sodium phosphate buffer at pH 7.0 and gel filtered on Sepharose CL-4B (Pharmacia, Piscataway, NJ). Regions of the CL-4B elution profile corresponding to the estimated molecular weight (about 900 kDa) of the native W-14 toxin complex were collected and concentrated and biological quantification was performed on the larvae. The collected 900 kDa fractions were found to have pesticidal activity with signs similar to those caused by the native W-14 toxin complex (see 31 below). This fraction was sensitive to proteinase K and heat treatment and in both cases activity was lost or decreased, providing evidence that the activity was in fact proteinaceous. In addition, the active fractions were tested immunologically positive for TcbA and TcbA _iii peptides in immunoblot assays when tested with anti-TcbA _iii monoclonal antibodies (Table 31).

Immunoblot and SCR Biological Quantification Results Fraction SCR activity Immunoblot Natural size % Lethality % Growth inhibition Peptide Detected Size estimated by CL-4B TcbA Badge 1.0 M +++ +++ TcbA Ion exchange TcbA Badge CL-4B +++ +++ TcbA, TcbA _iii To 900 kDa TcbA medium CL-4B + proteinase K + +++ NT TcbA Medium CL-4B + Heat Treatment － － NT TcbA Cell Supernatant CL-4B － +++ NT To 900 kDa PK = 2 hours of proteinase K treatment, heat treatment = 10 minutes at 100 ° C., ND = not detected, NT = not tested. Scoring system of mortality and growth inhibition compared to control sample; 5-24% = +, 25-49% = + +, 50-100% = + + +.

The cell pellet was resuspended in 10 mM sodium phosphate buffer at pH 7.0 and passed through a trademark Bio-Neb cell nebulizer (Glas-Col Inc., Terahort, IN). Lysate. DNA was removed by treating the pellet with DNase, and the cell supernatant and the cell pellet were separated by centrifugation at 17000 × g. The supernatant fractions were decanted and filtered through a 0.2 micron filter to remove large particles and anion exchange chromatography was performed. The attached protein was dialyzed by eluting with 1.0 M NaCl and concentrated using a trademark Biomax concentrator (Millipoa Corporation, Bedford, Mass.) With a molecular size cut off of 50000 Daltons. The concentrated fractions were subjected to gel filtration chromatography using a matrix of Sepharose CL-4B beads. Biological quantitative data of materials prepared in this manner is illustrated in Table 30 and represented as TcbA cell supernatant.

In another method of handling large amounts of material, the cell pellet was resuspended in 10 mM sodium phosphate buffer at pH 7.0 and used a 40 ml tissue grinder from Kontes Glass Company, Vineland, NJ. Complete homogenization. Cell lysates were pelleted by centrifugation at 25000 × g, the cell supernatant was decanted and passed through a 0.2 micron filter and subjected to anion exchange chromatography using a Pharmacia 10/10 column packed with porous HQ 50 beads. The attached protein was eluted by performing a NaCl concentration gradient of 0.0 to 1.0 M. Fractions containing TcbA protein were combined and concentrated using a 50 kDa concentrator, followed by gel filtration chromatography using a matrix of Pharmacia CL-4B beads. Fractions containing TcbA oligomers having a molecular size of about 900 kDa were collected and subjected to anion exchange chromatography using a Pharmacia mono Q 10/10 column equilibrated with 20 mM Tris buffer at pH 7.3. A 0.0 to 1.0 M linear gradient of NaCl was used to elute the recombinant TcbA protein. Recombinant TcbA was eluted from the column at about 0.3 to 0.4 M NaCl salt concentration, the same molar concentration as that of the native TcbA oligomer eluted from the mono Q 10/10 column. Recombinant TcbA fractions were found to induce SCR lethality in biological quantitative experiments similar to the experiments in Table 31.

A second set of expression constructs was prepared and tested for expression of TcbA protein toxins.

Construction of pDAB2030: Expression Plasmid for tcbA Coding Region

Plasmid pDAB2028 (see herein) contains the tcbA coding region in commercial vector pET15 (Novagen, Madison, Wisconsin) and encodes an ampicillin selection marker. Plasmid pDAB2030 was prepared to express the tcbA coding region from the plasmid encoding the kanamycin selection marker. This preparation is performed by cleaving pET27 (Novagen, Wisconsin Madison) kanamycin selective plasmid and pDAB2028 with Xba I and Xho I. This gives the entire multiplex cloning site including the tcbA coding region from plasmid pDAB2028. Two cleaved plasmids were mixed and ligated. Recombinant plasmids were selected on kanamycin and those containing pDAB2028 fragments were identified by restriction analysis. The new recombinant plasmid is called pDAB2030.

Composition of Plasmid pDAB2031: Correction of Mutations in tcbA _i

Two mutations at the N-terminus of the tcbA coding region as described in Example 19 (SEQ ID NO: 11; at 212 G changes asparagine 71 to serine 71; at 229 G> A alanine 77 to threonine Change to 77) was corrected as follows. PCR products were generated using primers TH50 (5 'ACC GTC TTC TTT ACG ATC AGT G 3') and S1Ac51 (5 'TTT AAA CCA TGG GAA ACT CAT TAT CAA GCA CTA TC 3') and pDAB2025 as a template to generate 1778 bp The product was obtained. This PCR product was cloned into plasmid pCR2.1 (Invitrogen, San Diego, Calif.) And clones were isolated and sequenced. Clones were cleaved with NCO I and Pin AI and 1670 bp fragment was purified from 1% agarose gel. Plasmids containing the mutated tcbA coding region (pDAB 2030) were digested with NcoI and Not I and purified from 1670 bp fragment in 0.8% agarose with Qiaex II (Qiagen, Chatsworth, CA). The corrected Nco I / Pin AI fragment was then ligated with pDAB2030. The ligated DNA was transformed with DH5α E. coli. Clones were isolated, sequenced and confirmed to be corrected. The plasmid containing this corrected tcbA coding region is called pDAB2031.

Construction of pDAB2033 and pDAB2034: Expression Plasmid for tcbA

Expression plasmids pDAB2025 and pDAB2027 both depend on the bacteriophage T7 expression system. Additional vector systems were used for the bacterial expression of the tcbA gene and its derivatives. The expression vector Trc99a (Pharmacia Biotech, Pittscataway, NJ) contains a strong trc promoter upstream of the 5 ′ Nco I site and multiple cloning sites compatible with the tcbA coding region from pDAB2030 and 2031. However, the plasmid does not have a compatible 3 'site. Therefore, the Hind III site of Trc99a was cut and blunted by treatment with T4 DNA polymerase (Boehringer Mannheim, Indianapolis, Indiana). The vector plasmid was then digested with Nco I and treated with alkaline phosphatase. Plasmids pDAB2030 and pDAB2031 were cut with Xho I (cut at 3 'end of tcbA coding region) respectively and treated with T4 DNA polymerase to blunt the ends. The plasmid was then digested with Nco I, the DNA extracted with phenol, ethanol precipitated and resuspended in buffer. Trc99a and pDAB2030 and pDAB2031 plasmids were mixed separately, ligated, transformed into DH5α cells and plated on LB medium containing ampicillin and 50 mM glucose. Recombinant plasmids were identified by restriction cleavage. The new plasmids are called pDAB2033 (containing the tcbA coding sequence with two mutations in tcbA _i ) and pDAB2034 (containing the corrected version of tcbA from pDAB2031).

Construction of Plasmid pDAB2032: Expression Plasmid for tcbA _ii A _iii

A plasmid encoding the tcbA _ii A _iii portion of TcbA was prepared in the same manner as plasmid pDAB2031. PCR products were generated using pDAB2025 as TH42 (5 'TAG GTC TCC ATG GCT TTT ATA CAA GGT TAT AGT GAT CTG 3') and TH50 (5 'ACC GTC TTC TTT ACG ATC AGT G 3') primers and templates. This gave 1521 bp of product with initial codons at the beginning of the coding sequence of tcbA _ii . This PCR product was isolated and purified in 1% agarose gel. The purified product was cloned into pCR2.1 as above and the corrected clones were confirmed by DNA sequencing. This clone was cleaved with Nco I and Pin AI, isolated in 1414 bp fragment 1% agarose, ligated to Nco I and Pin AI sites of plasmid pDAB2030 and transformed with DH5α E. coli. This new plasmid designated to express TcbA _ii A _iii is called pDAB2032.

Expression of tcbA and tcbA _ii A _iii from plasmids pDAB2030, pDAB2031 and pDAB2032

Expression of tcbA in E. coli from plasmids pDAB2030, pDAB2031 and pDAB2032 was performed as described herein except that tcbA _ii A _iii was performed in E. coli strain HMS174 (DE3) (Novagen, Madison, Wisconsin). .

Expression of tcbA from plasmid pDAB2033

Plasmid pDAB2033 was transformed into BL21 cells (Medicine, Novagen, Wisconsin) and plated on LB containing 100 μg / ml ampicillin and 50 mM glucose. The plates were spread out and incubated overnight at 30 ° C. or 37 ° C. such that there were several hundred wells of isolated colony on each plate. Colonies were scraped from the plates and suspended in LB containing 100 μg / ml ampicillin but no glucose. Typical culture volume was 250 ml in a single 1 l baffle bottom flask. The culture was induced when the density of the culture reached 0.3-0.6 OD600 nm. This concentration is most often reached immediately after suspending cells from the plate and does not require a growth period in the liquid medium. Two induction methods were used. Method 1: Cells were induced with 1 mM IPTG at 37 ° C. The culture was shaken for 5 hours at 200 rpm on a platform shaker and harvested. Method 2: Cultures were induced with 25 μM IPTG at 30 ° C. and shaken at 200 rpm at 20 ° C. or 30 ° C. for 15 hours. Cultures were stored at 4 ° C. until used for purification.

Purification of TcbA from E. coli

Purification, bioassay and immunoblot analysis of TcbA and TcbA _ii A _iii were performed as described herein. The results of some representative E. coli expression experiments are shown in Table 32. All materials shown in Table 32 were purified from culture medium fractions. The predicted intrinsic molecular weight is approximately 900 kD as described herein. The purity of the sample, and the amount of TcbA for contaminating proteins, varied with each preparation.

this. Bioassay Activity and Immunoblot Analysis of TcbA and Derivatives Produced from Coli and Purified from Culture Media Plasmid Coding region E. coli strain Southern Corn Roots Biologically Active Peptides Detected by Immune Blot Protein administered to the food (μg) Growth inhibition % Mortality pDAB2030 tcbA BL21 (DE3) - +++ TcbA + TcbA _iii 1-8 pDAB2031 tcbA BL21 (DE3) - +++ TcbA + TcbA _iii 1-10 pDAB2033 tcbA BL21 - +++ TcbA + TcbA _iii 1-2 pDAB2032 tcbA _ii A _iii HMS174 (DE3) +++ + TcbA _ii A _iii + TcbA _iii 13-27 Scoring system for mortality and growth inhibition in southern corn rootworms compared to control samples: 5-24% = +, 25-49% = ++, 50-100% = +++

<Example 20>

Characterization of Toxin Peptides Using Matrix-Assisted Laser Absorption Ionization Time-of-Flight Mass Spectroscopy

Toxins isolated from W-14 broth were isolated as described in Example 15. In some cases the TcaB protein toxin was pretreated with protease (Example 16) isolated from W-14 broth as described above (Example 15). Protein molecular mass was determined using matrix-assisted laser absorption ionization time-of-flight mass spectroscopy (hereinafter referred to as MALDI-TOF) as a delayed extraction technique on a VOYAGER BIOSPECTROMETRY workstation. Usually the main protein (100-500 pmoles in 5 μl) was mixed with 1 μl of acetonitrile and dialyzed on a Millipore VS filter (Millipore Corp. Bedford, Mass.) With a pore size of 0.025 μM for 0.5 to 1 hour. Dialysis was performed by suspending the filter against water and dropping the protein-acetonitrile mixture onto the filter surface (with the glossy side up). After dialysis, the dialyzed protein was removed using a pipette and mixed according to the manufacturer's instructions with a matrix consisting of cinafinic acid and trifluoroacetic acid. Proteins and matrices were cocrystallized on about 3 cm 2 gold plated sample plates (PerSeptive Corp.). The crystals were excited using the following conditions and subjected to subsequent mass spectrometry. Laser setting 3050; Pressure 4.55e-07; Low mass gate 1500.0; Anion off; Acceleration voltage 25,000; Grid voltage; 90.0%; Guide wire voltage 0.010%; Linear mode; And pulse delay time 350 ns.

Protein mass spectrometry data is presented in Table 33. Data obtained from MALDI-TOF was compared with the assumptions made from gene sequence information and those previously determined by SDS-PAGE.

Molecular analysis of peptides by MALDI-TOF, SDS-PAGE and predictive measurements based on gene sequence Peptide Prediction (Gene) SDS PAGE MALDI-TOF TcbATcbA _{i / ii} TcbA _ii TcbA _iii 80,634 Da217,710 Da207,698 Da62,943 Da 240,000 Da Not interpreted 201,000 Da58,000 Da 281,040 Da216,812 Da206,473 Da63,520 Da TcbA _ii TcbA _iii 209,218 Da63,943 Da 280,634 Da280,634 Da 280,634 Da280,634 Da TcbA _ii TcbA _iii Protease Produced Protease 280,634 Da280,634 Da 280,634 Da280,634 Da280,634 Da280,634 Da280,634 Da Generalized TcbA data, multiple fragments observed on * TcbA _{i / ii}

<Example 21>

Production of Peptide Specific Polyclonal Antibodies

Nine peptide components of the W-14 toxin complex, namely TcaA, TcaA _iii , TcaB, TcaB _ii , TcaC, TcbA _ii , TcbA _iii , TcdA _ii and TcdA _iii , were selected and antibodies were produced against them. Complementary DNA and derived amino acid sequence data for these peptides indicated that sequence identity between several of the peptides was real. If the entire peptide was used as an immunogen to induce antibody production, the resulting antibody would bind to several peptides in toxin production. To avoid this problem, antibodies were generated that specifically bind to the unique region of each desired peptide. The unique region (subpeptide) of each target peptide was selected based on the assay described below.

Each entire peptide sequence was analyzed using the MacVector ™ Protein Analysis Tool (IBI Wequence Analysis Software, International Biotechnologies, Inc., New Haven P. Box. 9558, Connecticut) and its antigen index was determined. The program is designed to locate as far as possible the amino acid sequence, ie the region that may be the antigenic site. This method combines information from skeletal flexibility predictions based on hydrophilicity, surface likelihood, and secondary structure predictions, resulting in complex predictions of the surface contours of proteins. Scores for each analysis were normalized to values between -1.0 and +1.0 (MacVector ™ Manual). Antigen index values were obtained for the entire sequence of the target peptide. From each peptide, the region covering at least 19 amino acids showing a high antigenic index from the original sequence was reanalyzed to determine the antigenic index of the subpeptide without flanking residues. This reanalysis is essential because the antigenic index of the peptide can be affected by flanking amino acid residues. If the isolated subpeptide sequence did not maintain a high antigenic index, a new region was selected and the analysis repeated.

Each selected subpeptide sequence was aligned and compared against all seven target peptide sequences using the MacVector ™ alignment program. If the selected subpeptide sequence showed identity (more than 20%) for other target peptides, new 19 or more amino acid regions were isolated and reanalyzed. Unique subpeptide sequences covering at least 19 amino acids showing high antigenic index were selected from all target peptides.

Seven subpeptide sequences were generated by the Genemid Biotechnology Inc. (Genemed Biotechnology Inc.). The last amino acid residue on each subpeptide was deleted because it did not show a clear effect on the antigen index. Cysteine residues were added to the N-terminus of each subpeptide sequence except TcaBi-syn containing internal cysteine residues. The presence of cysteine residues promotes the conjugation of the carrier protein (KLH). The final peptide product and SEQ ID NO. Corresponding to the appropriate toxin peptide are shown in Table 34.

Each conjugated synthetic peptide was injected into two rabbits according to the Genemed Acceleration Program. After 1 month pre and post immune serum were obtained for testing.

The primary test of both pre and post immune sera from each rabbit was performed by Genemid Biotechnology Inc. Genemed reported using ELISA and Western blot techniques to retrieve the response of post immune serum to each synthetic peptide. Serum was then tested with the total target peptide according to Western blot analysis. Two batches of partially purified Photoradbus strain W-14 toxin complex were used as antigens. Two samples showed activity against Southern corn root worms. Their pattern for SDS-PAGE gels was slightly different.

A pre-cast SDS-polyacrylamide gel (Integrated Separation Systems, 01760 Natick, Mass.) With a 4-20% gradient was used. 1-8 μg of protein was applied to each gel well. Electrophoresis was performed and proteins were electroblotted onto Hybond-ECL ™ Nitrocellulose membrane (Amersham International). The membrane was blocked with 10% milk in TBST (25 mM Tris HCl, pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) for 1 hour at room temperature. The serum of each rabbit was diluted 1: 500 in 10% milk / TBST. Other dilutions of 1:50 to 1: 1000 were also used. Serum was added to the membrane and placed on a 1 hour moving plate shaker. The membrane was washed thoroughly with blocking solution or TBST. Secondary antigen (goat anti-rat IgG conjugated to horse radish peroxidase) in 10% milk / TBST was applied to the membrane placed on a plateform shaker for 1 hour. The membrane was then washed with excess TBST. Search of proteins was performed using an ECL (enhanced chemifluorescence) search kit (Amersham International).

Western blot analysis was performed to confirm the binding specificity of each anti-synthetic peptide antibody. All synthetic polyclonal antibodies showed specificity for the processed and unprocessed target peptides (if available) from protein fractions derived from photolabose culture broth. Various antibodies appear to recognize processed or unprocessed recombinant proteins derived from heterologous expression systems such as bacteria or insect cells using baculosvirus expression constructs. In some cases, the anti-TcbA _iii- syn antibody showed some cross reactivity with the anti-TcdA _iii peptide. In the second case, the anti-TcaC-syn antibody recognized an unidentified 190 kDa peptide in the W-14 toxin complex fraction.

<Example 22>

Characterization of Photorapduus Strains

In order to confirm that the collections described herein include photolabduus strains, the strains of the present application were characterized in terms of photorapids in bacteria and evaluated in terms of recognized microbiological characteristics and other enterobacterial and genorabdus sp. (Farmer, JJ 1984. Bergey's Manual of Systemic Bacteriology, Vol 1. pp. 510-511. (Ed. Kreig NR and Holt, JG). Williams & Wilkins, Baltimore .; Akhurst and Boemare, 1988, J. Gen. Microbiol 134, 1835-1845; Forst and Nealson, 1996. Microbiol. Rev. 60, 21-43). These characteristic properties are as follows: Gram stained negative rod, bio size, 0.3-2 μm in width and 2-10 μm in length (with filaments (15-50 μm) and spherical shape), yellow to orange on nutrient agar incubators Red colony coloring, crystalline inclusions present, catalyst present, nitrate reduction not available, bioluminescence present, dye uptake from growth medium, positive for protease production, growth at temperatures below 37 ° C., survival under anaerobic conditions, magnetic viability (table 33). The test method was checked using Escherichia coli, Genorabdus and Photorabdus strains. The overall results were constant for all strains belonging to the genus Bacteria family and Photorapdus. It should be noted that DEP1, DEP2, and DEP3 refer to photolabose strains obtained from American Type Curlture Colection 12301 Parklawn Drive, Rockville, MD 20852 USA (# 29304, 29999 and 51583).

Luminometers were used to identify bioluminescence associated with these photolapse strains. To determine the presence or absence of relative luminescent units, broths from each strain (cells and media) were measured at three intervals of inoculation in liquid culture (24, 48, 72 hours) and background luminescence (uninoculated) Medium). Several genoviruses strains were tested as negative controls of luminescence. Prior to measuring luminescence from various broths, cipher cells were used to determine (560 nM) cell density by measuring absorption on a spectrophotometer (Gilford Systems, Oberlin, Ohio). The resulting luminescent units were then normalized to the density of the cells. Aliquots of broth were placed in 96-well microtitrator plates (100 μl each) and read on a Packard Lumicount ™ luminometer (Packard Instrument Co., Meriden, CT). The measurement period for each sample was 0.1 to 1.0 second. The sample was stirred in the light emitter for 10 seconds before reading. Positive testing was determined by 5-fold background luminescence (about 1-15 relative light units). In addition, colony luminescence was confirmed visually after adaptation in the photographic film overlay and in the dark. Grams staining characterization of each strain was confirmed with a commercial Grams staining kit (BBL, Cockiesville, MD) used with Grams staining control slides (Fisher Scientific, Pittsburgh, Pennsylvania). Subsequently, microscopic evaluation was performed using a Zeiss microscope (Carl Zeiss, Germany) 100X oil immersion objective lens (10X alternative and 2X objective magnification). Microscopic examination of individual strains for organism size, cell morphology and inclusion bodies (cell morphology and inclusion bodies observed after logarithmic growth) was performed in μm using wet mounting slides (10X alternative, 2X objective and 40X objective magnification) and phage control microscopy. (Akhurst, RJ and Boemare, NE 1990. Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R. and Kaya, H.). Pp 75-90.CRC Press, Boca Raton, USA .; Baghdiguin S., Boyer -Giglio MH, Thaler, JO, Bonnot G., Boemare N. 1993. Biol. Cell 79, 177-185). Colony staining was observed after inoculation on Bacto nutrient agar media (Difco Lboratories, Detroit, Michigan) prepared according to label instructions. Incubate at 28 ° C. and produce item on day 5. Colonies of test organisms were removed on small plugs from nutrient agar media plates and placed at the bottom of glass test tubes to test for the presence of enzyme catalysts. 1 ml of household hydrogen peroxide solution was added gently along the tube wall. Positive responses were recorded immediately after the appearance of bubbles (estimated by oxygen) or within 5 seconds. Controls of uninoculated nutrient agar medium and hydrogen peroxide solution were also examined. Each culture was inoculated in 10 ml of Bacto Nitrate broth (Difco Laboratories, Detroit, Michigan) to test for nitrate reduction. After incubation for 24 hours with gentle stirring at 28 ° C., 2 drops of sulfuric acid reagent and 2 drops of alpha-naphthylamine reagent were added to examine nitrate production (Difco Manual, 10th edition, Difco Laboratories, Detroit, MI, 1984). Reference). A pronounced pink or red color indicates the formation of nitrile from nitrate while the lack of color formation indicates that the strain is nitrate reduction negative. In later cases, fine powder zinc was added to further confirm the presence of unreduced nitrate; Formation of nitrate and redness were observed. Bacto MacConkey agar medium containing natural red dyes, with the ability of each strain to absorb dyes from the growth medium; Bacto Tergitol-7 agar medium containing bromothymol blue dye and Bacto EMB agar medium containing eosin-Y dye (agar medium formulated from Difco Laboratories, Detroit, Michigan, all prepared according to label instructions) . After inoculation on these media, dye uptake was recorded after incubation at 28 ° C. for 5 hours. Growth on these media is characteristic of organisms with enteric bacteria. The automated force of each strain was tested using a solution of Bacto automated force test medium (Difco Laboratories, Detroit, MI) prepared according to label instructions. Butt-stab inoculation was performed with each strain and macroscopic force was determined macroscopically as the propagation region of growth spread from the inoculation line. The production of proteases was examined by observing the hydrolysis of gelatin using Bacto gelatin (Difco Laboratories, Detroit, Mich.) Prepared according to label instructions. Cultures were inoculated and the tubes or plates were incubated at 28 ° C. for 5 days. The gelatin hydrolysis was then checked at room temperature, ie below 22 ° C. To assess growth at various temperatures, agar medium plates (2% protease peptone # 3 with 2% Bacto-Agar (Difco, Detroit, MI) in tarion water) were streaked from conventional inoculum. Plates were incubated at 20, 28 and 37 ° C. for up to 3 days. Incubator temperature levels were checked with an electrical thermocouple and measured for sure temperature setting. Oxygen conditions for the Photorabdus strain were examined in the following manner. Butt-stab inoculation into fluid thioglycobalt broth medium (Difco, Detroit, MI). Tubes were incubated for 1 week at room temperature and cultures tested for growth type extent. Indicator resazurin demonstrates the presence of medium cutting or aerobic zones (Difco Manual, 10th edition, Difco Laboratories, Detroit, Michigan). The growth zone results obtained for the tested photolabduus strains were consistent with those of the growable anaerobic microorganisms. In case of uncertainty, the final agar medium concentration of the fluid thioglycolate broth medium was raised to 0.75% and the growth properties were checked again.

*-A = gram strain, B = crystal inclusion body, C = bioluminescence, D = cell morphology, E = motility, F = nitrate reduction, G = presentation of catalase, H = gelatin hydrolysis, I = dye absorption, J = Coloration, growth on K = EMB agar, growth on L = MacConkey agar, growth on M = Tergitol-7 agar, N = passive anaerobic bacteria, growth at O = 20 ° C., growth at P = 28 ° C., Q = growth at 37 ° C., † − + / − = positive or negative of the characteristic, rd = battery, S = size within genus range, W = white, CR = cream, Y = yellow, YT = pale yellow, T = Brown, PO = pale orange, O = orange, PR = pale red, R = red.

The evolutionary diversity of the Photorabdus strains in the collection of the present invention was determined by analysis of PCR (polymerase chain reaction) mediated genomic fingerprinting using the genomic DNA of each strain. This technique is based on a group of repeating DNA sequences that exist throughout the genome of various bacterial species (Versalovic, J., Schneider, M., DE Bruijn, FJ and Lupski, JR 1994. Methods Mol. Cell. Biol. , 5, 25-40.). Of these sequences, the repeat extragenic palindromic sequence (REP), the enteric bacterial repeat gene common sequence (ERIC), and the BOX component are believed to play an important role in the organization of the bacterial genome. Genomic tissue is believed to be shaped by selection and different dispersions of these components in the genome of closely related bacterial strains can be used for identification of these strains (eg Louws, FJ, Fulbright, DW, Stephens, CT and DE Bruijn, FJ 1994. Appl. Environ. Micro. 60, 2286-2295.). Rep-PCR uses oligonucleotide primers complementary to the repeat sequence to amplify DNA fragments of different sizes that exist between the repeat sequences. The product is separated by electrophoresis to establish a DNA "fingerprint" for each strain.

To isolate genomic DNA from strains of the invention, the cell pellet was resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH8.0) to a final dose of 10 ml and 12 ml of 5 M NaCl was added. This mixture was centrifuged at 15,000 xg for 20 minutes. The resulting pellet was resuspended in 5.7 ml TE and 300 μl 10% SDS and 60 μl 20 mg / ml proteinase K (Gibco BRL, Grand Island, NY) was added. The mixture was incubated at 37 ° C. for 1 hour before about 10 mg of lysozyme was added and the mixture was incubated for an additional 45 minutes. 1 ml of 5M NaCl and 800 μl of CTAB / NaCl (10% w / v CTAB, 0.7 M NaCl) were added and the mixture was incubated at 65 ° C. for 10 minutes, gently shaken, incubated and shaken for an additional 20 minutes. Helped clear the cell material. Equal amounts of chloroform / isoamyl alcohol solution (24: 1, v / v) were added and mixed gently before centrifugation. Then extracted twice using the same volume of phenol / chloroform / isoamyl alcohol (50: 49: 1). Genomic DNA was precipitated using 0.6 doses of isopropanol. The precipitated DAN was collected with glass sticks, washed twice with 70% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1 mM EDTA). The DNA was then quantified by optical density at 260 nm. The following livers used primers to perform rep-PCR analysis of the Photoractus genomic DNA: REP1R-I; 5'-IIIICGICGICATCIGGC-3 'and REP2-I; 5'-ICGICTTATCIGGCCTAC-3 '. PCR was performed using the following 25 μl reactions: 7.75 μl H ₂ O, 2.5 μl 10 × LA buffer (PanVera Corp., Madison, Wis.), 16 μl dNTP mix (2.5 mM each), 1 μl of each primer (50 pM / μl), DMSO μl, 1.5 μl genomic DNA (concentration 0.075-0.480 μg / μl) and 0.25 μl TaKaRa EX Taq (PanVera Corp., Madison, Wis.). PCR amplification was carried out in a Perkin Elmer DNA thermal cycler (Norwalk, CT) using the following conditions: 95 ° C./7 min, followed by 94 ° C./1 min, 44 ° C./1 min, 65 ° C./8 min. Of 35 repetitions followed by 15 minutes at 65 ° C. After cycling 25 μl of the reaction was added to 5 μl of 6 × gel loading buffer (0.25% bromophenol blue, 40% sucrose). A 15 × 20 cm 1% agarose gel was then developed in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA) using 8 μl of each reaction. The gel was run at 45V for about 16 hours. The gel was then stained for 1 hour in 20 μg / ml ethidium bromide and destained for about 3 hours in TBE buffer. Polaroid pictures of the gel were printed under UV light.

The presence or absence of a band at a size specific to each stain was recorded from the photographs and entered as a similarity matrix into the numerical classification software program NTSYS-pc (Exeter Software, Setauket, NY). Teeth analyzed at the same time. E. coli strain HB101 and Xanthomonas duck fib. A control of duck (Xanthomonas oryzae pv. Oryzae) produced a "fingerprint" according to published literature (Versalovic, J., Koeuth, T. and Lupski, JR 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, CM, Halda-Alija, L., Louws, F., Skinner, DZ, George, ML, Nelson, RJ, DE Bruijn, FJ, Rice, C. and Leach, JE 1995. Int.Rice Res.Notes , 20, 23-24 .; Vera Cruz, CM, Ardales, EY, Skinner, DZ, Talag, J., Nelson, RJ, Louws, FJ, Leung, H., Mew, TW and Leach, JE 1996. Phytopathology) . Then a series of programs in NTSYS-pc; SIMQUAL (similarity to quantitative data) and related strains to generate similarity coefficients (using Jaccard coefficients) of the matrix and SAHN (continuous, cohesive, which can be expressed as a phenogram (Figure 5)) Data from photolapidus strains were analyzed using the Heraical and Nested clustering method (UPGMA (using Unweighted Pair-Group method using arithmetic mean)). COPH (cophenetic values) and MXCOMP (matrix comparison) programs were used to generate cophenetic matrixes and to compare the relationship between this and the original matrix on which clustering was based. The resulting normalized Mantel statistic (r) was generated, which is a measure of goodness suitable for cluster analysis (r = 0.8-0.9 indicates very good suitability). R is 0.919 for the present invention. Thus, the collection of the present invention consists of a diverse group of representative easily identifiable strains of the genus Photorapdus.

<Example 23>

Insecticidal Utility of Toxin (s) Produced by Various Photoractus Strains

In 175 ml of 2% Proteose Peptone # 3 (PP3, Difco Laboratories, Detroit, MI) liquid medium in a 500 ml Tribaffle flask with Kaput Primary strain subclones were inoculated to prepare initial seed cultures of the various photolapse strains. Inoculum for each seed culture was derived from an oil covered agar slope culture or plate culture. After inoculation, the flasks were incubated at 28 ° C. for 16 hours at 150 rpm on a rotary shaker. The seed culture was then used as a uniform inoculum for a given fermentation of each strain. The post-log seed cultures are also covered with sterile mineral oil, a sterile magnetic stir bar is added for future resuspension, and the culture is stored in the dark, at room temperature, thereby inoculating the inoculum toxin production. The state was stored for a long time. Production broth was inoculated by adding 1% of actively growing seed culture (eg, 1.75 ml per 175 ml of fresh culture) to 2% PP3 fresh medium. The broth was produced in a 500 ml tribaffle flask (see above) or a 2800 ml baffle uneven bottom flask (500 ml capacity) sealed with a silicone foam stopper. Production flasks were incubated for 24 to 72 hours under these conditions. After incubation, the broth was dispensed into cells and lysate pellets by dispensing into sterile 1 L polyethylene bottles and spinning at 2600 × g for 1 hour at 10 ° C. The liquid broth was then vacuum filtered through Whatman GF / D (2.7 μm retention) and GF / B (1.0 μm retention) glass filters to remove debris. The broth was further purified using a 0.5 μm open conduit filter with a tangential outflow microfiltration device (Pall Filtron, Northboro, Mass.). If necessary, the broth can be further clarified by cooling (up to 4 ° C.) and centrifuging at 2600 × g for several hours. After this procedure, the broth was filtered and sterilized using a 0.2 μm nitrocellulose membrane filter. Sterile broth was then used either directly for biological analysis, biochemical analysis or with a 10000 cutoff M12 ultrafiltration device (Amicon, Beverly, Mass.) Or a centrifugal concentrator (Millipore, Bedford, Mass.) With a pore size of 10000. Concentrated (up to 10 times). For centrifugal concentrators, the broth was centrifuged at 2000 × g for about 2 hours. A molecular weight 10000 permeate was added to the corresponding residue to give the desired concentrate of components with molecular weight greater than 10000. The treated broth sample was heat inactivated by heating the sample at 100 ° C. for 10 minutes in a sand filled heat block.

Broth (s) and toxin complex (s) from different photolabose strains are useful for reducing insect populations, and insects consisting of using the described active substances in insect habitats in an amount effective to inactivate insects. It was used in the population suppression method. Evidence of the range of pesticidal activity observed from broths of the select group of photolabduus strains fermented as above is shown in Table 36. Increasing the broth concentration or using other fermentation methods allows detection of additional pesticidal activity in the strain. Consistent with the activity associated with the protein, the pesticidal activity of all strains tested was heat labile.

Culture broth (s) from various photolabose strains exhibit differential pesticidal activity (decrease in mortality and / or growth inhibition, reduction in adult appearance) with respect to insect numbers. More specifically, activity is shown against corn rootworm larvae that are members of the Coleoptera insect throat. Other members of the beetle neck include cotton weevils, millipedes, worms, pollen beetles, jog beetles, seed beetles, and Colorado potato beetles. The broth and purified toxin complex (es) also have activity against gray tobacco moth, horn moth and European corn borer, which are members of the Lepidoptera. Other typical members of the neck include beetroot maggots, cabbage bugs, black beetles, corn ear beetles, apple moths, antagonists, gallery moths, leaf moths, cabbage beetles, cotton beetle moths, salamanders, eastern tent moths and grass. There are insect moths and pole matrix maggots. Activity is also observed for German cockroaches, a member of the Dictoptera (or Blatodea) neck. Other members of this neck have oriental cockroaches and American cockroaches.

Activity against corn rootworm larvae was tested as follows. Photolabose culture broth (s) (0-15 fold concentration, filtration sterilization), 2% proteose peptone # 3, purified toxin complex (s) (0.23 mg / ml) or 10 mM sodium phosphate buffer at pH 7.0 Was applied directly to the surface of the artificial feed (approximately 1.5 cm ² ) by 40 μl [Rose, RI and McCabe, JM (1973). J. Econ. Entomol. 66, (389-400). Toxin complexes were diluted in 10 mM sodium phosphate buffer at pH 7.0. The food plate was air dried in a sterile flow hood and wells were infested with a single, newborn Diabrotica undecimpunctata howardi (Sudden Corn Root, SCR) hatched from surface sterilized eggs. The plates were sealed and placed in a wet growth chamber and kept at 27 ° C. for a suitable period (3 to 5 days). Mortality and larval weight measurements were then recorded. In general, 16 insects were used per treatment in all studies. Control mortality was generally less than 5%.

Activity against Lepidoptera larvae was tested as follows. Concentrated (10-fold) photolabose culture broth (s), control medium (2% proteose peptone # 3), purified toxin complex (s) (0.23 mg / ml) or 10 mM sodium phosphate buffer at pH 7.0 40 μl was applied directly to the surface of standard artificial Lepidoptera food (Stoneville yellow food, approximately 1.5 cm ² ). The food plate was air dried in a sterile flow hood and each well was ramped up with one newborn larva. European corn borer (Ostrinia nubilalis) and Manuca sexta eggs were purchased and hatched, and gray tobacco moth (Heliothis virescens) larvae were supplied internally. After spreading with the larvae, the food plate was sealed and placed in the wet growth chamber and kept in the dark at 27 ° C. for an appropriate period. Lethality and weight measurements were performed on day 5. In general, 16 insects were used per treatment in all studies. Control mortality of control medium was generally 0-12.5% and control mortality of phosphate buffer was less than 10%.

The activity against cockroaches was tested as follows. Concentrated (10-fold) photolabose culture broth (s), control medium (2% proteose peptone # 3) were added directly to the surface of standard artificial lepidoptera feed (Stoneville yellow feed, approximately 1.5 cm ² ) by 40 μl. Applied. The food plate was air dried in a sterile flow hood and each well was ramped up with a first ₂ German cockroach (Blatella Germanica). After spreading with the larvae, the food plate was sealed and placed in the wet growth chamber and kept in the dark at 27 ° C. for an appropriate period. Lethality and weight measurements were performed on day 5. In general, 16 insects were used per treatment in all studies. Control mortality was less than 10%.

Insecticidal Spectrum of Broth from Different Photoractus Strains Photolapux strain Susceptibility ^* Insect Species P. Zealandicopy. Hapialus HB-ArgHB Oswego HB Leviston K-122HMGD Indicus GDPWH-5 Megidis HF-85A. Mouse MP1MP2MP3MP4MP5GL98GL101GL138GL155GL217GL257DEP1DEP2DEP3 1 ^** , 2, 41, 2, 41, 2, 41, 2, 41, 2, 41, 41, 41, 2, 42, 41, 2, 41, 2, 41, 2, 41, 41, 2, 41, 2, 441, 441, 41, 4, 51, 2, 41, 41, 2, 41, 41, 41, 2, 3, 44 * = 25% motility and / or growth inhibition rate relative to the control group ** = 1; Gray tobacco moth, 2; Beetle moth, 3; European Corn Borer, 4; Southern Corn Roots, 5; German cockroach.

<Example 24>

Southern analysis of non-W-14 photoladus using W-14 gene probe

Photolabduus strains were grown on 2% proeoze peptone # 3 agar (Difco Laboratories, Detroit, Michigan) and pesticidal toxins were maintained after repeated bioassay. 50 ml shaker cultures were generated in a 175 ml baffle flask with 2% protease peptone # 3 medium and grown for about 24 hours at 28 ° C. and 150 rpm. 15 ml of this culture were centrifuged (700 × g, 30 min) and frozen in medium at −20 ° C. until thawed (slow in ice water) for DNA isolation. Thawed W-14 cultures were centrifuged (900 × g, 15 min, 4 ° C.) and the floating orange mucopolysaccharide material was removed. Residual cellular material was centrifuged (25,000 × g, 4 ° C.) to pellet bacterial cells, remove media and discard.

Total DNA was isolated using the CTAB method described in Section 2.4.1 of Ausubel, 1994 et al. The modifications included high salt shanks and all volumes increased to 10 times the "miniprep" recommended volume. All centrifugation was carried out at 4 ° C unless otherwise indicated. The pelleted bacterial cells were resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) to a final volume of 10 ml, followed by the addition of 12 ml 5 M NaCl. This mixture was centrifuged at 15,000 x g for 20 minutes. The pellet was resuspended in 5.7 ml TE and 300 μl 10% SDS and 60 μl 20 mg / ml proteinase K (sterile distilled water), Gibco BRL Products, Grand Island, NY) were added to the suspension. The mixture was incubated at 37 ° C. for 1 hour and then about 10 mg lysozyme (Worthington Biochemical Corp., Freehold, NJ) was added. After further incubation for 45 minutes, 1 ml of 5 M NaCl and 800 μl of CTAB / NaCl solution (10% w / v CTAB, 0.7 M NaCl) were added. This precipitate was incubated at 65 ° C. for 10 minutes, then gently stirred and further incubated, and stirred for about 20 minutes to clear the foamed material. Equivalent volumes of chloroform / isoamyl alcohol solution (24: 1 v / v) were added, mixed very gently and centrifuged at 12,000 × g for 15 minutes to separate the phases. The upper layer (aqueous phase) was gently removed with a null bore pipette, PCI (phenol / chloroform.isoamyl alcohol; 50: 49: 1, v / v / v; 1 M Tris-HCl (pH 8.0, Intermountain Scientific Corporateion) , Kayville, UT)), extracted twice as above in the equivalent volume of). DNA precipitated with 0.6 volume of isopropanol was gently removed with a glass rod, washed twice with 70% ethanol and dissolved in 2 ml STE (10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 8). This precipitate contained 2.5 mg / ml as measured by optical density at 260 nm.

[Identification of Bgl II / Hind III Fragments Hybridized with tc-gene Specific Probes]

About 10 μg of genomic DNA was digested into about 30 units of each of Bgl II / Hind III (NEB) for 180 minutes, frozen overnight, then heated at 65 ° C. for 5 minutes, and 0.8% of agarose gel ( Electrophoresis on Seakem LE, 1X TEA, 80 volts, 90 minutes). DNA was stained with ethidium bromide (50 μg / ml) as described above and photographed under ultraviolet light. DNA fragments in agarose gels are clarified (5 minutes in 0.2 M HCl), denatured (0.5 M NaOH, 15 minutes in 1.5 M NaCl), neutralized (15 in 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl). Min), rinsed three times with distilled water between each step. DNA was transferred from gels to NYTRAN nylon membranes (Amersham, Arlington Heights, IL) using Southern Blot (20X SSC) protocol as described in Section 2.9 of Ausubel, CPMB, op., Et al. The transferred DNA was then UV crosslinked with the nylon membrane using a Stratagene UV Stratalinker adapted for automatic crosslinking. The membranes were stored dry at 25 ° C. until used.

Hybridization was performed using a detection system following the ECL® Direct (Amersham, Arlington Heights, IL) labeling and protocol provided by the manufacturer. Briefly, probes were prepared by covalently binding the denatured DNA to the enzyme hose radish peroxidase. Once labeled probes were used under hybridization conditions to maintain enzymatic activity. The non-hybrid probe was removed by gentle wash twice at 42 ° C. for 20 minutes each in 0.5 × SSC, 0.4% SDS and 6 M urea. Then it was washed twice for 5 minutes each at room temperature in 2 × SSC. As indicated by the manufacturer, hybridized DNA bands were detected using ECL reagents. There are several factors that affect the ability to detect genetic relationships between various photolabose strains and strain W-14. First, very stringent conditions were used for these hybridizations. Changing the stringency and washing conditions of hybridization is known to affect the pattern and strength of the hybridization band. Second, the blot versus blot modification of the Southern blot affects the fluidity of the hybridization band and molecular weight measurements. Thus, W-14 was included as standard on all Southern blots.

Gene specific probes derived from the W-14 virulence gene were used for these hybridizations. The probes listed below are specific coordination within each corresponding gene sequence. probes specific for tcaBi / B _ii : 1174 to 3642 of SEQ ID NO: 25, probes specific for tcaC: 3637 to 6005 of SEQ ID NO: 25, probes specific for tcbA: 2097 to 4964 of SEQ ID NO: 11, tcdA _i Probes specific for: 1660 to 4191 of SEQ ID NO: 46. The following table summarizes the Southern blot analysis of the Photorabdus strains. When hybridization of the probes occurred, the hybridized fragments appeared to be the same or different from the pattern observed for the W-14 strain.

Southern analysis of photolapux strain Strain tcdA tcbA tcaC tcaB _{i / ii} WX-1WX-2WX-3WX-4WX-5WX-6WX-7WX-8WX-9WX-10WX-11WX-12WX-14WX-15HP88HmHbH9B2NC-1WIRW30W-14 DDDDDDDDNDNDNDDDDDDDDDDDDI DDDDDDDDDDDDDD ------- DI D-DNDDDNDDDDDDDDDDDIDDDDI DDDDDDDDDDDDDDDD-D-DDDI ND = no measurement; -= Unable to detect hybridize product; I = same fragment pattern; D = different fragment patterns

Southern analysis of photolapux strain Strain tcdA tcbA tcaC tcaB _{i / ii} K-122PWH-5 indicus megidis GDHF-85MP3MP1A. Mouse HB-ArgHMGDHB Leviston HB Oswego W-14 3.3, 2.8 + DDDDDDDDDDDI DDDDDD-++ NDDDDI -D3.0DDDDDDDDDDI ND-I ---------- I ND = no measurement; -= Unable to detect hybridize product; I = same fragment pattern; D = different fragment patterns

Southern analysis of photolapux strain Strain tcdA tcbA tcaC tcaB _{i / ii} GL98GL101GL138GL155GL217GL257MP4MP5p. Hephyaluspi. ZealandiaDEP1DEP2DEP3W-14 + --- ++-++ 3.8, 2.8 +++-++ --- 2.8 DDD-DD--D11.02.8 ND = no measurement; -= Unable to detect hybridize product; I = same fragment pattern; D = different fragment pattern, + = unmeasured hybridization fragment pattern.

From these analyzes, it is evident that homologues of the W-14 gene, as evidenced by differences in gene fragment size between W-14 and other strains, are dispersed throughout these inverse photoladus.

<Example 25>

N-terminal amino acid sequence of toxin complex peptides from different photolabose strains

As described in Example 14, the relationship of peptides isolated from different photolabose strains was introduced in N-terminal amino acid sequencing. Toxin peptide N-terminal amino acid sequences in several strains were compared to W-14 toxin. In Table 40, comparisons of the toxin peptides so far compared showed that the same or similar (at least 40% similarity to the W-14 gene / peptide) toxin peptides were present in all strains. For example, the N-terminal amino acid sequence of TcaC, SEQ ID NO: 2, was found to be identical to that for the 160 kDa peptide in HP88, and analogues were also present in strains WIR, H9, Hb, WX-1 and Hm. Some W-14 peptides or analogs were not found in other strains, but not all peptides were sequenced for toxin complexes from other strains due to N-terminal blockade or low abundance. In addition, many other N-terminal amino acid sequences (SEQ ID NOS: 82-88) were obtained for toxin complex peptides from other strains that were not similar to peptides from W-14, and in some cases were identical to each other. For example, the same amino acid sequence, SEQ ID NO: 82, was obtained for 64 kDa peptides present in HP88 and Hb strains, and similar sequences were obtained for 70 kDa peptides in NC-1 strain (SEQ ID NO: 83).

Comparison of amino terminal sequence homology between proteins isolated from non-W-14 strains W-14 peptide W-14 gene W-14 sequence order Strain sameness Homology TcaAiiTcaAiiiTcaBiTcaBiiTcaCTcbAiiTcbAiiTccATccBTcdAiiTcdAiii? tcaAtcaAtcaBtcaAtcaAtcbAtcbAtccAtccBtcdAtcdA? 15435214087419 7676727374758077787981 H9HmH9HmHbHP88WIRH9HmWX-1HbWX-1WX-2WX-14WIRH9NC-1HmHbH9HbHP88 --61 KDa61 KDa160 KDa ----- 170 KDa180 KDa180 KDa170 KDa-140 KDa-57 KDa-86 KDa86 KDa 74 KDa71 KDa--160 KDa-170 KDa180 KDa170 KDa170 KDa81 KDa ---- 170 KDa-190 KDa-69 KDa--

Homologous refers to an amino acid sequence having at least 40% similarity to the W14 gene / peptide. Similar residues are characterized in one of five groups (P, A, G, S, T); (Q, N, E, B, D, Z), (H, K, R), (L, I, V, M), and (F, Y, W)

<Example 26>

Immunological Analysis of Photorabdus Strains

Arrangement broth of the Photorabdus strains were concentrated 10-15 times using Centriprep-10 ultrafiltration apparatus (Ameicon, Inc. Beverly, MA 01915). The concentration of protein ranges from 0.3 to 3.0 mg per ml. 10-20 μg of total protein was filled into each well of a precast 4-20% polyacrylamide gel (Integrated Separation Systems, Natick, MA 01760). Gel electrophoresis was performed for 1.25 hours using a constant current set at 25 ma per gel. Gels were electroblotted onto a Hybond-ECL ™ Nitrocellulose Cell (Amersham Corporation, Arlington Hts, Il 60005) using a semi-dry electric blotter (Pharmacia Biotech Inc., Piscataway, NJ 08854). A constant current was applied at 0.75 ma per cm for 2.5 hours. The membrane was blocked for 1 hour at room temperature with 10% milk in TBST (25 mM Tris HCl pH 7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20). Each primary antibody was diluted 1: 500 in 10% wheat / TBST. Other dilutions between 1:50 and 1: 1000 were also used. Then it was washed thoroughly with blocking solution or TBST. A 1: 2000 dilution of a secondary antibody (goat anti-rat IgG or goat anti-rabbit TgG conjugated to horseradish peroxidase in 10% wheat / TBST; BioRad Laboratories, Hercules, CA 94547) was applied to the membrane and this was applied. Placed on a flat shaker for 1 hour. The membrane was subsequently washed with excess TBST. Detection of the protein was washed using an ECL Enhanced Chemiluminescence Detection Device (Amersham International).

A panel of peptide specific antibodies generated against the W-14 peptide was used and the Western blot analysis was used to characterize the protein composition of the broth from nine non-W-14 photolabose strains. In addition, one monoclonal antibody (MAb-C5F2) that recognizes the TcbA _iii protein in the W-14 induced toxin complex was used. The results (Table 39) showed the mutual recognition of antibodies to some of the proteins in these broths. In some cases, the protein recognized by the antibody was the same size as the W-14 target peptide. In other cases, the protein recognized by the antibody was smaller than the W-14 target peptide. This data shows that some of the non-W-14 photolabdus can produce proteins similar to the W-14 strain. These differences are due to detection or protein processing or degradation processes. Some of the strains did not contain proteins that could be recognized by some antibodies, but the concentrations were significantly lower than those observed for W-14 peptides. When compared to the various toxin peptide analogs, these results showed peptide diversity between the Photorabdus strains.

Additional non-W-14 photolabose strains were characterized by western blot analysis using culture broth and / or partially purified protein fractions as antibodies. Panels of antibodies include MAb-C5F2, MAb-DE1 (recognized TcdA _ii ), PAb-DE2 (recognized TcaB), PAb-TcbA-syn, PAb-, TcaC-syn, PAb TcaB _ii -syn, PAb-TcbA _iii -syn , PAb-TcaB _i -syn. These antibodies showed cross-reactivity with proteins in partially stagnant fractions of broth and non-W-14 strains.

The data indicate that antibodies can be used to identify proteins in broth as well as partially purified protein fractions.

<Example 27>

Bacterial Expression of the tcdA Coding Region

Engineering the tcdA Gene for Bacterial Expression

The 5 ^' and 3 ^' ends of the tcdA coding region (SEQ ID NO: 46) were modified to add a cloning site useful for inserting segments into heterogeneous expression vectors. The ends were modified using a single primer in the polymerase chain reaction (PCR), which was performed basically as described in Example 8. A primer set as described below was used in combination with cosmid 21D2.4 as a template to produce a product with suitably modified ends.

The 5 ^' terminus of the gene was modified using the first primer set to use the preceding primer A0F1 (5 ^' GAT CGA TCG ATC CAT GGC CAA CGA CGA GTC TGT AAA AGA GAT ACC TGA TG TAT TAA AAA GCC AGT GTG 3 ^' ) Insert a single nCO i site in the initiator codon and use reverse primer A0R1 (5 ^' GAT CGA TCG TAC GCG GCC GCT GAT TCG ATC GTC GAC CCA TTG ATT TGA GAT CTG GGC GGC GGG TAT CCA GAT AAT AAA CGG AGT CAC 3 ^' ) A single Bgl II, Sal I and Not I was added to facilitate insertion of the rest of the gene.

Another PCR reaction was designed to modify the three ends of the gene by adding additional stop codons. Using the preceding primer A0F2 (5 ^'ACT GGC TGC GTC GTC GAC TGG CGG CGA TTT ACT ^3') was amplified across a single Sal I site of the gene by using the latter was cloned into a modified 3 ^'end. Second stop codon (TGA) and cloning site Xho I, Stu I and using reverse primer A0R2 (5 ^' CGA TGC ATG CTG CGG CCG CAG GCC TTC CTC GAG TCA TTA TTT AAT GGT GTA GCG AAT ATG CAA AAT 3 ^' ) Not I was inserted. The bacterial expression vector pET27b (Novagen, Madison, Wis.) Was modified to remove the Bgl II site at position 446 using standard molecular biology techniques.

The 497 bp PCR product from the first amplification reaction (A0F1 + AOR1) to modify the 5 ^' end of the gene was ligated to the modified pET27b according to the supplier's instructions. DNA sequences of the amplified portions of the three isolates were determined using the recommended primers from the supplier and by arranging the methods described above. The sequences of all isolates were identical.

Then, one isolate was used as the cloning vector to insert the middle portion of the tcdA gene on the 6341 bp Bgl II to Sal I fragment. The resulting clones were called MC4 and contained all but most of the 3 ^′ of the tcdA coding sequence. Finally, to complete the full length coding region, 832 bp PCR product from the second PCR amplification (A0F2 + A0F2) for modifying the 3 ^' end of the gene was ligated according to standard molecular biology techniques to Sal I to MC4 was isolated on Not I fragment. The tcdA coding region was sequenced and found to be complete, and the resulting plasmid was called pDAB2035.

Constitution of plasmids pDAB2036, pDAB2037 and pDAB2038 for bacterial expression of tcdA

The tcdA coding region was cleaved from plasmid pDAB2035 with restriction enzymes Nco I and Xho I and the gel was purified. The fragment was ligated to the Nco I and Xho I sites of the expression vector pET15 to produce plasmid pDAB2036. In addition, pDAB2035 was cleaved with Nco I and Not I to release the tcdA coding region and ligated to the Nco I and Not I positions of the expression vector pET28b to generate plasmid pDAB2037. Finally, plasmid pDAB2035 was cleaved with Nco I and Stu I to release the tcdA region. This fragment was ligated with the expression vector Trc99a, cleaved with Hind III and treated with T4 DNA polymerase to blunt the ends. The vector was then cleaved with Nco I and the tcdA fragment was ligated with the cleaved Nco I / Stu I. The resulting plasmid is called pDAB2038.

Expression of tcdA from plasmid pDAB2038

Plasmid pDAB2038 was transcribed into BL21 cells and expressed as described above for plasmid pDAB2033 in Example 19.

Purification of tcdA from E. coli

Expression cultures were centrifuged at 10,300 g for 30 minutes and the supernatants were collected. 7.5 ml / min flow rate on a pore 50 HQ (Perspective System, MA) column (1.6 cm x 10 cm) diluted with 2 volumes H ₂ O and equilibrated with 10 mM sodium phosphate buffer (pH 7.0, buffer A). Walked by. The column was washed with Buffer A at 280 nm until the optical density returned to baseline. The protein bound to the column was then eluted with 1 M NaCl in Buffer A.

Fractions were charged to a gel filtration column, Sepharose CL-4B (2.6 x 100 cm), equilibrated with Buffer A with 20 ml aliquots. Protein was eluted with Buffer A at a flow rate of 0.75 mL / min. Fractions with retention times between 260 and 460 minutes were collected and walked at 1 ml / min on a mono Q 5/5 column equilibrated with 20 mM Tris-HCl (pH 7.0, Buffer B). The column was washed with Buffer A at 280 nm until the optical density returned to baseline. The protein bound to the column was then eluted with 1 M NaCl in Buffer B. The protein bound to the column was eluted with a linear gradient of 0-1 M NaCl in Buffer B at 1 mL / min for 30 minutes. One milliliter fractions were collected, serially diluted and introduced into SCR bioassay. The fraction eluted between 0.1 and 0.3 M NaCl was found to have the highest pesticidal activity. The pAb TcdA _iii -syn antibody and the presence of Tcd _iii peptides in Western analysis of the active fractions using an antibody -syn TcdA _iii and _iii TcdA were confirmed.

DNA SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Ensign, Jerald C

Bowen, David J

Petell, James

Fatig, Raymond

Schoonover, Sue

ffrench-Constant, Richard

Orr, Gregory L

Merlo, Donald J

Roberts, Jean L

Rocheleau, Thomas A

(ii) TITLE OF A: Insecticidal Protein Toxins from

Photorhabdus

(iii) NUMBER OF SEQUENCES: 88

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DowElanco

(B) STREET: 9330 Zionsville Road

(C) CITY: Indianapolis

(D) STATE: IN

(E) COUNTRY: US

(F) ZIP: 46268

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent In Release # 1.0, Version # 1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08 / 063,615

(B) FILING DATE: 18-MAY-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08 / 395,497

(B) FILING DATE: 28-FEB-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60 / 007,255

(B) FILING DATE: 06-NOV-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08 / 608,423

(B) FILING DATE: 28-FEB-1996

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08 / 705,484

(B) FILING DATE: 28-AUG-1996

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08 / 743,699

(B) FILING DATE: 06-NOV-1996

(viii) ATTORNEY / AGENT INFORMATION:

(A) NAME: Borucki, Andrea T.

(B) REGISTRATION NUMBER: 33651

(C) REFERENCE / DOCKET NUMBER: 50301E

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 317-337-4846

(B) TELEFAX: 317-337-4847

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 (TcbAii N-terminus):

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn

1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 (TcaC N-terminus):

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Trp

1 5 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 (TcaBi N-terminus):

Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp Ala

1 5 10 15

Leu val ala

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 (TcaAiii N-terminus):

Ala Ser Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn

1 5 10

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 (TcaBii N-terminus):

Ala Gly Asp Thr Ala Asn Ile Gly Asp

1 5

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Leu Gly Gly Ala Ala Thr Leu Leu Asp Leu Leu Leu Pro Gln Ile

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 (TccB N-terminus):

Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 (TccA N-terminus):

Met Asn Leu Ala Ser Pro Leu Ile Ser

1 5

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Ile Asn Leu Asp Ile Asn Glu Gln Asn Lys Ile Met Val Val Ser

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Ala Ala Lys Asp Val Lys Phe Gly Ser Asp Ala Arg Val Lys Met Leu

1 5 10 15

Arg Gly Val Asn

20

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7515 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..7515

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 (tcbA gene):

ATG CAA AAC TCA TTA TCA AGC ACT ATC GAT ACT ATT TGT CAG AAA CTG 48

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu

1 5 10 15

CAA TTA ACT TGT CCG GCG GAA ATT GCT TTG TAT CCC TTT GAT ACT TTC 96

Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe

20 25 30

CGG GAA AAA ACT CGG GGA ATG GTT AAT TGG GGG GAA GCA AAA CGG ATT 144

Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile

35 40 45

TAT GAA ATT GCA CAA GCG GAA CAG GAT AGA AAC CTA CTT CAT GAA AAA 192

Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys

50 55 60

CGT ATT TTT GCC TAT GCT AAT CCG CTG CTG AAA AAC GCT GTT CGG TTG 240

Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu

65 70 75 80

GGT ACC CGG CAA ATG TTG GGT TTT ATA CAA GGT TAT AGT GAT CTG TTT 288

Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe

85 90 95

GGT AAT CGT GCT GAT AAC TAT GCC GCG CCG GGC TCG GTT GCA TCG ATG 336

Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met

100 105 110

TTC TCA CCG GCG GCT TAT TTG ACG GAA TTG TAC CGT GAA GCC AAA AAC 384

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn

115 120 125

TTG CAT GAC AGC AGC TCA ATT TAT TAC CTA GAT AAA CGT CGC CCG GAT 432

Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp

130 135 140

TTA GCA AGC TTA ATG CTC AGC CAG AAA AAT ATG GAT GAG GAA ATT TCA 480

Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser

145 150 155 160

ACG CTG GCT CTC TCT AAT GAA TTG TGC CTT GCC GGG ATC GAA ACA AAA 528

Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys

165 170 175

ACA GGA AAA TCA CAA GAT GAA GTG ATG GAT ATG TTG TCA ACT TAT CGT 576

Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg

180 185 190

TTA AGT GGA GAG ACA CCT TAT CAT CAC GCT TAT GAA ACT GTT CGT GAA 624

Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu

195 200 205

ATC GTT CAT GAA CGT GAT CCA GGA TTT CGT CAT TTG TCA CAG GCA CCC 672

Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro

210 215 220

ATT GTT GCT GCT AAG CTC GAT CCT GTG ACT TTG TTG GGT ATT AGC TCC 720

Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser

225 230 235 240

CAT ATT TCG CCA GAA CTG TAT AAC TTG CTG ATT GAG GAG ATC CCG GAA 768

His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu

245 250 255

AAA GAT GAA GCC GCG CTT GAT ACG CTT TAT AAA ACA AAC TTT GGC GAT 816

Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp

260 265 270

ATT ACT ACT GCT CAG TTA ATG TCC CCA AGT TAT CTG GCC CGG TAT TAT 864

Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr

275 280 285

GGC GTC TCA CCG GAA GAT ATT GCC TAC GTG ACG ACT TCA TTA TCA CAT 912

Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His

290 295 300

GTT GGA TAT AGC AGT GAT ATT CTG GTT ATT CCG TTG GTC GAT GGT GTG 960

Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val

305 310 315 320

GGT AAG ATG GAA GTA GTT CGT GTT ACC CGA ACA CCA TCG GAT AAT TAT 1008

Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr

325 330 335

ACC AGT CAG ACG AAT TAT ATT GAG CTG TAT CCA CAG GGT GGC GAC AAT 1056

Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn

340 345 350

TAT TTG ATC AAA TAC AAT CTA AGC AAT AGT TTT GGT TTG GAT GAT TTT 1104

Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe

355 360 365

TAT CTG CAA TAT AAA GAT GGT TCC GCT GAT TGG ACT GAG ATT GCC CAT 1152

Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His

370 375 380

AAT CCC TAT CCT GAT ATG GTC ATA AAT CAA AAG TAT GAA TCA CAG GCG 1200

Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala

385 390 395 400

ACA ATC AAA CGT AGT GAC TCT GAC AAT ATA CTC AGT ATA GGG TTA CAA 1248

Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln

405 410 415

AGA TGG CAT AGC GGT AGT TAT AAT TTT GCC GCC GCC AAT TTT AAA ATT 1296

Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile

420 425 430

GAC CAA TAC TCC CCG AAA GCT TTC CTG CTT AAA ATG AAT AAG GCT ATT 1344

Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile

435 440 445

CGG TTG CTC AAA GCT ACC GGC CTC TCT TTT GCT ACG TTG GAG CGT ATT 1392

Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile

450 455 460

GTT GAT AGT GTT AAT AGC ACC AAA TCC ATC ACG GTT GAG GTA TTA AAC 1440

Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn

465 470 475 480

AAG GTT TAT CGG GTA AAA TTC TAT ATT GAT CGT TAT GGC ATC AGT GAA 1488

Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu

485 490 495

GAG ACA GCC GCT ATT TTG GCT AAT ATT AAT ATC TCT CAG CAA GCT GTT 1536

Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val

500 505 510

GGC AAT CAG CTT AGC CAG TTT GAG CAA CTA TTT AAT CAC CCG CCG CTC 1584

Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu

515 520 525

AAT GGT ATT CGC TAT GAA ATC AGT GAG GAC AAC TCC AAA CAT CTT CCT 1632

Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro

530 535 540

AAT CCT GAT CTG AAC CTT AAA CCA GAC AGT ACC GGT GAT GAT CAA CGC 1680

Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg

545 550 555 560

AAG GCG GTT TTA AAA CGC GCG TTT CAG GTT AAC GCC AGT GAG TTG TAT 1728

Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr

565 570 575

CAG ATG TTA TTG ATC ACT GAT CGT AAA GAA GAC GGT GTT ATC AAA AAT 1776

Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn

580 585 590

AAC TTA GAG AAT TTG TCT GAT CTG TAT TTG GTT AGT TTG CTG GCC CAG 1824

Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln

595 600 605

ATT CAT AAC CTG ACT ATT GCT GAA TTG AAC ATT TTG TTG GTG ATT TGT 1872

Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys

610 615 620

GGC TAT GGC GAC ACC AAC ATT TAT CAG ATT ACC GAC GAT AAT TTA GCC 1920

Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala

625 630 635 640

AAA ATA GTG GAA ACA TTG TTG TGG ATC ACT CAA TGG TTG AAG ACC CAA 1968

Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln

645 650 655

AAA TGG ACA GTT ACC GAC CTG TTT CTG ATG ACC ACG GCC ACT TAC AGC 2016

Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser

660 665 670

ACC ACT TTA ACG CCA GAA ATT AGC AAT CTG ACG GCT ACG TTG TCT TCA 2064

Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser

675 680 685

ACT TTG CAT GGC AAA GAG AGT CTG ATT GGG GAA GAT CTG AAA AGA GCA 2112

Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala

690 695 700

ATG GCG CCT TGC TTC ACT TCG GCT TTG CAT TTG ACT TCT CAA GAA GTT 2160

Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val

705 710 715 720

GCG TAT GAC CTG CTG TTG TGG ATA GAC CAG ATT CAA CCG GCA CAA ATA 2208

Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile

725 730 735

ACT GTT GAT GGG TTT TGG GAA GAA GTG CAA ACA ACA CCA ACC AGC TTG 2256

Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu

740 745 750

AAG GTG ATT ACC TTT GCT CAG GTG CTG GCA CAA TTG AGC CTG ATC TAT 2304

Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr

755 760 765

CGT CGT ATT GGG TTA AGT GAA ACG GAA CTG TCA CTG ATC GTG ACT CAA 2352

Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln

770 775 780

TCT TCT CTG CTA GTG GCA GGC AAA AGC ATA CTG GAT CAC GGT CTG TTA 2400

Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu

785 790 795 800

ACC CTG ATG GCC TTG GAA GGT TTT CAT ACC TGG GTT AAT GGC TTG GGG 2448

Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly

805 810 815

CAA CAT GCC TCC TTG ATA TTG GCG GCG TTG AAA GAC GGA GCC TTG ACA 2496

Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr

820 825 830

GTT ACC GAT GTA GCA CAA GCT ATG AAT AAG GAG GAA TCT CTC CTA CAA 2544

Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln

835 840 845

ATG GCA GCT AAT CAG GTG GAG AAG GAT CTA ACA AAA CTG ACC AGT TGG 2592

Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp

850 855 860

ACA CAG ATT GAC GCT ATT CTG CAA TGG TTA CAG ATG TCT TCG GCC TTG 2640

Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu

865 870 875 880

GCG GTT TCT CCA CTG GAT CTG GCA GGG ATG ATG GCC CTG AAA TAT GGG 2688

Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly

885 890 895

ATA GAT CAT AAC TAT GCT GCC TGG CAA GCT GCG GCG GCT GCG CTG ATG 2736

Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met

900 905 910

GCT GAT CAT GCT AAT CAG GCA CAG AAA AAA CTG GAT GAG ACG TTC AGT 2784

Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser

915 920 925

AAG GCA TTA TGT AAC TAT TAT ATT AAT GCT GTT GTC GAT AGT GCT GCT 2832

Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala

930 935 940

GGA GTA CGT GAT CGT AAC GGT TTA TAT ACC TAT TTG CTG ATT GAT AAT 2880

Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn

945 950 955 960

CAG GTT TCT GCC GAT GTG ATC ACT TCA CGT ATT GCA GAA GCT ATC GCC 2928

Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala

965 970 975

GGT ATT CAA CTG TAC GTT AAC CGG GCT TTA AAC CGA GAT GAA GGT CAG 2976

Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln

980 985 990

CTT GCA TCG GAC GTT AGT ACC CGT CAG TTC TTC ACT GAC TGG GAA CGT 3024

Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg

995 1000 1005

TAC AAT AAA CGT TAC AGT ACT TGG GCT GGT GTC TCT GAA CTG GTC TAT 3072

Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr

1010 1015 1020

TAT CCA GAA AAC TAT GTT GAT CCC ACT CAG CGC ATT GGG CAA ACC AAA 3120

Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys

1025 1030 1035 1040

ATG ATG GAT GCG CTG TTG CAA TCC ATC AAC CAG AGC CAG CTA AAT GCG 3168

Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala

1045 1050 1055

GAT ACG GTG GAA GAT GCT TTC AAA ACT TAT TTG ACC AGC TTT GAG CAG 3216

Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln

1060 1065 1070

GTA GCA AAT CTG AAA GTA ATT AGT GCT TAC CAC GAT AAT GTG AAT GTG 3264

Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val

1075 1080 1085

GAT CAA GGA TTA ACT TAT TTT ATC GGT ATC GAC CAA GCA GCT CCG GGT 3312

Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly

1090 1095 1100

ACG TAT TAC TGG CGT AGT GTT GAT CAC AGC AAA TGT GAA AAT GGC AAG 3360

Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys

1105 1110 1115 1120

TTT GCC GCT AAT GCT TGG GGT GAG TGG AAT AAA ATT ACC TGT GCT GTC 3408

Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val

1125 1130 1135

AAT CCT TGG AAA AAT ATC ATC CGT CCG GTT GTT TAT ATG TCC CGC TTA 3456

Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu

1140 1145 1150

TAT CTG CTA TGG CTG GAG CAG CAA TCA AAG AAA AGT GAT GAT GGT AAA 3504

Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys

1155 1160 1165

ACC ACG ATT TAT CAA TAT AAC TTA AAA CTG GCT CAT ATT CGT TAC GAC 3552

Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp

1170 1175 1180

GGT AGT TGG AAT ACA CCA TTT ACT TTT GAT GTG ACA GAA AAG GTA AAA 3600

Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys

1185 1190 1195 1200

AAT TAC ACG TCG AGT ACT GAT GCT GCT GAA TCT TTA GGG TTG TAT TGT 3648

Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys

1205 1210 1215

ACT GGT TAT CAA GGG GAA GAC ACT CTA TTA GTT ATG TTC TAT TCG ATG 3696

Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met

1220 1225 1230

CAG AGT AGT TAT AGC TCC TAT ACC GAT AAT AAT GCG CCG GTC ACT GGG 3744

Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly

1235 1240 1245

CTA TAT ATT TTC GCT GAT ATG TCA TCA GAC AAT ATG ACG AAT GCA CAA 3792

Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln

1250 1255 1260

GCA ACT AAC TAT TGG AAT AAC AGT TAT CCG CAA TTT GAT ACT GTG ATG 3840

Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met

1265 1270 1275 1280

GCA GAT CCG GAT AGC GAC AAT AAA AAA GTC ATA ACC AGA AGA GTT AAT 3888

Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn

1285 1290 1295

AAC CGT TAT GCG GAG GAT TAT GAA ATT CCT TCC TCT GTG ACA AGT AAC 3936

Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn

1300 1305 1310

AGT AAT TAT TCT TGG GGT GAT CAC AGT TTA ACC ATG CTT TAT GGT GGT 3984

Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly

1315 1320 1325

AGT GTT CCT AAT ATT ACT TTT GAA TCG GCG GCA GAA GAT TTA AGG CTA 4032

Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu

1330 1335 1340

TCT ACC AAT ATG GCA TTG AGT ATT ATT CAT AAT GGA TAT GCG GGA ACC 4080

Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr

1345 1350 1355 1360

CGC CGT ATA CAA TGT AAT CTT ATG AAA CAA TAC GCT TCA TTA GGT GAT 4128

Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp

1365 1370 1375

AAA TTT ATA ATT TAT GAT TCA TCA TTT GAT GAT GCA AAC CGT TTT AAT 4176

Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn

1380 1385 1390

CTG GTG CCA TTG TTT AAA TTC GGA AAA GAC GAG AAC TCA GAT GAT AGT 4224

Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser

1395 1400 1405

ATT TGT ATA TAT AAT GAA AAC CCT TCC TCT GAA GAT AAG AAG TGG TAT 4272

Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr

1410 1415 1420

TTT TCT TCG AAA GAT GAC AAT AAA ACA GCG GAT TAT AAT GGT GGA ACT 4320

Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr

1425 1430 1435 1440

CAA TGT ATA GAT GCT GGA ACC AGT AAC AAA GAT TTT TAT TAT AAT CTC 4368

Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu

1445 1450 1455

CAG GAG ATT GAA GTA ATT AGT GTT ACT GGT GGG TAT TGG TCG AGT TAT 4416

Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr

1460 1465 1470

AAA ATA TCC AAC CCG ATT AAT ATC AAT ACG GGC ATT GAT AGT GCT AAA 4464

Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys

1475 1480 1485

GTA AAA GTC ACC GTA AAA GCG GGT GGT GAC GAT CAA ATC TTT ACT GCT 4512

Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala

1490 1495 1500

GAT AAT AGT ACC TAT GTT CCT CAG CAA CCG GCA CCC AGT TTT GAG GAG 4560

Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu

1505 1510 1515 1520

ATG ATT TAT CAG TTC AAT AAC CTG ACA ATA GAT TGT AAG AAT TTA AAT 4608

Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn

1525 1530 1535

TTC ATC GAC AAT CAG GCA CAT ATT GAG ATT GAT TTC ACC GCT ACG GCA 4656

Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala

1540 1545 1550

CAA GAT GGC CGA TTC TTG GGT GCA GAA ACT TTT ATT ATC CCG GTA ACT 4704

Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr

1555 1560 1565

AAA AAA GTT CTC GGT ACT GAG AAC GTG ATT GCG TTA TAT AGC GAA AAT 4752

Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn

1570 1575 1580

AAC GGT GTT CAA TAT ATG CAA ATT GGC GCA TAT CGT ACC CGT TTG AAT 4800

Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn

1585 1590 1595 1600

ACG TTA TTC GCT CAA CAG TTG GTT AGC CGT GCT AAT CGT GGC ATT GAT 4848

Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp

1605 1610 1615

GCA GTG CTC AGT ATG GAA ACT CAG AAT ATT CAG GAA CCG CAA TTA GGA 4896

Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly

1620 1625 1630

GCG GGC ACA TAT GTG CAG CTT GTG TTG GAT AAA TAT GAT GAG TCT ATT 4944

Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile

1635 1640 1645

CAT GGC ACT AAT AAA AGC TTT GCT ATT GAA TAT GTT GAT ATA TTT AAA 4992

His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys

1650 1655 1660

GAG AAC GAT AGT TTT GTG ATT TAT CAA GGA GAA CTT AGC GAA ACA AGT 5040

Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser

1665 1670 1675 1680

CAA ACT GTT GTG AAA GTT TTC TTA TCC TAT TTT ATA GAG GCG ACT GGA 5088

Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly

1685 1690 1695

AAT AAG AAC CAC TTA TGG GTA CGT GCT AAA TAC CAA AAG GAA ACG ACT 5136

Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr

1700 1705 1710

GAT AAG ATC TTG TTC GAC CGT ACT GAT GAG AAA GAT CCG CAC GGT TGG 5184

Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp

1715 1720 1725

TTT CTC AGC GAC GAT CAC AAG ACC TTT AGT GGT CTC TCT TCC GCA CAG 5232

Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln

1730 1735 1740

GCA TTA AAG AAC GAC AGT GAA CCG ATG GAT TTC TCT GGC GCC AAT GCT 5280

Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala

1745 1750 1755 1760

CTC TAT TTC TGG GAA CTG TTC TAT TAC ACG CCG ATG ATG ATG GCT CAT 5328

Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His

1765 1770 1775

CGT TTG TTG CAG GAA CAG AAT TTT GAT GCG GCG AAC CAT TGG TTC CGT 5376

Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg

1780 1785 1790

TAT GTC TGG AGT CCA TCC GGT TAT ATC GTT GAT GGT AAA ATT GCT ATC 5424

Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile

1795 1800 1805

TAC CAC TGG AAC GTG CGA CCG CTG GAA GAA GAC ACC AGT TGG AAT GCA 5472

Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala

1810 1815 1820

CAA CAA CTG GAC TCC ACC GAT CCA GAT GCT GTA GCC CAA GAT GAT CCG 5520

Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro

1825 1830 1835 1840

ATG CAC TAC AAG GTG GCT ACC TTT ATG GCG ACG TTG GAT CTG CTA ATG 5568

Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met

1845 1850 1855

GCC CGT GGT GAT GCT GCT TAC CGC CAG TTA GAG CGT GAT ACG TTG GCT 5616

Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala

1860 1865 1870

GAA GCT AAA ATG TGG TAT ACA CAG GCG CTT AAT CTG TTG GGT GAT GAG 5664

Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu

1875 1880 1885

CCA CAA GTG ATG CTG AGT ACG ACT TGG GCT AAT CCA ACA TTG GGT AAT 5712

Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn

1890 1895 1900

GCT GCT TCA AAA ACC ACA CAG CAG GTT CGT CAG CAA GTG CTT ACC CAG 5760

Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln

1905 1910 1915 1920

TTG CGT CTC AAT AGC AGG GTA AAA ACC CCG TTG CTA GGA ACA GCC AAT 5808

Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn

1925 1930 1935

TCC CTG ACC GCT TTA TTC CTG CCG CAG GAA AAT AGC AAG CTC AAA GGC 5856

Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly

1940 1945 1950

TAC TGG CGG ACA CTG GCG CAG CGT ATG TTT AAT TTA CGT CAT AAT CTG 5904

Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu

1955 1960 1965

TCG ATT GAC GGC CAG CCG CTC TCC TTG CCG CTG TAT GCT AAA CCG GCT 5952

Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala

1970 1975 1980

GAT CCA AAA GCT TTA CTG AGT GCG GCG GTT TCA GCT TCT CAA GGG GGA 6000

Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly

1985 1990 1995 2000

GCC GAC TTG CCG AAG GCG CCG CTG ACT ATT CAC CGC TTC CCT CAA ATG 6048

Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met

2005 2010 2015

CTA GAA GGG GCA CGG GGC TTG GTT AAC CAG CTT ATA CAG TTC GGT AGT 6096

Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser

2020 2025 2030

TCA CTA TTG GGG TAC AGT GAG CGT CAG GAT GCG GAA GCT ATG AGT CAA 6144

Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln

2035 2040 2045

CTA CTG CAA ACC CAA GCC AGC GAG TTA ATA CTG ACC AGT ATT CGT ATG 6192

Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met

2050 2055 2060

CAG GAT AAC CAA TTG GCA GAG CTG GAT TCG GAA AAA ACC GCC TTG CAA 6240

Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln

2065 2070 2075 2080

GTC TCT TTA GCT GGA GTG CAA CAA CGG TTT GAC AGC TAT AGC CAA CTG 6288

Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu

2085 2090 2095

TAT GAG GAG AAC ATC AAC GCA GGT GAG CAG CGA GCG CTG GCG TTA CGC 6336

Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg

2100 2105 2110

TCA GAA TCT GCT ATT GAG TCT CAG GGA GCG CAG ATT TCC CGT ATG GCA 6384

Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala

2115 2120 2125

GGC GCG GGT GTT GAT ATG GCA CCA AAT ATC TTC GGC CTG GCT GAT GGC 6432

Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly

2130 2135 2140

GGC ATG CAT TAT GGT GCT ATT GCC TAT GCC ATC GCT GAC GGT ATT GAG 6480

Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu

2145 2150 2155 2160

TTG AGT GCT TCT GCC AAG ATG GTT GAT GCG GAG AAA GTT GCT CAG TCG 6528

Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser

2165 2170 2175

GAA ATA TAT CGC CGT CGC CGT CAA GAA TGG AAA ATT CAG CGT GAC AAC 6576

Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn

2180 2185 2190

GCA CAA GCG GAG ATT AAC CAG TTA AAC GCG CAA CTG GAA TCA CTG TCT 6624

Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser

2195 2200 2205

ATT CGC CGT GAA GCC GCT GAA ATG CAA AAA GAG TAC CTG AAA ACC CAG 6672

Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln

2210 2215 2220

CAA GCT CAG GCG CAG GCA CAA CTT ACT TTC TTA AGA AGC AAA TTC AGT 6720

Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser

2225 2230 2235 2240

AAT CAA GCG TTA TAT AGT TGG TTA CGA GGG CGT TTG TCA GGT ATT TAT 6768

Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr

2245 2250 2255

TTC CAG TTC TAT GAC TTG GCC GTA TCA CGT TGC CTG ATG GCA GAG CAA 6816

Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln

2260 2265 2270

TCC TAT CAA TGG GAA GCT AAT GAT AAT TCC ATT AGC TTT GTC AAA CCG 6864

Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro

2275 2280 2285

GGT GCA TGG CAA GGA ACT TAC GCC GGC TTA TTG TGT GGA GAA GCT TTG 6912

Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu

2290 2295 2300

ATA CAA AAT CTG GCA CAA ATG GAA GAG GCA TAT CTG AAA TGG GAA TCT 6960

Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser

2305 2310 2315 2320

CGC GCT TTG GAA GTA GAA CGC ACG GTT TCA TTG GCA GTG GTT TAT GAT 7008

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp

2325 2330 2335

TCA CTG GAA GGT AAT GAT CGT TTT AAT TTA GCG GAA CAA ATA CCT GCA 7056

Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala

2340 2345 2350

TTA TTG GAT AAG GGG GAG GGA ACA GCA GGA ACT AAA GAA AAT GGG TTA 7104

Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu

2355 2360 2365

TCA TTG GCT AAT GCT ATC CTG TCA GCT TCG GTC AAA TTG TCC GAC TTG 7 152

Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu

2370 2375 2380

AAA CTG GGA ACG GAT TAT CCA GAC AGT ATC GTT GGT AGC AAC AAG GTT 7200

Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val

2385 2390 2395 2400

CGT CGT ATT AAG CAA ATC AGT GTT TCG CTA CCT GCA TTG GTT GGG CCT 7248

Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro

2405 2410 2415

TAT CAG GAT GTT CAG GCT ATG CTC AGC TAT GGT GGC AGT ACT CAA TTG 7296

Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu

2420 2425 2430

CCG AAA GGT TGT TCA GCG TTG GCT GTG TCT CAT GGT ACC AAT GAT AGT 7344

Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser

2435 2440 2445

GGT CAG TTC CAG TTG GAT TTC AAT GAC GGC AAA TAC CTG CCA TTT GAA 7392

Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu

2450 2455 2460

GGT ATT GCT CTT GAT GAT CAG GGT ACA CTG AAT CTT CAA TTT CCG AAT 7440

Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn

2465 2470 2475 2480

GCT ACC GAC AAG CAG AAA GCA ATA TTG CAA ACT ATG AGC GAT ATT ATT 7488

Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile

2485 2490 2495

TTG CAT ATT CGT TAT ACC ATC CGT TAA 7515

Leu His Ile Arg Tyr Thr Ile Arg *

2500 2505

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2504 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 (TcbA protein):

Met Gln Asn Ser Leu Ser Ser Thr Ile Asp Thr Ile Cys Gln Lys Leu

1 5 10 15

Gln Leu Thr Cys Pro Ala Glu Ile Ala Leu Tyr Pro Phe Asp Thr Phe

20 25 30

Arg Glu Lys Thr Arg Gly Met Val Asn Trp Gly Glu Ala Lys Arg Ile

35 40 45

Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn Leu Leu His Glu Lys

50 55 60

Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn Ala Val Arg Leu

65 70 75 80

Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp Leu Phe

85 90 95

Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala Ser Met

100 105 110

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Lys Asn

115 120 125

Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg Pro Asp

130 135 140

Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu Ile Ser

145 150 155 160

Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu Thr Lys

165 170 175

Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu Ser Thr Tyr Arg

180 185 190

Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu Thr Val Arg Glu

195 200 205

Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu Ser Gln Ala Pro

210 215 220

Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu Gly Ile Ser Ser

225 230 235 240

His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu Glu Ile Pro Glu

245 250 255

Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys Thr Asn Phe Gly Asp

260 265 270

Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr

275 280 285

Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr Thr Ser Leu Ser His

290 295 300

Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro Leu Val Asp Gly Val

305 310 315 320

Gly Lys Met Glu Val Val Arg Val Thr Arg Thr Pro Ser Asp Asn Tyr

325 330 335

Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr Pro Gln Gly Gly Asp Asn

340 345 350

Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser Phe Gly Leu Asp Asp Phe

355 360 365

Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp Trp Thr Glu Ile Ala His

370 375 380

Asn Pro Tyr Pro Asp Met Val Ile Asn Gln Lys Tyr Glu Ser Gln Ala

385 390 395 400

Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile Leu Ser Ile Gly Leu Gln

405 410 415

Arg Trp His Ser Gly Ser Tyr Asn Phe Ala Ala Ala Asn Phe Lys Ile

420 425 430

Asp Gln Tyr Ser Pro Lys Ala Phe Leu Leu Lys Met Asn Lys Ala Ile

435 440 445

Arg Leu Leu Lys Ala Thr Gly Leu Ser Phe Ala Thr Leu Glu Arg Ile

450 455 460

Val Asp Ser Val Asn Ser Thr Lys Ser Ile Thr Val Glu Val Leu Asn

465 470 475 480

Lys Val Tyr Arg Val Lys Phe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu

485 490 495

Glu Thr Ala Ala Ile Leu Ala Asn Ile Asn Ile Ser Gln Gln Ala Val

500 505 510

Gly Asn Gln Leu Ser Gln Phe Glu Gln Leu Phe Asn His Pro Pro Leu

515 520 525

Asn Gly Ile Arg Tyr Glu Ile Ser Glu Asp Asn Ser Lys His Leu Pro

530 535 540

Asn Pro Asp Leu Asn Leu Lys Pro Asp Ser Thr Gly Asp Asp Gln Arg

545 550 555 560

Lys Ala Val Leu Lys Arg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr

565 570 575

Gln Met Leu Leu Ile Thr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn

580 585 590

Asn Leu Glu Asn Leu Ser Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln

595 600 605

Ile His Asn Leu Thr Ile Ala Glu Leu Asn Ile Leu Leu Val Ile Cys

610 615 620

Gly Tyr Gly Asp Thr Asn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala

625 630 635 640

Lys Ile Val Glu Thr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln

645 650 655

Lys Trp Thr Val Thr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser

660 665 670

Thr Thr Leu Thr Pro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser

675 680 685

Thr Leu His Gly Lys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala

690 695 700

Met Ala Pro Cys Phe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val

705 710 715 720

Ala Tyr Asp Leu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile

725 730 735

Thr Val Asp Gly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu

740 745 750

Lys Val Ile Thr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr

755 760 765

Arg Arg Ile Gly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln

770 775 780

Ser Ser Leu Leu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu

785 790 795 800

Thr Leu Met Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly

805 810 815

Gln His Ala Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr

820 825 830

Val Thr Asp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln

835 840 845

Met Ala Ala Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp

850 855 860

Thr Gln Ile Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu

865 870 875 880

Ala Val Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly

885 890 895

Ile Asp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met

900 905 910

Ala Asp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser

915 920 925

Lys Ala Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala

930 935 940

Gly Val Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn

945 950 955 960

Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala

965 970 975

Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln

980 985 990

Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg

995 1000 1005

Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr

1010 1015 1020

Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln Thr Lys

1025 1030 1035 1040

Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu Asn Ala

1045 1050 1055

Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe Glu Gln

1060 1065 1070

Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Val Asn Val

1075 1080 1085

Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp Gln Ala Ala Pro Gly

1090 1095 1100

Thr Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Cys Glu Asn Gly Lys

1105 1110 1115 1120

Phe Ala Ala Asn Ala Trp Gly Glu Trp Asn Lys Ile Thr Cys Ala Val

1125 1130 1135

Asn Pro Trp Lys Asn Ile Ile Arg Pro Val Val Tyr Met Ser Arg Leu

1140 1145 1150

Tyr Leu Leu Trp Leu Glu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys

1155 1160 1165

Thr Thr Ile Tyr Gln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp

1170 1175 1180

Gly Ser Trp Asn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys

1185 1190 1195 1200

Asn Tyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys

1205 1210 1215

Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met

1220 1225 1230

Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val Thr Gly

1235 1240 1245

Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn Ala Gln

1250 1255 1260

Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr Val Met

1265 1270 1275 1280

Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg Val Asn

1285 1290 1295

Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Thr Ser Asn

1300 1305 1310

Ser Asn Tyr Ser Trp Gly Asp His Ser Leu Thr Met Leu Tyr Gly Gly

1315 1320 1325

Ser Val Pro Asn Ile Thr Phe Glu Ser Ala Ala Glu Asp Leu Arg Leu

1330 1335 1340

Ser Thr Asn Met Ala Leu Ser Ile Ile His Asn Gly Tyr Ala Gly Thr

1345 1350 1355 1360

Arg Arg Ile Gln Cys Asn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp

1365 1370 1375

Lys Phe Ile Ile Tyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn

1380 1385 1390

Leu Val Pro Leu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser

1395 1400 1405

Ile Cys Ile Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr

1410 1415 1420

Phe Ser Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr

1425 1430 1435 1440

Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu

1445 1450 1455

Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser Ser Tyr

1460 1465 1470

Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys

1475 1480 1485

Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe Thr Ala

1490 1495 1500

Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro Ser Phe Glu Glu

1505 1510 1515 1520

Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp Cys Lys Asn Leu Asn

1525 1530 1535

Phe Ile Asp Asn Gln Ala His Ile Glu Ile Asp Phe Thr Ala Thr Ala

1540 1545 1550

Gln Asp Gly Arg Phe Leu Gly Ala Glu Thr Phe Ile Ile Pro Val Thr

1555 1560 1565

Lys Lys Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn

1570 1575 1580

Asn Gly Val Gln Tyr Met Gln Ile Gly Ala Tyr Arg Thr Arg Leu Asn

1585 1590 1595 1600

Thr Leu Phe Ala Gln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp

1605 1610 1615

Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly

1620 1625 1630

Ala Gly Thr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile

1635 1640 1645

His Gly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys

1650 1655 1660

Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser

1665 1670 1675 1680

Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala Thr Gly

1685 1690 1695

Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu Thr Thr

1700 1705 1710

Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp Pro His Gly Trp

1715 1720 1725

Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly Leu Ser Ser Ala Gln

1730 1735 1740

Ala Leu Lys Asn Asp Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ala

1745 1750 1755 1760

Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro Met Met Met Ala His

1765 1770 1775

Arg Leu Leu Gln Glu Gln Asn Phe Asp Ala Ala Asn His Trp Phe Arg

1780 1785 1790

Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val Asp Gly Lys Ile Ala Ile

1795 1800 1805

Tyr His Trp Asn Val Arg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala

1810 1815 1820

Gln Gln Leu Asp Ser Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro

1825 1830 1835 1840

Met His Tyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met

1845 1850 1855

Ala Arg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala

1860 1865 1870

Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu

1875 1880 1885

Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu Gly Asn

1890 1895 1900

Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu Thr Gln

1905 1910 1915 1920

Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr Ala Asn

1925 1930 1935

Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn Ser Lys Leu Lys Gly

1940 1945 1950

Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn Leu Arg His Asn Leu

1955 1960 1965

Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu Tyr Ala Lys Pro Ala

1970 1975 1980

Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser Ala Ser Gln Gly Gly

1985 1990 1995 2000

Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His Arg Phe Pro Gln Met

2005 2010 2015

Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser

2020 2025 2030

Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln

2035 2040 2045

Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met

2050 2055 2060

Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln

2065 2070 2075 2080

Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu

2085 2090 2095

Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala Leu Arg

2100 2105 2110

Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg Met Ala

2115 2120 2125

Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala Asp Gly

2130 2135 2140

Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala Asp Gly Ile Glu

2145 2150 2155 2160

Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu Lys Val Ala Gln Ser

2165 2170 2175

Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys Ile Gln Arg Asp Asn

2180 2185 2190

Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln Leu Glu Ser Leu Ser

2195 2200 2205

Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu Tyr Leu Lys Thr Gln

2210 2215 2220

Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu Arg Ser Lys Phe Ser

2225 2230 2235 2240

Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr

2245 2250 2255

Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln

2260 2265 2270

Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro

2275 2280 2285

Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu

2290 2295 2300

Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser

2305 2310 2315 2320

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val Tyr Asp

2325 2330 2335

Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile Pro Ala

2340 2345 2350

Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys Glu Asn Gly Leu

2355 2360 2365

Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val Lys Leu Ser Asp Leu

2370 2375 2380

Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val Gly Ser Asn Lys Val

2385 2390 2395 2400

Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro Ala Leu Val Gly Pro

2405 2410 2415

Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly Gly Ser Thr Gln Leu

2420 2425 2430

Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His Gly Thr Asn Asp Ser

2435 2440 2445

Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu

2450 2455 2460

Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn

2465 2470 2475 2480

Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile

2485 2490 2495

Leu His Ile Arg Tyr Thr Ile Arg *

2500 2505

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 (TcdAii N-terminus):

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Xaa Ala

1 5 10

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14 (TcdB N-terminus):

Met Gln Asn Ser Gln Thr Phe Ser Val Gly Glu Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15 (TcaAii N-terminus):

Ala Gln Asp Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr

1 5 10

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16 (TcbA N-terminus):

Met Gln Asn Ser Leu

1 5

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17 (TcdAii-PTlll internal

peptide):

Ala Phe Asn Ile Asp Asp Val Ser Leu Phe

1 5 10

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18 (TcdAii-PT79 internal

peptide):

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19 (TcaBi-PT158 internal

peptide):

Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile Gly Ser

1 5 10 15

Leu Gln Leu Phe Ile

20

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20 (TcaBi-PT 108

internal peptide):

Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro

1 5 10

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21 (TcbAii-PT103

internal peptide):

Gly Ile Asp Ala Val Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro

1 5 10 15

Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu

20 25

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22 (TcbAii- PT56 internal

peptide):

Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser Ala Lys

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23 (TcbA-PT81 (a)

internal peptide):

Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 (TcbAii-PT81 (b)

internal peptide):

Val Leu Gly Thr Glu Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly

1 5 10 15

Val Gln Tyr Met Gln Ile

20

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6054 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..43

(D) OTHER INFORMATION: / product = &quot; end of TcaAiii &quot;

(ix) FEATURE:

(A) NAME / KEY: RBS

(B) LOCATION: 51 58

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 65 3634

(D) OTHER INFORMATION: / product = &quot; TcaBi &quot;

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

A GTA GCC CAA AAC TTA AGT GCC GCA ATC AGC AAT CGT CAG TAACCGGATA 50

Val Ala Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln ???

AAGAAGGAAT TGATT ATG TCT GAA TCT TTA TTT ACA CAA ACG TTG AAA GAA 100

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu

1 5 10

GCG CGC CGT GAT GCA TTG GTT GCT CAT TAT ATT GCT ACT CAG GTG CCC 148

Ala Arg Arg Asp Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro

15 20 25

GCA GAT TTA AAA GAG AGT ATC CAG ACC GCG GAT GAT CTG TAC GAA TAT 196

Ala Asp Leu Lys Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr

30 35 40

CTG TTG CTG GAT ACC AAA ATT AGC GAT CTG GTT ACT ACT TCA CCG CTG 244

Leu Leu Leu Asp Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu

45 50 55 60

TCC GAA GCG ATT GGC AGT CTG CAA TTG TTT ATT CAT CGT GCG ATA GAG 292

Ser Glu Ala Ile Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu

65 70 75

GGC TAT GAC GGC ACG CTG GCA GAC TCA GCA AAA CCC TAT TTT GCC GAT 340

Gly Tyr Asp Gly Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp

80 85 90

GAA CAG TTT TTA TAT AAC TGG GAT AGT TTT AAC CAC CGT TAT AGC ACT 388

Glu Gln Phe Leu Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr

95 100 105

TGG GCT GGC AAG GAA CGG TTG AAA TTC TAT GCC GGG GAT TAT ATT GAT 436

Trp Ala Gly Lys Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp

110 115 120

CCA ACA TTG CGA TTG AAT AAG ACC GAG ATA TTT ACC GCA TTT GAA CAA 484

Pro Thr Leu Arg Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln

125 130 135 140

GGT ATT TCT CAA GGG AAA TTA AAA AGT GAA TTA GTC GAA TCT AAA TTA 532

Gly Ile Ser Gln Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu

145 150 155

CGT GAT TAT CTA ATT AGT TAT GAC ACT TTA GCC ACC CTT GAT TAT ATT 580

Arg Asp Tyr Leu Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile

160 165 170

ACT GCC TGC CAA GGC AAA GAT AAT AAA ACC ATC TTC TTT ATT GGC CGT 628

Thr Ala Cys Gln Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg

175 180 185

ACA CAG AAT GCA CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT TTA GTC 676

Thr Gln Asn Ala Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val

190 195 200

ACT GAT GGC GGT AAG TTG AAA CCA GAT CAA TGG TCA GAG TGG CGA GCA 724

Thr Asp Gly Gly Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala

205 210 215 220

ATT AAT GCC GGG ATT AGT GAG GCA TAT TCA GGG CAT GTC GAG CCT TTC 772

Ile Asn Ala Gly Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe

225 230 235

TGG GAA AAT AAC AAG CTG CAC ATC CGT TGG TTT ACT ATC TCG AAA GAA 820

Trp Glu Asn Asn Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu

240 245 250

GAT AAA ATA GAT TTT GTT TAT AAA AAC ATC TGG GTG ATG AGT AGC GAT 868

Asp Lys Ile Asp Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp

255 260 265

TAT AGC TGG GCA TCA AAG AAA AAA ATC TTG GAA CTT TCT TTT ACT GAC 916

Tyr Ser Trp Ala Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp

270 275 280

TAC AAT AGA GTT GGA GCA ACA GGA TCA TCA AGC CCG ACT GAA GTA GCT 964

Tyr Asn Arg Val Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala

285 290 295 300

TCA CAA TAT GGT TCT GAT GCT CAG ATG AAT ATT TCT GAT GAT GGG ACT 1012

Ser Gln Tyr Gly Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr

305 310 315

GTA CTT ATT TTT CAG AAT GCC GGC GGA GCT ACT CCC AGT ACT GGA GTG 1060

Val Leu Ile Phe Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val

320 325 330

ACG TTA TGT TAT GAC TCT GGC AAC GTG ATT AAG AAC CTA TCT AGT ACA 1108

Thr Leu Cys Tyr Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr

335 340 345

GGA AGT GCA AAT TTA TCG TCA AAG GAT TAT GCC ACA ACT AAA TTA CGC 1156

Gly Ser Ala Asn Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg

350 355 360

ATG TGT CAT GGA CAA AGT TAC AAT GAT AAT AAC TAC TGC AAT TTT ACA 1204

Met Cys His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr

365 370 375 380

CTC TCT ATT AAT ACA ATA GAA TTC ACC TCC TAC GGC ACA TTC TCA TCA 1252

Leu Ser Ile Asn Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser

385 390 395

GAT GGA AAA CAA TTT ACA CCA CCT TCT GGT TCT GCC ATT GAT TTA CAC 1300

Asp Gly Lys Gln Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His

400 405 410

CTC CCT AAT TAT GTA GAT CTC AAC GCG CTA TTA GAT ATT AGC CTC GAT 1348

Leu Pro Asn Tyr Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp

415 420 425

TCA CTA CTT AAT TAT GAC GTT CAG GGG CAG TTT GGC GGA TCT AAT CCG 1396

Ser Leu Leu Asn Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro

430 435 440

GTT GAT AAT TTC AGT GGT CCC TAT GGT ATT TAT CTA TGG GAA ATC TTC 1444

Val Asp Asn Phe Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe

445 450 455 460

TTC CAT ATT CCG TTC CTT GTT ACG GTC CGT ATG CAA ACC GAA CAA CGT 1492

Phe His Ile Pro Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg

465 470 475

TAC GAA GAC GCG GAC ACT TGG TAC AAA TAT ATT TTC CGC AGC GCC GGT 1540

Tyr Glu Asp Ala Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly

480 485 490

TAT CGC GAT GCT AAT GGC CAG CTC ATT ATG GAT GGC AGT AAA CCA CGT 1588

Tyr Arg Asp Ala Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg

495 500 505

TAT TGG AAT GTG ATG CCA TTG CAA CTG GAT ACC GCA TGG GAT ACC ACA 1636

Tyr Trp Asn Val Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr

510 515 520

CAG CCC GCC ACC ACT GAT CCA GAT GTG ATC GCT ATG GCG GAC CCG ATG 1684

Gln Pro Ala Thr Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met

525 530 535 540

CAT TAC AAG CTG GCG ATA TTC CTG CAT ACC CTT GAT CTA TTG ATT GCC 1732

His Tyr Lys Leu Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala

545 550 555

CGA GGC GAC AGC GCT TAC CGT CAA CTT GAA CGC GAT ACT CTA GTC GAA 1780

Arg Gly Asp Ser Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu

560 565 570

GCC AAA ATG TAC TAC ATT CAG GCA CAA CAG CTA CTG GGA CCG CGC CCT 1828

Ala Lys Met Tyr Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro

575 580 585

GAT ATC CAT ACC ACC AAT ACT TGG CCA AAT CCC ACC TTG AGT AAA GAA 1876

Asp Ile His Thr Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu

590 595 600

GCT GGC GCT ATT GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG 1924

Ala Gly Ala Ile Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met

605 610 615 620

ACG TTC GCT GCC TGG CTA AGC GCA GGC GAT ACC GCA AAT ATT GGC GAC 1972

Thr Phe Ala Ala Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp

625 630 635

GGT GAT TTC TTG CCA CCG TAC AAC GAT GTA CTA CTC GGT TAC TGG GAT 2020

Gly Asp Phe Leu Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp

640 645 650

AAA CTT GAG TTA CGC CTA TAC AAC CTG CGC CAC AAT CTG AGT CTG GAT 2068

Lys Leu Glu Leu Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp

655 660 665

GGT CAA CCG CTA AAT CTG CCA CTG TAT GCC ACG CCG GTA GAC CCG AAA 2 116

Gly Gln Pro Leu Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys

670 675 680

ACC CTG CAA CGC CAG CAA GCC GGA GGG GAC GGT ACA GGC AGT AGT CCG 2164

Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro

685 690 695 700

GCT GGT GGT CAA GGC AGT GTT CAG GGC TGG CGC TAT CCG TTA TTG GTA 2212

Ala Gly Gly Gln Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val

705 710 715

GAA CGC GCC CGC TCT GCC GTG AGT TTG TTG ACT CAG TTC GGC AAC AGC 2260

Glu Arg Ala Arg Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser

720 725 730

TTA CAA ACA ACG TTA GAA CAT CAG GAT AAT GAA AAA ATG ACG ATA CTG 2308

Leu Gln Thr Thr Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu

735 740 745

TTG CAG ACT CAA CAG GAA GCC ATC CTG AAA CAT CAG CAC GAT ATA CAA 2356

Leu Gln Thr Gln Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln

750 755 760

CAA AAT AAT CTA AAA GGA TTA CAA CAC AGC CTG ACC GCA TTA CAG GCT 2404

Gln Asn Asn Leu Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala

765 770 775 780

AGC CGT GAT GGC GAC ACA TTG CGG CAA AAA CAT TAC AGC GAC CTG ATT 2452

Ser Arg Asp Gly Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile

785 790 795

AAC GGT GGT CTA TCT GCG GCA GAA ATC GCC GGT CTG ACA CTA CGC AGC 2500

Asn Gly Gly Leu Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser

800 805 810

ACC GCC ATG ATT ACC AAT GGC GTT GCA ACG GGA TTG CTG ATT GCC GGC 2548

Thr Ala Met Ile Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly

815 820 825

GGA ATC GCC AAC GCG GTA CCT AAC GTC TTC GGG CTG GCT AAC GGT GGA 2596

Gly Ile Ala Asn Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly

830 835 840

TCG GAA TGG GGA GCG CCA TTA ATT GGC TCC GGG CAA GCA ACC CAA GTT 2644

Ser Glu Trp Gly Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val

845 850 855 860

GGC GCC GGC ATC CAG GAT CAG AGC GCG GGC ATT TCA GAA GTG ACA GCA 2692

Gly Ala Gly Ile Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala

865 870 875

GGC TAT CAG CGT CGT CAG GAA GAA TGG GCA TTG CAA CGG GAT ATT GCT 2740

Gly Tyr Gln Arg Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala

880 885 890

GAT AAC GAA ATA ACC CAA CTG GAT GCC CAG ATA CAA AGC CTG CAA GAG 2788

Asp Asn Glu Ile Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu

895 900 905

CAA ATC ACG ATG GCA CAA AAA CAG ATC ACG CTC TCT GAA ACC GAA CAA 2836

Gln Ile Thr Met Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln

910 915 920

GCG AAT GCC CAA GCG ATT TAT GAC CTG CAA ACC ACT CGT TTT ACC GGG 2884

Ala Asn Ala Gln Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly

925 930 935 940

CAG GCA CTG TAT AAC TGG ATG GCC GGT CGT CTC TCC GCG CTC TAT TAC 2932

Gln Ala Leu Tyr Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr

945 950 955

CAA ATG TAT GAT TCC ACT CTG CCA ATC TGT CTC CAG CCA AAA GCC GCA 2980

Gln Met Tyr Asp Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala

960 965 970

TTA GTA CAG GAA TTA GGC GAG AAA GAG AGC GAC AGT CTT TTC CAG GTT 3028

Leu Val Gln Glu Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val

975 980 985

CCG GTG TGG AAT GAT CTG TGG CAA GGG CTG TTA GCA GGA GAA GGT TTA 3076

Pro Val Trp Asn Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu

990 995 1000

AGT TCA GAG CTA CAG AAA CTG GAT GCC ATC TGG CTT GCA CGT GGT GGT 3124

Ser Ser Glu Leu Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly

1005 1010 1015 1020

ATT GGG CTA GAA GCC ATC CGC ACC GTG TCG CTG GAT ACC CTG TTT GGC 3172

Ile Gly Leu Glu Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly

1025 1030 1035

ACA GGG ACG TTA AGT GAA AAT ATC AAT AAA GTG CTT AAC GGG GAA ACG 3220

Thr Gly Thr Leu Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr

1040 1045 1050

GTA TCT CCA TCC GGT GGC GTC ACT CTG GCG CTG ACA GGG GAT ATC TTC 3268

Val Ser Pro Ser Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe

1055 1060 1065

CAA GCA ACA CTG GAT TTG AGT CAG CTA GGT TTG GAT AAC TCT TAC AAC 3316

Gln Ala Thr Leu Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn

1070 1075 1080

TTG GGT AAC GAG AAG AAA CGT CGT ATT AAA CGT ATC GCC GTC ACC CTG 3364

Leu Gly Asn Glu Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu

1085 1090 1095 1100

CCA ACA CTT CTG GGG CCA TAT CAA GAT CTT GAA GCC ACA CTG GTA ATG 3412

Pro Thr Leu Leu Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met

1105 1110 1115

GGT GCG GAA ATC GCC GCC TTA TCA CAC GGT GTG AAT GAC GGA GGC CGG 3460

Gly Ala Glu Ile Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg

1120 1125 1130

TTT GTT ACC GAC TTT AAC GAC AGC CGT TTT CTG CCT TTT GAA GGT CGA 3508

Phe Val Thr Asp Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg

1135 1140 1145

GAT GCA ACA ACC GGC ACA CTG GAG CTC AAT ATT TTC CAT GCG GGT AAA 3556

Asp Ala Thr Thr Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys

1150 1155 1160

GAG GGA ACG CAA CAC GAG TTG GTC GCG AAT CTG AGT GAC ATC ATT GTG 3604

Glu Gly Thr Gln His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val

1165 1170 1175 1180

CAT CTG AAT TAC ATC ATT CGA GAC GCG TAA ATTTCTTTTC TTTGTCGATT 3654

His Leu Asn Tyr Ile Ile Arg Asp Ala *

1185 1190

ACAGGTCCCT ATCAGGGGCC TGTTATTAAG GAGTACTTTA TGCAGGATTC ACCAGAAGTA 3714

TCGATTACAA CGCTGTCACT TCCCAAAGGT GGCGGTGCTA TCAATGGCAT GGGAGAAGCA 3774

CTGAATGCTG CCGGCCCTGA TGGAATGGCC TCCCTATCTC TGCCATTACC CCTTTCGACC 3834

GGCAGAGGGA CGGCTCCTGG ATTATCGCTG ATTTACAGCA ACAGTGCAGG TAATGGGCCT 3894

TTCGGCATCG GCTGGCAATG CGGTGTTATG TCCATTAGCC GACGCACCCA ACATGGCATT 3954

CCACAATACG GTAATGACGA CACGTTCCTA TCCCCACAAG GCGAGGTCAT GAATATCGCC 4014

CTGAATGACC AAGGGCAACC TGATATCCGT CAAGACGTTA AAACGCTGCA AGGCGTTACC 4074

TTGCCAATTT CCTATACCGT GACCCGCTAT CAAGCCCGCC AGATCCTGGA TTTCAGTAAA 4134

ATCGAATACT GGCAACCTGC CTCCGGTCAA GAAGGACGCG CTTTCTGGCT GATATCGACA 4194

CCGGACGGGC ATCTACACAT CTTAGGGAAA ACCGCGCAGG CTTGTCTGGC AAATCCGCAA 4254

AATGACCAAC AAATCGCCCA GTGGTTGCTG GAAGAAACTG TGACGCCAGC CGGTGAACAT 4314

GTCAGCTATC AATATCGAGC CGAAGATGAA GCCCATTGTG ACGACAATGA AAAAACCGCT 4374

CATCCCAATG TTACCGCACA GCGCTATCTG GTACAGGTGA ACTACAGGCA ACATCAAACC 4434

ACAAGCCAGC CTGTTCGTAC TGGATAACGC ACCTCCCGCA CCGGAAGAGT GGCTGTTTCA 4494

TCTGGTCTTT GACCACGGTG AGCGCGTACC TCACTTCATA CCGTGCCAAC ATGGGATGCA 4554

GGTACAGCGC AATGGTCTGT ACGCCCGGAT ATCTTCTCTC GCTATGAATA TGGTTTTGAA 4614

GTGCGTACTC GCCGCTTATG TCAACAAGTG CTGATGTTTC ACCGCACCGC GCTCATGGCC 4674

GGAGAAGCCA GTACCAATGA CGCCCCGGAA CTGGTTGGAC GCTTAATACT GGAATATGAC 4634

AAAAACGCCA GCGTCACCAC GTTGATTACC ATCCGTCAAT TAAGCCATGA ATCGGACGGG 4794

AGGCCAGTCA CCCAGCCACC ACTAGAACTA GCCTGGCAAC GGTTTGATCT GGAGAAAATC 4854

CCGACATGGC AACGCTTTGA CGCACTAGAT AATTTTAACT CGCAGCAACG TTATCAACTG 4865

GTTGATCTGC GGGGAGAAGG GTTGCCAGGT ATGCTGTATC AAGATCGAGG CGCTTGGTGG 4914

TATAAAGCTC CGCAACGTCA GGAAGACGGA GACAGCAATG CCGTCACTTA CGACAAAATC 4974

GCCCCACTGC CTACCCTACC CAATTTGCAG GATAATGCCT CATTGATGGA TATCAACGGA 5034

GACGGCCAAC TGGATTGGGT TGTTACCGCC TCCGGTATTC GCGGATACCA TAGTCAGCAA 5094

CCCGATGGAA AGTGGACGCA CTTTACGCCA ATCAATGCCT TGCCCGTGGA ATATTTTCAT 5214

CCAAGCATCC AGTTCGCTGA CCTTACCGGG GCAGGCTTAT CTGATTTAGT GTTGATCGGG 5274

CCGAAAAGCG TGCGTCTATA TGCCAACCAG CGAAACGGCT GGCGTAAAGG AGAAGATGTC 5334

CCCCAATCCA CAGGTATCAC CCTGCCTGTC ACAGGGACCG ATGCCCGCAA ACTGGTGGCT 5394

TTCAGTGATA TGCTCGGTTC CGGTCAACAA CATCTGGTGG AAATCAAGGG TAATCGCGTC 5454

ACCTGTTGGC CGAATCTAGG GCATGGCCGT TTCGGTCAAC CACTAACTCT GTCAGGATTT 5514

AGCCAGCCCG AAAATAGCTT CAATCCCGAA CGGCTGTTTC TGGCGGATAT CGACGGCTCC 5574

GGCACCACCG ACCTTATCTA TGCGCAATCC GGCTCTTTGC TCATTTATCT CAACCAAAGT 5634

GGTAATCAGT TTGATGCCCC GTTGACATTA GCGTTGCCAG AAGGCGTACA ATTTGACAAC 5694

ACTTGCCAAC TTCAAGTCGC CGATATTCAG GGATTAGGGA TAGCCAGCTT GATTCTGACT 5754

GTGCCACATA TCGCGCCACA TCACTGGCGT TGTGACCTGT CACTGACCAA ACCCTGGTTG 5814

TTGAATGTAA TGAACAATAA CCGGGGCGCA CATCACACGC TACATTATCG TAGTTCCGCG 5874

CAATTCTGGT TGGATGAAAA ATTACAGCTC ACCAAAGCAG GCAAATCTCC GGCTTGTTAT 5934

CTGCCGTTTC CAATGCATTT GCTATGGTAT ACCGAAATTC AGGATGAAAT CAGCGGCAAC 5994

CGGCTCACCA GTGAAGTCAA CTACAGCCAC GGCGTCTGGG ATGGTAAAGA GCGGGAATTC 6054

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1189 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26 (TcaB protein):

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys

20 25 30

Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp

35 40 45

Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile

50 55 60

Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly

65 70 75 80

Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu

85 90 95

Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys

100 105 110

Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg

115 120 125

Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln

130 135 140

Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu

145 150 155 160

Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln

165 170 175

Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala

180 185 190

Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly

195 200 205

Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly

210 215 220

Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn

225 230 235 240

Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp

245 250 255

Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala

260 265 270

Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val

275 280 285

Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly

290 295 300

Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe

305 310 315 320

Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr

325 330 335

Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn

340 345 350

Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly

355 360 365

Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn

370 375 380

Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln

385 390 395 400

Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr

405 410 415

Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn

420 425 430

Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe

435 440 445

Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro

450 455 460

Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala

465 470 475 480

Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala

485 490 495

Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val

500 505 510

Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr

515 520 525

Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu

530 535 540

Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser

545 550 555 560

Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr

565 570 575

Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr

580 585 590

Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile

595 600 605

Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala

610 615 620

Trp Leu Ser Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu

625 630 635 640

Pro Pro Tyr Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu

645 650 655

Arg Leu Tyr Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu

660 665 670

Asn Leu Pro Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg

675 680 685

Gln Gln Ala Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln

690 695 700

Gly Ser Val Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg

705 710 715 720

Ser Ala Val Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr

725 730 735

Leu Glu His Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln

740 745 750

Gln Glu Ala Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu

755 760 765

Lys Gly Leu Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly

770 775 780

Asp Thr Leu Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu

785 790 795 800

Ser Ala Ala Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile

805 810 815

Thr Asn Gly Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn

820 825 830

Ala Val Pro Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly

835 840 845

Ala Pro Leu Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile

850 855 860

Gln Asp Gln Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg

865 870 875 880

Arg Gln Glu Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile

885 890 895

Thr Gln Leu Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met

900 905 910

Ala Gln Lys Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln

915 920 925

Ala Ile Tyr Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr

930 935 940

Asn Trp Met Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp

945 950 955 960

Ser Thr Leu Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu

965 970 975

Leu Gly Glu Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn

980 985 990

Asp Leu Trp Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu

995 1000 1005

Gln Lys Leu Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu

1010 1015 1020

Ala Ile Arg Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu

1025 1030 1035 1040

Ser Glu Asn Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser

1045 1050 1055

Gly Gly Val Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu

1060 1065 1070

Asp Leu Ser Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu

1075 1080 1085

Lys Lys Arg Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu

1090 1095 1100

Gly Pro Tyr Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile

1105 1110 1115 1120

Ala Ala Leu Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp

1125 1130 1135

Phe Asn Asp Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr

1140 1145 1150

Gly Thr Leu Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln

1155 1160 1165

His Glu Leu Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr

1170 1175 1180

Ile Ile Arg Asp Ala *

1185 1190

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1881 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..1881

(D) OTHER INFORMATION: tcaBi

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27 (tcaBi coding region):

ATG TCT GAA TCT TTA TTT ACA CAA ACG TTG AAA GAA GCG CGC CGT GAT 48

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

GCA TTG GTT GCT CAT TAT ATT GCT ACT CAG GTG CCC GCA GAT TTA AAA 96

Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys

20 25 30

GAG AGT ATC CAG ACC GCG GAT GAT CTG TAC GAA TAT CTG TTG CTG GAT 144

Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp

35 40 45

ACC AAA ATT AGC GAT CTG GTT ACT ACT TCA CCG CTG TCC GAA GCG ATT 192

Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile

50 55 60

GGC AGT CTG CAA TTG TTT ATT CAT CGT GCG ATA GAG GGC TAT GAC GGC 240

Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly

65 70 75 80

ACG CTG GCA GAC TCA GCA AAA CCC TAT TTT GCC GAT GAA CAG TTT TTA 288

Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu

85 90 95

TAT AAC TGG GAT AGT TTT AAC CAC CGT TAT AGC ACT TGG GCT GGC AAG 336

Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys

100 105 110

GAA CGG TTG AAA TTC TAT GCC GGG GAT TAT ATT GAT CCA ACA TTG CGA 384

Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg

115 120 125

TTG AAT AAG ACC GAG ATA TTT ACC GCA TTT GAA CAA GGT ATT TCT CAA 432

Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln

130 135 140

GGG AAA TTA AAA AGT GAA TTA GTC GAA TCT AAA TTA CGT GAT TAT CTA 480

Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu

145 150 155 160

ATT AGT TAT GAC ACT TTA GCC ACC CTT GAT TAT ATT ACT GCC TGC CAA 528

Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln

165 170 175

GGC AAA GAT AAT AAA ACC ATC TTC TTT ATT GGC CGT ACA CAG AAT GCA 576

Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala

180 185 190

CCC TAT GCA TTT TAT TGG CGA AAA TTA ACT TTA GTC ACT GAT GGC GGT 624

Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly

195 200 205

AAG TTG AAA CCA GAT CAA TGG TCA GAG TGG CGA GCA ATT AAT GCC GGG 672

Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly

210 215 220

ATT AGT GAG GCA TAT TCA GGG CAT GTC GAG CCT TTC TGG GAA AAT AAC 720

Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn

225 230 235 240

AAG CTG CAC ATC CGT TGG TTT ACT ATC TCG AAA GAA GAT AAA ATA GAT 768

Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp

245 250 255

TTT GTT TAT AAA AAC ATC TGG GTG ATG AGT AGC GAT TAT AGC TGG GCA 816

Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala

260 265 270

TCA AAG AAA AAA ATC TTG GAA CTT TCT TTT ACT GAC TAC AAT AGA GTT 864

Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val

275 280 285

GGA GCA ACA GGA TCA TCA AGC CCG ACT GAA GTA GCT TCA CAA TAT GGT 912

Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly

290 295 300

TCT GAT GCT CAG ATG AAT ATT TCT GAT GAT GGG ACT GTA CTT ATT TTT 960

Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe

305 310 315 320

CAG AAT GCC GGC GGA GCT ACT CCC AGT ACT GGA GTG ACG TTA TGT TAT 1008

Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr

325 330 335

GAC TCT GGC AAC GTG ATT AAG AAC CTA TCT AGT ACA GGA AGT GCA AAT 1056

Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn

340 345 350

TTA TCG TCA AAG GAT TAT GCC ACA ACT AAA TTA CGC ATG TGT CAT GGA 1104

Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly

355 360 365

CAA AGT TAC AAT GAT AAT AAC TAC TGC AAT TTT ACA CTC TCT ATT AAT 1152

Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn

370 375 380

ACA ATA GAA TTC ACC TCC TAC GGC ACA TTC TCA TCA GAT GGA AAA CAA 1200

Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln

385 390 395 400

TTT ACA CCA CCT TCT GGT TCT GCC ATT GAT TTA CAC CTC CCT AAT TAT 1248

Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr

405 410 415

GTA GAT CTC AAC GCG CTA TTA GAT ATT AGC CTC GAT TCA CTA CTT AAT 1296

Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn

420 425 430

TAT GAC GTT CAG GGG CAG TTT GGC GGA TCT AAT CCG GTT GAT AAT TTC 1344

Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe

435 440 445

AGT GGT CCC TAT GGT ATT TAT CTA TGG GAA ATC TTC TTC CAT ATT CCG 1392

Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro

450 455 460

TTC CTT GTT ACG GTC CGT ATG CAA ACC GAA CAA CGT TAC GAA GAC GCG 1440

Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala

465 470 475 480

GAC ACT TGG TAC AAA TAT ATT TTC CGC AGC GCC GGT TAT CGC GAT GCT 1488

Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala

485 490 495

AAT GGC CAG CTC ATT ATG GAT GGC AGT AAA CCA CGT TAT TGG AAT GTG 1536

Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val

500 505 510

ATG CCA TTG CAA CTG GAT ACC GCA TGG GAT ACC ACA CAG CCC GCC ACC 1584

Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr

515 520 525

ACT GAT CCA GAT GTG ATC GCT ATG GCG GAC CCG ATG CAT TAC AAG CTG 1632

Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu

530 535 540

GCG ATA TTC CTG CAT ACC CTT GAT CTA TTG ATT GCC CGA GGC GAC AGC 1680

Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser

545 550 555 560

GCT TAC CGT CAA CTT GAA CGC GAT ACT CTA GTC GAA GCC AAA ATG TAC 1728

Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr

565 570 575

TAC ATT CAG GCA CAA CAG CTA CTG GGA CCG CGC CCT GAT ATC CAT ACC 1776

Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr

580 585 590

ACC AAT ACT TGG CCA AAT CCC ACC TTG AGT AAA GAA GCT GGC GCT ATT 1824

Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile

595 600 605

GCC ACA CCG ACA TTC CTC AGT TCA CCG GAG GTG ATG ACG TTC GCT GCC 1872

Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala

610 615 620

TGG CTA AGC 1881

Trp Leu Ser

625

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 627 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28 (TcaBi protein):

Met Ser Glu Ser Leu Phe Thr Gln Thr Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

Ala Leu Val Ala His Tyr Ile Ala Thr Gln Val Pro Ala Asp Leu Lys

20 25 30

Glu Ser Ile Gln Thr Ala Asp Asp Leu Tyr Glu Tyr Leu Leu Leu Asp

35 40 45

Thr Lys Ile Ser Asp Leu Val Thr Thr Ser Pro Leu Ser Glu Ala Ile

50 55 60

Gly Ser Leu Gln Leu Phe Ile His Arg Ala Ile Glu Gly Tyr Asp Gly

65 70 75 80

Thr Leu Ala Asp Ser Ala Lys Pro Tyr Phe Ala Asp Glu Gln Phe Leu

85 90 95

Tyr Asn Trp Asp Ser Phe Asn His Arg Tyr Ser Thr Trp Ala Gly Lys

100 105 110

Glu Arg Leu Lys Phe Tyr Ala Gly Asp Tyr Ile Asp Pro Thr Leu Arg

115 120 125

Leu Asn Lys Thr Glu Ile Phe Thr Ala Phe Glu Gln Gly Ile Ser Gln

130 135 140

Gly Lys Leu Lys Ser Glu Leu Val Glu Ser Lys Leu Arg Asp Tyr Leu

145 150 155 160

Ile Ser Tyr Asp Thr Leu Ala Thr Leu Asp Tyr Ile Thr Ala Cys Gln

165 170 175

Gly Lys Asp Asn Lys Thr Ile Phe Phe Ile Gly Arg Thr Gln Asn Ala

180 185 190

Pro Tyr Ala Phe Tyr Trp Arg Lys Leu Thr Leu Val Thr Asp Gly Gly

195 200 205

Lys Leu Lys Pro Asp Gln Trp Ser Glu Trp Arg Ala Ile Asn Ala Gly

210 215 220

Ile Ser Glu Ala Tyr Ser Gly His Val Glu Pro Phe Trp Glu Asn Asn

225 230 235 240

Lys Leu His Ile Arg Trp Phe Thr Ile Ser Lys Glu Asp Lys Ile Asp

245 250 255

Phe Val Tyr Lys Asn Ile Trp Val Met Ser Ser Asp Tyr Ser Trp Ala

260 265 270

Ser Lys Lys Lys Ile Leu Glu Leu Ser Phe Thr Asp Tyr Asn Arg Val

275 280 285

Gly Ala Thr Gly Ser Ser Ser Pro Thr Glu Val Ala Ser Gln Tyr Gly

290 295 300

Ser Asp Ala Gln Met Asn Ile Ser Asp Asp Gly Thr Val Leu Ile Phe

305 310 315 320

Gln Asn Ala Gly Gly Ala Thr Pro Ser Thr Gly Val Thr Leu Cys Tyr

325 330 335

Asp Ser Gly Asn Val Ile Lys Asn Leu Ser Ser Thr Gly Ser Ala Asn

340 345 350

Leu Ser Ser Lys Asp Tyr Ala Thr Thr Lys Leu Arg Met Cys His Gly

355 360 365

Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu Ser Ile Asn

370 375 380

Thr Ile Glu Phe Thr Ser Tyr Gly Thr Phe Ser Ser Asp Gly Lys Gln

385 390 395 400

Phe Thr Pro Pro Ser Gly Ser Ala Ile Asp Leu His Leu Pro Asn Tyr

405 410 415

Val Asp Leu Asn Ala Leu Leu Asp Ile Ser Leu Asp Ser Leu Leu Asn

420 425 430

Tyr Asp Val Gln Gly Gln Phe Gly Gly Ser Asn Pro Val Asp Asn Phe

435 440 445

Ser Gly Pro Tyr Gly Ile Tyr Leu Trp Glu Ile Phe Phe His Ile Pro

450 455 460

Phe Leu Val Thr Val Arg Met Gln Thr Glu Gln Arg Tyr Glu Asp Ala

465 470 475 480

Asp Thr Trp Tyr Lys Tyr Ile Phe Arg Ser Ala Gly Tyr Arg Asp Ala

485 490 495

Asn Gly Gln Leu Ile Met Asp Gly Ser Lys Pro Arg Tyr Trp Asn Val

500 505 510

Met Pro Leu Gln Leu Asp Thr Ala Trp Asp Thr Thr Gln Pro Ala Thr

515 520 525

Thr Asp Pro Asp Val Ile Ala Met Ala Asp Pro Met His Tyr Lys Leu

530 535 540

Ala Ile Phe Leu His Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp Ser

545 550 555 560

Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Val Glu Ala Lys Met Tyr

565 570 575

Tyr Ile Gln Ala Gln Gln Leu Leu Gly Pro Arg Pro Asp Ile His Thr

580 585 590

Thr Asn Thr Trp Pro Asn Pro Thr Leu Ser Lys Glu Ala Gly Ala Ile

595 600 605

Ala Thr Pro Thr Phe Leu Ser Ser Pro Glu Val Met Thr Phe Ala Ala

610 615 620

Trp Leu Ser

625

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1689 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..1689

(D) OTHER INFORMATION: tcaBii

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29 (tcaBii coding

regaion):

GCA GGC GAT ACC GCA AAT ATT GGC GAC GGT GAT TTC TTG CCA CCG TAC 48

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr

1 5 10 15

AAC GAT GTA CTA CTC GGT TAC TGG GAT AAA CTT GAG TTA CGC CTA TAC 96

Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr

20 25 30

AAC CTG CGC CAC AAT CTG AGT CTG GAT GGT CAA CCG CTA AAT CTG CCA 144

Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro

35 40 45

CTG TAT GCC ACG CCG GTA GAC CCG AAA ACC CTG CAA CGC CAG CAA GCC 192

Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala

50 55 60

GGA GGG GAC GGT ACA GGC AGT AGT CCG GCT GGT GGT CAA GGC AGT GTT 240

Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val

65 70 75 80

CAG GGC TGG CGC TAT CCG TTA TTG GTA GAA CGC GCC CGC TCT GCC GTG 288

Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val

85 90 95

AGT TTG TTG ACT CAG TTC GGC AAC AGC TTA CAA ACA ACG TTA GAA CAT 336

Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His

100 105 110

CAG GAT AAT GAA AAA ATG ACG ATA CTG TTG CAG ACT CAA CAG GAA GCC 384

Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala

115 120 125

ATC CTG AAA CAT CAG CAC GAT ATA CAA CAA AAT AAT CTA AAA GGA TTA 432

Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu

130 135 140

CAA CAC AGC CTG ACC GCA TTA CAG GCT AGC CGT GAT GGC GAC ACA TTG 480

Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu

145 150 155 160

CGG CAA AAA CAT TAC AGC GAC CTG ATT AAC GGT GGT CTA TCT GCG GCA 528

Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala

165 170 175

GAA ATC GCC GGT CTG ACA CTA CGC AGC ACC GCC ATG ATT ACC AAT GGC 576

Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly

180 185 190

GTT GCA ACG GGA TTG CTG ATT GCC GGC GGA ATC GCC AAC GCG GTA CCT 624

Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro

195 200 205

AAC GTC TTC GGG CTG GCT AAC GGT GGA TCG GAA TGG GGA GCG CCA TTA 672

Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu

210 215 220

ATT GGC TCC GGG CAA GCA ACC CAA GTT GGC GCC GGC ATC CAG GAT CAG 720

Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln

225 230 235 240

AGC GCG GGC ATT TCA GAA GTG ACA GCA GGC TAT CAG CGT CGT CAG GAA 768

Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu

245 250 255

GAA TGG GCA TTG CAA CGG GAT ATT GCT GAT AAC GAA ATA ACC CAA CTG 816

Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu

260 265 270

GAT GCC CAG ATA CAA AGC CTG CAA GAG CAA ATC ACG ATG GCA CAA AAA 864

Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys

275 280 285

CAG ATC ACG CTC TCT GAA ACC GAA CAA GCG AAT GCC CAA GCG ATT TAT 912

Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr

290 295 300

GAC CTG CAA ACC ACT CGT TTT ACC GGG CAG GCA CTG TAT AAC TGG ATG 960

Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met

305 310 315 320

GCC GGT CGT CTC TCC GCG CTC TAT TAC CAA ATG TAT GAT TCC ACT CTG 1008

Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu

325 330 335

CCA ATC TGT CTC CAG CCA AAA GCC GCA TTA GTA CAG GAA TTA GGC GAG 1056

Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu

340 345 350

AAA GAG AGC GAC AGT CTT TTC CAG GTT CCG GTG TGG AAT GAT CTG TGG 1104

Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp

355 360 365

CAA GGG CTG TTA GCA GGA GAA GGT TTA AGT TCA GAG CTA CAG AAA CTG 1152

Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu

370 375 380

GAT GCC ATC TGG CTT GCA CGT GGT GGT ATT GGG CTA GAA GCC ATC CGC 1200

Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg

385 390 395 400

ACC GTG TCG CTG GAT ACC CTG TTT GGC ACA GGG ACG TTA AGT GAA AAT 1248

Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn

405 410 415

ATC AAT AAA GTG CTT AAC GGG GAA ACG GTA TCT CCA TCC GGT GGC GTC 1296

Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val

420 425 430

ACT CTG GCG CTG ACA GGG GAT ATC TTC CAA GCA ACA CTG GAT TTG AGT 1344

Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser

435 440 445

CAG CTA GGT TTG GAT AAC TCT TAC AAC TTG GGT AAC GAG AAG AAA CGT 1392

Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg

450 455 460

CGT ATT AAA CGT ATC GCC GTC ACC CTG CCA ACA CTT CTG GGG CCA TAT 1440

Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr

465 470 475 480

CAA GAT CTT GAA GCC ACA CTG GTA ATG GGT GCG GAA ATC GCC GCC TTA 1488

Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu

485 490 495

TCA CAC GGT GTG AAT GAC GGA GGC CGG TTT GTT ACC GAC TTT AAC GAC 1536

Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp

500 505 510

AGC CGT TTT CTG CCT TTT GAA GGT CGA GAT GCA ACA ACC GGC ACA CTG 1584

Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu

515 520 525

GAG CTC AAT ATT TTC CAT GCG GGT AAA GAG GGA ACG CAA CAC GAG TTG 1632

Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu

530 535 540

GTC GCG AAT CTG AGT GAC ATC ATT GTG CAT CTG AAT TAC ATC ATT CGA 1680

Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg

545 550 555 560

GAC GCG TAA 1689

Asp Ala *

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 562 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30 (TcaBii protein):

Ala Gly Asp Thr Ala Asn Ile Gly Asp Gly Asp Phe Leu Pro Pro Tyr

1 5 10 15

Asn Asp Val Leu Leu Gly Tyr Trp Asp Lys Leu Glu Leu Arg Leu Tyr

20 25 30

Asn Leu Arg His Asn Leu Ser Leu Asp Gly Gln Pro Leu Asn Leu Pro

35 40 45

Leu Tyr Ala Thr Pro Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala

50 55 60

Gly Gly Asp Gly Thr Gly Ser Ser Pro Ala Gly Gly Gln Gly Ser Val

65 70 75 80

Gln Gly Trp Arg Tyr Pro Leu Leu Val Glu Arg Ala Arg Ser Ala Val

85 90 95

Ser Leu Leu Thr Gln Phe Gly Asn Ser Leu Gln Thr Thr Leu Glu His

100 105 110

Gln Asp Asn Glu Lys Met Thr Ile Leu Leu Gln Thr Gln Gln Glu Ala

115 120 125

Ile Leu Lys His Gln His Asp Ile Gln Gln Asn Asn Leu Lys Gly Leu

130 135 140

Gln His Ser Leu Thr Ala Leu Gln Ala Ser Arg Asp Gly Asp Thr Leu

145 150 155 160

Arg Gln Lys His Tyr Ser Asp Leu Ile Asn Gly Gly Leu Ser Ala Ala

165 170 175

Glu Ile Ala Gly Leu Thr Leu Arg Ser Thr Ala Met Ile Thr Asn Gly

180 185 190

Val Ala Thr Gly Leu Leu Ile Ala Gly Gly Ile Ala Asn Ala Val Pro

195 200 205

Asn Val Phe Gly Leu Ala Asn Gly Gly Ser Glu Trp Gly Ala Pro Leu

210 215 220

Ile Gly Ser Gly Gln Ala Thr Gln Val Gly Ala Gly Ile Gln Asp Gln

225 230 235 240

Ser Ala Gly Ile Ser Glu Val Thr Ala Gly Tyr Gln Arg Arg Gln Glu

245 250 255

Glu Trp Ala Leu Gln Arg Asp Ile Ala Asp Asn Glu Ile Thr Gln Leu

260 265 270

Asp Ala Gln Ile Gln Ser Leu Gln Glu Gln Ile Thr Met Ala Gln Lys

275 280 285

Gln Ile Thr Leu Ser Glu Thr Glu Gln Ala Asn Ala Gln Ala Ile Tyr

290 295 300

Asp Leu Gln Thr Thr Arg Phe Thr Gly Gln Ala Leu Tyr Asn Trp Met

305 310 315 320

Ala Gly Arg Leu Ser Ala Leu Tyr Tyr Gln Met Tyr Asp Ser Thr Leu

325 330 335

Pro Ile Cys Leu Gln Pro Lys Ala Ala Leu Val Gln Glu Leu Gly Glu

340 345 350

Lys Glu Ser Asp Ser Leu Phe Gln Val Pro Val Trp Asn Asp Leu Trp

355 360 365

Gln Gly Leu Leu Ala Gly Glu Gly Leu Ser Ser Glu Leu Gln Lys Leu

370 375 380

Asp Ala Ile Trp Leu Ala Arg Gly Gly Ile Gly Leu Glu Ala Ile Arg

385 390 395 400

Thr Val Ser Leu Asp Thr Leu Phe Gly Thr Gly Thr Leu Ser Glu Asn

405 410 415

Ile Asn Lys Val Leu Asn Gly Glu Thr Val Ser Pro Ser Gly Gly Val

420 425 430

Thr Leu Ala Leu Thr Gly Asp Ile Phe Gln Ala Thr Leu Asp Leu Ser

435 440 445

Gln Leu Gly Leu Asp Asn Ser Tyr Asn Leu Gly Asn Glu Lys Lys Arg

450 455 460

Arg Ile Lys Arg Ile Ala Val Thr Leu Pro Thr Leu Leu Gly Pro Tyr

465 470 475 480

Gln Asp Leu Glu Ala Thr Leu Val Met Gly Ala Glu Ile Ala Ala Leu

485 490 495

Ser His Gly Val Asn Asp Gly Gly Arg Phe Val Thr Asp Phe Asn Asp

500 505 510

Ser Arg Phe Leu Pro Phe Glu Gly Arg Asp Ala Thr Thr Gly Thr Leu

515 520 525

Glu Leu Asn Ile Phe His Ala Gly Lys Glu Gly Thr Gln His Glu Leu

530 535 540

Val Ala Asn Leu Ser Asp Ile Ile Val His Leu Asn Tyr Ile Ile Arg

545 550 555 560

Asp Ala *

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4458 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..4458

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31 (tcac gene):

ATG CAG GAT TCA CCA GAA GTA TCG ATT ACA ACG CTG TCA CTT CCC AAA 48

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys

1 5 10 15

GGT GGC GGT GCT ATC AAT GGC ATG GGA GAA GCA CTG AAT GCT GCC GGC 96

Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly

20 25 30

CCT GAT GGA ATG GCC TCC CTA TCT CTG CCA TTA CCC CTT TCG ACC GGC 144

Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly

35 40 45

AGA GGG ACG GCT CCT GGA TTA TCG CTG ATT TAC AGC AAC AGT GCA GGT 192

Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly

50 55 60

AAT GGG CCT TTC GGC ATC GGC TGG CAA TGC GGT GTT ATG TCC ATT AGC 240

Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser

65 70 75 80

CGA CGC ACC CAA CAT GGC ATT CCA CAA TAC GGT AAT GAC GAC ACG TTC 288

Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe

85 90 95

CTA TCC CCA CAA GGC GAG GTC ATG AAT ATC GCC CTG AAT GAC CAA GGG 336

Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly

100 105 110

CAA CCT GAT ATC CGT CAA GAC GTT AAA ACG CTG CAA GGC GTT ACC TTG 384

Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu

115 120 125

CCA ATT TCC TAT ACC GTG ACC CGC TAT CAA GCC CGC CAG ATC CTG GAT 432

Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp

130 135 140

TTC AGT AAA ATC GAA TAC TGG CAA CCT GCC TCC GGT CAA GAA GGA CGC 480

Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg

145 150 155 160

GCT TTC TGG CTG ATA TCG ACA CCG GAC GGG CAT CTA CAC ATC TTA GGG 528

Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly

165 170 175

AAA ACC GCG CAG GCT TGT CTG GCA AAT CCG CAA AAT GAC CAA CAA ATC 576

Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile

180 185 190

GCC CAG TGG TTG CTG GAA GAA ACT GTG ACG CCA GCC GGT GAA CAT GTC 624

Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val

195 200 205

AGC TAT CAA TAT CGA GCC GAA GAT GAA GCC CAT TGT GAC GAC AAT GAA 672

Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu

210 215 220

AAA ACC GCT CAT CCC AAT GTT ACC GCA CAG CGC TAT CTG GTA CAG GTG 720

Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val

225 230 235 240

AAC TAC GGC AAC ATC AAA CCA CAA GCC AGC CTG TTC GTA CTG GAT AAC 768

Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn

245 250 255

GCA CCT CCC GCA CCG GAA GAG TGG CTG TTT CAT CTG GTC TTT GAC CAC 816

Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His

260 265 270

GGT GAG CGC GAT ACC TCA CTT CAT ACC GTG CCA ACA TGG GAT GCA GGT 864

Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly

275 280 285

ACA GCG CAA TGG TCT GTA CGC CCG GAT ATC TTC TCT CGC TAT GAA TAT 912

Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr

290 295 300

GGT TTT GAA GTG CGT ACT CGC CGC TTA TGT CAA CAA GTG CTG ATG TTT 960

Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe

305 310 315 320

CAC CGC ACC GCG CTC ATG GCC GGA GAA GCC AGT ACC AAT GAC GCC CCG 1008

His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro

325 330 335

GAA CTG GTT GGA CGC TTA ATA CTG GAA TAT GAC AAA AAC GCC AGC GTC 1056

Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val

340 345 350

ACC ACG TTG ATT ACC ATC CGT CAA TTA AGC CAT GAA TCG GAC GGG AGG 1104

Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg

355 360 365

CCA GTC ACC CAG CCA CCA CTA GAA CTA GCC TGG CAA CGG TTT GAT CTG 1152

Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu

370 375 380

GAG AAA ATC CCG ACA TGG CAA CGC TTT GAC GCA CTA GAT AAT TTT AAC 1200

Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn

385 390 395 400

TCG CAG CAA CGT TAT CAA CTG GTT GAT CTG CGG GGA GAA GGG TTG CCA 1248

Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro

405 410 415

GGT ATG CTG TAT CAA GAT CGA GGC GCT TGG TGG TAT AAA GCT CCG CAA 1296

Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln

420 425 430

CGT CAG GAA GAC GGA GAC AGC AAT GCC GTC ACT TAC GAC AAA ATC GCC 1344

Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala

435 440 445

CCA CTG CCT ACC CTA CCC AAT TTG CAG GAT AAT GCC TCA TTG ATG GAT 1392

Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp

450 455 460

ATC AAC GGA GAC GGC CAA CTG GAT TGG GTT GTT ACC GCC TCC GGT ATT 1440

Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile

465 470 475 480

CGC GGA TAC CAT AGT CAG CAA CCC GAT GGA AAG TGG ACG CAC TTT ACG 1488

Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr

485 490 495

CCA ATC AAT GCC TTG CCC GTG GAA TAT TTT CAT CCA AGC ATC CAG TTC 1536

Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe

500 505 510

GCT GAC CTT ACC GGG GCA GGC TTA TCT GAT TTA GTG TTG ATC GGG CCG 1584

Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro

515 520 525

AAA AGC GTG CGT CTA TAT GCC AAC CAG CGA AAC GGC TGG CGT AAA GGA 1632

Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly

530 535 540

GAA GAT GTC CCC CAA TCC ACA GGT ATC ACC CTG CCT GTC ACA GGG ACC 1680

Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr

545 550 555 560

GAT GCC CGC AAA CTG GTG GCT TTC AGT GAT ATG CTC GGT TCC GGT CAA 1728

Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln

565 570 575

CAA CAT CTG GTG GAA ATC AAG GGT AAT CGC GTC ACC TGT TGG CCG AAT 1776

Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn

580 585 590

CTA GGG CAT GGC CGT TTC GGT CAA CCA CTA ACT CTG TCA GGA TTT AGC 1824

Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser

595 600 605

CAG CCC GAA AAT AGC TTC AAT CCC GAA CGG CTG TTT CTG GCG GAT ATC 1872

Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile

610 615 620

GAC GGC TCC GGC ACC ACC GAC CTT ATC TAT GCG CAA TCC GGC TCT TTG 1920

Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu

625 630 635 640

CTC ATT TAT CTC AAC CAA AGT GGT AAT CAG TTT GAT GCC CCG TTG ACA 1968

Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr

645 650 655

TTA GCG TTG CCA GAA GGC GTA CAA TTT GAC AAC ACT TGC CAA CTT CAA 2016

Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln

660 665 670

GTC GCC GAT ATT CAG GGA TTA GGG ATA GCC AGC TTG ATT CTG ACT GTG 2064

Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val

675 680 685

CCA CAT ATC GCG CCA CAT CAC TGG CGT TGT GAC CTG TCA CTG ACC AAA 2112

Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys

690 695 700

CCC TGG TTG TTG AAT GTA ATG AAC AAT AAC CGG GGC GCA CAT CAC ACG 2160

Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr

705 710 715 720

CTA CAT TAT CGT AGT TCC GCG CAA TTC TGG TTG GAT GAA AAA TTA CAG 2208

Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln

725 730 735

CTC ACC AAA GCA GGC AAA TCT CCG GCT TGT TAT CTG CCG TTT CCA ATG 2256

Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met

740 745 750

CAT TTG CTA TGG TAT ACC GAA ATT CAG GAT GAA ATC AGC GGC AAC CGG 2304

His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg

755 760 765

CTC ACC AGT GAA GTC AAC TAC AGC CAC GGC GTC TGG GAT GGT AAA GAG 2352

Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu

770 775 780

CGG GAA TTC AGA GGA TTT GGC TGC ATC AAA CAG ACA GAT ACC ACA ACG 2400

Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr

785 790 795 800

TTT TCT CAC GGC ACC GCC CCC GAA CAG GCG GCA CCG TCG CTG AGT ATT 2448

Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile

805 810 815

AGC TGG TTT GCC ACC GGC ATG GAT GAA GTA GAC AGC CAA TTA GCT ACG 2496

Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr

820 825 830

GAA TAT TGG CAG GCA GAC ACG CAA GCT TAT AGC GGA TTT GAA ACC CGT 2544

Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg

835 840 845

TAT ACC GTC TGG GAT CAC ACC AAC CAG ACA GAC CAA GCA TTT ACC CCC 2592

Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro

850 855 860

AAT GAG ACA CAA CGT AAC TGG CTG ACG CGA GCG CTT AAA GGC CAA CTG 2640

Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu

865 870 875 880

CTA CGC ACT GAG CTC TAC GGT CTG GAC GGA ACA GAT AAG CAA ACA GTG 2688

Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val

885 890 895

CCT TAT ACC GTC AGT GAA TCG CGC TAT CAG GTA CGC TCT ATT CCC GTA 2736

Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val

900 905 910

AAT AAA GAA ACT GAA TTA TCT GCC TGG GTG ACT GCT ATT GAA AAT CGC 2784

Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg

915 920 925

AGC TAC CAC TAT GAA CGT ATC ATC ACT GAC CCA CAG TTC AGC CAG AGT 2832

Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser

930 935 940

ATC AAG TTG CAA CAC GAT ATC TTT GGT CAA TCA CTG CAA AGT GTC GAT 2880

Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp

945 950 955 960

ATT GCC TGG CCG CGC CGC GAA AAA CCA GCA GTG AAT CCC TAC CCG CCT 2928

Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro

965 970 975

ACC CTG CCG GAA ACG CTA TTT GAC AGC AGC TAT GAT GAT CAA CAA CAA 2976

Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln

980 985 990

CTA TTA CGT CTG GTG AGA CAA AAA AAT AGC TGG CAT CAC CTG ACT GAT 3024

Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp

995 1000 1005

GGG GAA AAC TGG CGA TTA GGT TTA CCG AAT GCA CAA CGC CGT GAT GTT 3072

Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val

1010 1015 1020

TAT ACT TAT GAC CGG AGC AAA ATT CCA ACC GAA GGG ATT TCC CTT GAA 3120

Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu

1025 1030 1035 1040

ATC TTG CTG AAA GAT GAT GGC CTG CTA GCA GAT GAA AAA GCG GCC GTT 3168

Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val

1045 1050 1055

TAT CTG GGA CAA CAA CAG ACG TTT TAC ACC GCC GGT CAA GCG GAA GTC 3216

Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val

1060 1065 1070

ACT CTA GAA AAA CCC ACG TTA CAA GCA CTG GTC GCG TTC CAA GAA ACC 3264

Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr

1075 1080 1085

GCC ATG ATG GAC GAT ACC TCA TTA CAG GCG TAT GAA GGC GTG ATT GAA 3312

Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu

1090 1095 1100

GAG CAA GAG TTG AAT ACC GCG CTG ACA CAG GCC GGT TAT CAG CAA GTC 3360

Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val

1105 1110 1115 1120

GCG CGG TTG TTT AAT ACC AGA TCA GAA AGC CCG GTA TGG GCG GCA CGG 3408

Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg

1125 1130 1135

CAA GGT TAT ACC GAT TAC GGT GAC GCC GCA CAG TTC TGG CGG CCT CAG 3456

Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln

1140 1145 1150

GCT CAG CGT AAC TCG TTG CTG ACA GGG AAA ACC ACA CTG ACC TGG GAT 3504

Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp

1155 1160 1165

ACC CAT CAT TGT GTA ATA ATA CAG ACT CAA GAT GCC GCT GGA TTA ACG 3552

Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr

1170 1175 1180

ACG CAA GCC CAT TAC GAT TAT CGT TTC CTT ACA CCG GTA CAA CTG ACA 3600

Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr

1185 1190 1195 1200

GAT ATT AAT GAT AAT CAA CAT ATT GTG ACT CTG GAC GCG CTA GGT CGC 3648

Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg

1205 1210 1215

GTA ACC ACC AGC CGG TTC TGG GGC ACA GAG GCA GGA CAA GCC GCA GGC 3696

Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly

1220 1225 1230

TAT TCC AAC CAG CCC TTC ACA CCA CCG GAC TCC GTA GAT AAA GCG CTG 3744

Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu

1235 1240 1245

GCA TTA ACC GGC GCA CTC CCT GTT GCC CAA TGT TTA GTC TAT GCC GTT 3792

Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val

1250 1255 1260

GAT AGC TGG ATG CCG TCG TTA TCT TTG TCT CAG CTT TCT CAG TCA CAA 3840

Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln

1265 1270 1275 1280

GAA GAG GCA GAA GCG CTA TGG GCG CAA CTG CGT GCC GCT CAT ATG ATT 3888

Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile

1285 1290 1295

ACC GAA GAT GGG AAA GTG TGT GCG TTA AGC GGG AAA CGA GGA ACA AGC 3936

Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser

1300 1305 1310

CAT CAG AAC CTG ACG ATT CAA CTT ATT TCG CTA TTG GCA AGT ATT CCC 3984

His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro

1315 1320 1325

CGT TTA CCG CCA CAT GTA CTG GGG ATC ACC ACT GAT CGC TAT GAT AGC 4032

Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser

1330 1335 1340

GAT CCG CAA CAG CAG CAC CAA CAG ACG GTG AGC TTT AGT GAC GGT TTT 4080

Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe

1345 1350 1355 1360

GGC CGG TTA CTC CAG AGT TCA GCT CGT CAT GAG TCA GGT GAT GCC TGG 4128

Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp

1365 1370 1375

CAA CGT AAA GAG GAT GGC GGG CTG GTC GTG GAT GCA AAT GGC GTT CTG 4176

Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu

1380 1385 1390

GTC AGT GCC CCT ACA GAC ACC CGA TGG GCC GTT TCC GGT CGC ACA GAA 4224

Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu

1395 1400 1405

TAT GAC GAC AAA GGC CAA CCT GTG CGT ACT TAT CAA CCC TAT TTT CTA 4272

Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu

1410 1415 1420

AAT GAC TGG CGT TAC GTT AGT GAT GAC AGC GCA CGA GAT GAC CTG TTT 4320

Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe

1425 1430 1435 1440

GCC GAT ACC CAC CTT TAT GAT CCA TTG GGA CGG GAA TAC AAA GTC ATC 4368

Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile

1445 1450 1455

ACT GCT AAG AAA TAT TTG CGA GAA AAG CTG TAC ACC CCG TGG TTT ATT 4416

Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile

1460 1465 1470

GTC AGT GAG GAT GAA AAC GAT ACA GCA TCA AGA ACC CCA TAG 4458

Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro *

1475 1480 1485

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1485 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32 (TcaC protein):

Met Gln Asp Ser Pro Glu Val Ser Ile Thr Thr Leu Ser Leu Pro Lys

1 5 10 15

Gly Gly Gly Ala Ile Asn Gly Met Gly Glu Ala Leu Asn Ala Ala Gly

20 25 30

Pro Asp Gly Met Ala Ser Leu Ser Leu Pro Leu Pro Leu Ser Thr Gly

35 40 45

Arg Gly Thr Ala Pro Gly Leu Ser Leu Ile Tyr Ser Asn Ser Ala Gly

50 55 60

Asn Gly Pro Phe Gly Ile Gly Trp Gln Cys Gly Val Met Ser Ile Ser

65 70 75 80

Arg Arg Thr Gln His Gly Ile Pro Gln Tyr Gly Asn Asp Asp Thr Phe

85 90 95

Leu Ser Pro Gln Gly Glu Val Met Asn Ile Ala Leu Asn Asp Gln Gly

100 105 110

Gln Pro Asp Ile Arg Gln Asp Val Lys Thr Leu Gln Gly Val Thr Leu

115 120 125

Pro Ile Ser Tyr Thr Val Thr Arg Tyr Gln Ala Arg Gln Ile Leu Asp

130 135 140

Phe Ser Lys Ile Glu Tyr Trp Gln Pro Ala Ser Gly Gln Glu Gly Arg

145 150 155 160

Ala Phe Trp Leu Ile Ser Thr Pro Asp Gly His Leu His Ile Leu Gly

165 170 175

Lys Thr Ala Gln Ala Cys Leu Ala Asn Pro Gln Asn Asp Gln Gln Ile

180 185 190

Ala Gln Trp Leu Leu Glu Glu Thr Val Thr Pro Ala Gly Glu His Val

195 200 205

Ser Tyr Gln Tyr Arg Ala Glu Asp Glu Ala His Cys Asp Asp Asn Glu

210 215 220

Lys Thr Ala His Pro Asn Val Thr Ala Gln Arg Tyr Leu Val Gln Val

225 230 235 240

Asn Tyr Gly Asn Ile Lys Pro Gln Ala Ser Leu Phe Val Leu Asp Asn

245 250 255

Ala Pro Pro Ala Pro Glu Glu Trp Leu Phe His Leu Val Phe Asp His

260 265 270

Gly Glu Arg Asp Thr Ser Leu His Thr Val Pro Thr Trp Asp Ala Gly

275 280 285

Thr Ala Gln Trp Ser Val Arg Pro Asp Ile Phe Ser Arg Tyr Glu Tyr

290 295 300

Gly Phe Glu Val Arg Thr Arg Arg Leu Cys Gln Gln Val Leu Met Phe

305 310 315 320

His Arg Thr Ala Leu Met Ala Gly Glu Ala Ser Thr Asn Asp Ala Pro

325 330 335

Glu Leu Val Gly Arg Leu Ile Leu Glu Tyr Asp Lys Asn Ala Ser Val

340 345 350

Thr Thr Leu Ile Thr Ile Arg Gln Leu Ser His Glu Ser Asp Gly Arg

355 360 365

Pro Val Thr Gln Pro Pro Leu Glu Leu Ala Trp Gln Arg Phe Asp Leu

370 375 380

Glu Lys Ile Pro Thr Trp Gln Arg Phe Asp Ala Leu Asp Asn Phe Asn

385 390 395 400

Ser Gln Gln Arg Tyr Gln Leu Val Asp Leu Arg Gly Glu Gly Leu Pro

405 410 415

Gly Met Leu Tyr Gln Asp Arg Gly Ala Trp Trp Tyr Lys Ala Pro Gln

420 425 430

Arg Gln Glu Asp Gly Asp Ser Asn Ala Val Thr Tyr Asp Lys Ile Ala

435 440 445

Pro Leu Pro Thr Leu Pro Asn Leu Gln Asp Asn Ala Ser Leu Met Asp

450 455 460

Ile Asn Gly Asp Gly Gln Leu Asp Trp Val Val Thr Ala Ser Gly Ile

465 470 475 480

Arg Gly Tyr His Ser Gln Gln Pro Asp Gly Lys Trp Thr His Phe Thr

485 490 495

Pro Ile Asn Ala Leu Pro Val Glu Tyr Phe His Pro Ser Ile Gln Phe

500 505 510

Ala Asp Leu Thr Gly Ala Gly Leu Ser Asp Leu Val Leu Ile Gly Pro

515 520 525

Lys Ser Val Arg Leu Tyr Ala Asn Gln Arg Asn Gly Trp Arg Lys Gly

530 535 540

Glu Asp Val Pro Gln Ser Thr Gly Ile Thr Leu Pro Val Thr Gly Thr

545 550 555 560

Asp Ala Arg Lys Leu Val Ala Phe Ser Asp Met Leu Gly Ser Gly Gln

565 570 575

Gln His Leu Val Glu Ile Lys Gly Asn Arg Val Thr Cys Trp Pro Asn

580 585 590

Leu Gly His Gly Arg Phe Gly Gln Pro Leu Thr Leu Ser Gly Phe Ser

595 600 605

Gln Pro Glu Asn Ser Phe Asn Pro Glu Arg Leu Phe Leu Ala Asp Ile

610 615 620

Asp Gly Ser Gly Thr Thr Asp Leu Ile Tyr Ala Gln Ser Gly Ser Leu

625 630 635 640

Leu Ile Tyr Leu Asn Gln Ser Gly Asn Gln Phe Asp Ala Pro Leu Thr

645 650 655

Leu Ala Leu Pro Glu Gly Val Gln Phe Asp Asn Thr Cys Gln Leu Gln

660 665 670

Val Ala Asp Ile Gln Gly Leu Gly Ile Ala Ser Leu Ile Leu Thr Val

675 680 685

Pro His Ile Ala Pro His His Trp Arg Cys Asp Leu Ser Leu Thr Lys

690 695 700

Pro Trp Leu Leu Asn Val Met Asn Asn Asn Arg Gly Ala His His Thr

705 710 715 720

Leu His Tyr Arg Ser Ser Ala Gln Phe Trp Leu Asp Glu Lys Leu Gln

725 730 735

Leu Thr Lys Ala Gly Lys Ser Pro Ala Cys Tyr Leu Pro Phe Pro Met

740 745 750

His Leu Leu Trp Tyr Thr Glu Ile Gln Asp Glu Ile Ser Gly Asn Arg

755 760 765

Leu Thr Ser Glu Val Asn Tyr Ser His Gly Val Trp Asp Gly Lys Glu

770 775 780

Arg Glu Phe Arg Gly Phe Gly Cys Ile Lys Gln Thr Asp Thr Thr Thr

785 790 795 800

Phe Ser His Gly Thr Ala Pro Glu Gln Ala Ala Pro Ser Leu Ser Ile

805 810 815

Ser Trp Phe Ala Thr Gly Met Asp Glu Val Asp Ser Gln Leu Ala Thr

820 825 830

Glu Tyr Trp Gln Ala Asp Thr Gln Ala Tyr Ser Gly Phe Glu Thr Arg

835 840 845

Tyr Thr Val Trp Asp His Thr Asn Gln Thr Asp Gln Ala Phe Thr Pro

850 855 860

Asn Glu Thr Gln Arg Asn Trp Leu Thr Arg Ala Leu Lys Gly Gln Leu

865 870 875 880

Leu Arg Thr Glu Leu Tyr Gly Leu Asp Gly Thr Asp Lys Gln Thr Val

885 890 895

Pro Tyr Thr Val Ser Glu Ser Arg Tyr Gln Val Arg Ser Ile Pro Val

900 905 910

Asn Lys Glu Thr Glu Leu Ser Ala Trp Val Thr Ala Ile Glu Asn Arg

915 920 925

Ser Tyr His Tyr Glu Arg Ile Ile Thr Asp Pro Gln Phe Ser Gln Ser

930 935 940

Ile Lys Leu Gln His Asp Ile Phe Gly Gln Ser Leu Gln Ser Val Asp

945 950 955 960

Ile Ala Trp Pro Arg Arg Glu Lys Pro Ala Val Asn Pro Tyr Pro Pro

965 970 975

Thr Leu Pro Glu Thr Leu Phe Asp Ser Ser Tyr Asp Asp Gln Gln Gln

980 985 990

Leu Leu Arg Leu Val Arg Gln Lys Asn Ser Trp His His Leu Thr Asp

995 1000 1005

Gly Glu Asn Trp Arg Leu Gly Leu Pro Asn Ala Gln Arg Arg Asp Val

1010 1015 1020

Tyr Thr Tyr Asp Arg Ser Lys Ile Pro Thr Glu Gly Ile Ser Leu Glu

1025 1030 1035 1040

Ile Leu Leu Lys Asp Asp Gly Leu Leu Ala Asp Glu Lys Ala Ala Val

1045 1050 1055

Tyr Leu Gly Gln Gln Gln Thr Phe Tyr Thr Ala Gly Gln Ala Glu Val

1060 1065 1070

Thr Leu Glu Lys Pro Thr Leu Gln Ala Leu Val Ala Phe Gln Glu Thr

1075 1080 1085

Ala Met Met Asp Asp Thr Ser Leu Gln Ala Tyr Glu Gly Val Ile Glu

1090 1095 1100

Glu Gln Glu Leu Asn Thr Ala Leu Thr Gln Ala Gly Tyr Gln Gln Val

1105 1110 1115 1120

Ala Arg Leu Phe Asn Thr Arg Ser Glu Ser Pro Val Trp Ala Ala Arg

1125 1130 1135

Gln Gly Tyr Thr Asp Tyr Gly Asp Ala Ala Gln Phe Trp Arg Pro Gln

1140 1145 1150

Ala Gln Arg Asn Ser Leu Leu Thr Gly Lys Thr Thr Leu Thr Trp Asp

1155 1160 1165

Thr His His Cys Val Ile Ile Gln Thr Gln Asp Ala Ala Gly Leu Thr

1170 1175 1180

Thr Gln Ala His Tyr Asp Tyr Arg Phe Leu Thr Pro Val Gln Leu Thr

1185 1190 1195 1200

Asp Ile Asn Asp Asn Gln His Ile Val Thr Leu Asp Ala Leu Gly Arg

1205 1210 1215

Val Thr Thr Ser Arg Phe Trp Gly Thr Glu Ala Gly Gln Ala Ala Gly

1220 1225 1230

Tyr Ser Asn Gln Pro Phe Thr Pro Pro Asp Ser Val Asp Lys Ala Leu

1235 1240 1245

Ala Leu Thr Gly Ala Leu Pro Val Ala Gln Cys Leu Val Tyr Ala Val

1250 1255 1260

Asp Ser Trp Met Pro Ser Leu Ser Leu Ser Gln Leu Ser Gln Ser Gln

1265 1270 1275 1280

Glu Glu Ala Glu Ala Leu Trp Ala Gln Leu Arg Ala Ala His Met Ile

1285 1290 1295

Thr Glu Asp Gly Lys Val Cys Ala Leu Ser Gly Lys Arg Gly Thr Ser

1300 1305 1310

His Gln Asn Leu Thr Ile Gln Leu Ile Ser Leu Leu Ala Ser Ile Pro

1315 1320 1325

Arg Leu Pro Pro His Val Leu Gly Ile Thr Thr Asp Arg Tyr Asp Ser

1330 1335 1340

Asp Pro Gln Gln Gln His Gln Gln Thr Val Ser Phe Ser Asp Gly Phe

1345 1350 1355 1360

Gly Arg Leu Leu Gln Ser Ser Ala Arg His Glu Ser Gly Asp Ala Trp

1365 1370 1375

Gln Arg Lys Glu Asp Gly Gly Leu Val Val Asp Ala Asn Gly Val Leu

1380 1385 1390

Val Ser Ala Pro Thr Asp Thr Arg Trp Ala Val Ser Gly Arg Thr Glu

1395 1400 1405

Tyr Asp Asp Lys Gly Gln Pro Val Arg Thr Tyr Gln Pro Tyr Phe Leu

1410 1415 1420

Asn Asp Trp Arg Tyr Val Ser Asp Asp Ser Ala Arg Asp Asp Leu Phe

1425 1430 1435 1440

Ala Asp Thr His Leu Tyr Asp Pro Leu Gly Arg Glu Tyr Lys Val Ile

1445 1450 1455

Thr Ala Lys Lys Tyr Leu Arg Glu Lys Leu Tyr Thr Pro Trp Phe Ile

1460 1465 1470

Val Ser Glu Asp Glu Asn Asp Thr Ala Ser Arg Thr Pro *

1475 1480 1485

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3288 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33 (tcaA gene):

ATG GTG ACT GTT ATG CAA AAT AAA ATA TCA TTT TTA TCA GGT ACA TCC 48

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser

1 5 10 15

GAA CAG CCC CTG CTT GAC GCC GGT TAT CAA AAC GTA TTT GAT ATC GCA 96

Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala

20 25 30

TCA ATC AGC CGG GCT ACT TTC GTT CAA TCC GTT CCC ACC CTG CCC GTT 144

Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val

35 40 45

AAA GAG GCT CAT ACC GTC TAT CGT CAG GCG CGG CAA CGT GCG GAA AAT 192

Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn

50 55 60

CTG AAA TCC CTC TAC CGA GCC TGG CAA TTG CGT CAG GAG CCG GTT ATT 240

Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile

65 70 75 80

AAA GGG CTG GCT AAA CTT AAC CTA CAA TCC AAC GTT TCT GTG CTT CAA 288

Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln

85 90 95

GAT GCT TTG GTA GAG AAT ATT GGC GGT GAT GGG GAT TTC AGC GAT TTA 336

Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu

100 105 110

ATG AAC CGT GCC AGT CAA TAT GCT GAC GCT GCC TCT ATT CAA TCC CTA 384

Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu

115 120 125

TTT TCA CCG GGC CGT TAT GCT TCC GCA CTC TAC AGA GTT GCT AAA GAT 432

Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp

130 135 140

CTG CAT AAA TCA GAT TCC AGT TTG CAT ATT GAT AAT CGC CGC GCT GAT 480

Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp

145 150 155 160

CTG AAG GAT CTG ATA TTA AGC GAA ACG ACG ATG AAT AAA GAG GTC ACT 528

Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr

165 170 175

TCC CTT GAT ATC TTG TTG GAT GTG CTA CAA AAA GGC GGT AAA GAT ATT 576

Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile

180 185 190

ACT GAG CTG TCC GGC GCA TTC TTC CCA ATG ACG TTA CCT TAT GAC GAT 624

Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp

195 200 205

CAT CTG TCG CAA ATC GAT TCC GCT TTA TCG GCA CAA GCC AGA ACG CTG 672

His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu

210 215 220

AAC GGT GTG TGG AAT ACT TTG ACA GAT ACC ACG GCA CAA GCG GTT TCA 720

Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser

225 230 235 240

GAA CAA ACC AGT AAT ACG AAT ACA CGC AAA CTG TTC GCT GCC CAA GAT 768

Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp

245 250 255

GGT AAT CAA GAT ACA TTT TTT TCC GGA AAC ACT TTT TAT TTC AAA GCG 816

Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala

260 265 270

GTG GGA TTC AGC GGG CAA CCT ATG GTT TAC CTG TCA CAG TAC ACC AGC 864

Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser

275 280 285

GGG AAC GGC ATT GTC GGC GCA CAA TTG ATT GCA GGT AAT CCA GAC CAA 912

Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln

290 295 300

GCC GCC GCC GCA ATA GTC GCA CCG TTG AAA CTC ACT TGG TCA ATG GCA 960

Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala

305 310 315 320

AAA CAG TGT TAC TAC CTC GTC GCT CCC GAT GGT ACA ACG ATG GGA GAC 1008

Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp

325 330 335

GGT AAT GTT CTG ACC GGC TGT TTC TTA AGA GGC AAC AGC CCA ACT AAC 1056

Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn

340 345 350

CCG GAT AAA GAC GGT ATT TTT GCT CAG GTA GCC AAC AAA TCA GGC AGT 1104

Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser

355 360 365

ACT CAG CCT TTG CCA AGC TTC CAT CTG CCG GTC ACA CTG GAA CAC AGC 1152

Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser

370 375 380

GAG AAT AAA GAT CAG TAC TAT CTG AAA ACA GAG CAG GGT TAT ATC ACG 1200

Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr

385 390 395 400

GTA GAT AGT TCC GGA CAG TCA AAT TGG AAA AAC GCG CTG GTT ATC AAT 1248

Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn

405 410 415

GGG ACA AAA GAC AAG GGG CTG TTA TTA ACC TTT TGC AGC GAT AGC TCA 1296

Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser

420 425 430

GGC ACT CCG ACA AAC CCT GAT GAT GTG ATT CCT CCC GCT ATC AAT GAT 1344

Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp

435 440 445

ATT CCA TCG CCG CCA GCC CGC GAA ACA CTG TCA CTG ACG CCG GTC AGT 1392

Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser

450 455 460

TAT CAA TTG ATG ACC AAT CCG GCA CCG ACA GAA GAT GAT ATT ACC AAC 1440

Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn

465 470 475 480

CAT TAT GGT TTT AAC GGC GCT AGC TTA CGG GCT TCT CCA TTG TCA ACC 1488

His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr

485 490 495

AGC GAG TTG ACC AGC AAA CTG AAT TCT ATC GAT ACT TTC TGT GAG AAG 1536

Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys

500 505 510

ACC CGG TTA AGC TTC AAT CAG TTA ATG GAT TTG ACC GCT CAG CAA TCT 1584

Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser

515 520 525

TAC AGT CAA AGC AGC ATT GAT GCG AAA GCA GCC AGC CGC TAT GTT CGT 1632

Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg

530 535 540

TTT GGG GAA ACC ACC CCA ACC CGC GTC AAT GTC TAC GGT GCC GCT TAT 1680

Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr

545 550 555 560

CTG AAC AGC ACA CTG GCA GAC GCG GCT GAT GGT CAA TAT CTG TGG ATT 1728

Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile

565 570 575

CAG ACT GAT GGC AAG AGC CTA AAT TTC ACT GAC GAT ACG GTA GTC GCC 1776

Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala

580 585 590

TTA GCC GGT CGC GCT GAA AAG CTG GTA CGT TTA TCA TCC CAG ACC GGG 1824

Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly

595 600 605

CTA TCA TTT GAA GAA TTG GAC TGG CTG ATT GCC AAT GCC AGT CGT AGT 1872

Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser

610 615 620

GTG CCG GAC CAC CAC GAC AAA ATT GTG CTG GAT AAG CCG GTC CTT GAA 1920

Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu

625 630 635 640

GCA CTG GCA GAG TAT GTC AGC CTA AAA CAG CGC TAT GGG CTT GAT GCC 1968

Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala

645 650 655

AAT ACC TTT GCG ACC TTC ATT AGT GCA GTA AAT CCT TAT ACG CCA GAT 2016

Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp

660 665 670

CAG ACA CCC AGT TTC TAT GAA ACC GCT TTC CGC TCT GCC GAC GGT AAT 2064

Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn

675 680 685

CAT GTC ATT GCG CTA GGT ACA GAG GTG AAA TAT GCA GAA AAT GAG CAG 2112

His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln

690 695 700

GAT GAG TTA GCC GCC ATA TGC TGC AAA GCA TTG GGT GTC ACC AGT GAT 2160

Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp

705 710 715 720

GAA CTG CTC CGT ATT GGT CGC TAT TGC TTC GGT AAT GCA GGC AGT TTT 2208

Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe

725 730 735

ACC TTG GAT GAA TAT ACC GCC AGT CAG TTG TAT CGC TTC GGC GCC ATT 2256

Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile

740 745 750

CCC CGT TTG TTT GGG CTG ACA TTT GCC CAA GCC GAA ATT TTA TGG CGT 2304

Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg

755 760 765

CTG ATG GAA GGC GGA AAA GAT ATC TTA TTG CAA CAG TTA GGT CAG GCA 2352

Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala

770 775 780

AAA TCC CTG CAA CCA CTG GCT ATT TTA CGC CGT ACC GAG CAG GTG CTG 2400

Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu

785 790 795 800

GAT TGG ATG TCG TCC GTA AAT CTA AGT CTG ACT TAT CTG CAA GGG ATG 2448

Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met

805 810 815

GTA AGT ACG CAA TGG AGC GGT ACC GCC ACC GCT GAG ATG TTC AAT TTC 2496

Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe

820 825 830

TTG GAA AAC GTT TGT GAC AGC GTG AAT AGT CAA GCT GCC ACT AAA GAA 2544

Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu

835 840 845

ACA ATG GAT TCG GCG TTA CAG CAG AAA GTG CTG CGG GCG CTA AGC GCC 2592

Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala

850 855 860

GGT TTC GGC ATT AAG AGC AAT GTG ATG GGT ATC GTC ACC TTC TGG CTG 2640

Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu

865 870 875 880

GAG AAA ATC ACA ATC GGT AGT GAT AAT CCT TTT ACA TTG GCA AAC TAC 2688

Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr

885 890 895

TGG CAT GAT ATT CAA ACC CTG TTT AGC CAT GAC AAT GCC ACG TTA GAG 2736

Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu

900 905 910

TCC TTA CAA ACC GAC ACT TCT CTG GTA ATT GCT ACT CAG CAA CTT AGC 2784

Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser

915 920 925

CAG CTA GTG TTA ATT GTG AAA TGG CTG AGC CTG ACC GAG CAG GAT CTG 2832

Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu

930 935 940

CAA TTA CTG ACA ACC TAT CCC GAA CGT TTA ATC AAC GGC ATC ACG AAT 2880

Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn

945 950 955 960

GTT CCT GTA CCC AAT CCG GAG CTA TTA CTC ACG CTA TCA CGT TTT AAG 2928

Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys

965 970 975

CAG TGG GAA ACT CAA GTC ACC GTT TCC CGT GAT GAA GCG ATG CGC TGT 2976

Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys

980 985 990

TTC GAT CAA TTA AAT GCC AAT GAT ATG ACG ACT GAA AAT GCA GGT TCA 3024

Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser

995 1000 1005

CTG ATC GCC ACA TTG TAT GAG ATG GAT AAA GGT ACG GGA GCG CAA GTT 3072

Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val

1010 1015 1020

AAT ACC TTG CTA TTA GGT GAA AAT AAC TGG CCG AAA AGT TTT ACC TCT 3120

Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser

1025 1030 1035 1040

CTC TGG CAA CTT CTG ACC TGG TTA CGC GTC GGG CAA AGA CTG AAT GTC 3168

Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val

1045 1050 1055

GGT AGT ACC ACT CTG GGC AAT CTG TTG TCC ATG ATG CAA GCA GAC CCT 3216

Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro

1060 1065 1070

GCT GCC GAG AGT AGC GCT TTA TTG GCA TCA GTA GCC CAA AAC TTA AGT 3264

Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser

1075 1080 1085

GCC GCA ATC AGC AAT CGT CAG TAA 3288

Ala Ala Ile Ser Asn Arg Gln

1090 1095

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1095 amino acids

(B) TYPE: amino acids

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34 (TcaA protein):

Features From To Description

254 267 SEQ ID NO: 15

254 492 TcaAii peptide

Met Val Thr Val Met Gln Asn Lys Ile Ser Phe Leu Ser Gly Thr Ser

1 5 10 15

Glu Gln Pro Leu Leu Asp Ala Gly Tyr Gln Asn Val Phe Asp Ile Ala

20 25 30

Ser Ile Ser Arg Ala Thr Phe Val Gln Ser Val Pro Thr Leu Pro Val

35 40 45

Lys Glu Ala His Thr Val Tyr Arg Gln Ala Arg Gln Arg Ala Glu Asn

50 55 60

Leu Lys Ser Leu Tyr Arg Ala Trp Gln Leu Arg Gln Glu Pro Val Ile

65 70 75 80

Lys Gly Leu Ala Lys Leu Asn Leu Gln Ser Asn Val Ser Val Leu Gln

85 90 95

Asp Ala Leu Val Glu Asn Ile Gly Gly Asp Gly Asp Phe Ser Asp Leu

100 105 110

Met Asn Arg Ala Ser Gln Tyr Ala Asp Ala Ala Ser Ile Gln Ser Leu

115 120 125

Phe Ser Pro Gly Arg Tyr Ala Ser Ala Leu Tyr Arg Val Ala Lys Asp

130 135 140

Leu His Lys Ser Asp Ser Ser Leu His Ile Asp Asn Arg Arg Ala Asp

145 150 155 160

Leu Lys Asp Leu Ile Leu Ser Glu Thr Thr Met Asn Lys Glu Val Thr

165 170 175

Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Lys Gly Gly Lys Asp Ile

180 185 190

Thr Glu Leu Ser Gly Ala Phe Phe Pro Met Thr Leu Pro Tyr Asp Asp

195 200 205

His Leu Ser Gln Ile Asp Ser Ala Leu Ser Ala Gln Ala Arg Thr Leu

210 215 220

Asn Gly Val Trp Asn Thr Leu Thr Asp Thr Thr Ala Gln Ala Val Ser

225 230 235 240

Glu Gln Thr Ser Asn Thr Asn Thr Arg Lys Leu Phe Ala Ala Gln Asp

245 250 255

Gly Asn Gln Asp Thr Phe Phe Ser Gly Asn Thr Phe Tyr Phe Lys Ala

260 265 270

Val Gly Phe Ser Gly Gln Pro Met Val Tyr Leu Ser Gln Tyr Thr Ser

275 280 285

Gly Asn Gly Ile Val Gly Ala Gln Leu Ile Ala Gly Asn Pro Asp Gln

290 295 300

Ala Ala Ala Ala Ile Val Ala Pro Leu Lys Leu Thr Trp Ser Met Ala

305 310 315 320

Lys Gln Cys Tyr Tyr Leu Val Ala Pro Asp Gly Thr Thr Met Gly Asp

325 330 335

Gly Asn Val Leu Thr Gly Cys Phe Leu Arg Gly Asn Ser Pro Thr Asn

340 345 350

Pro Asp Lys Asp Gly Ile Phe Ala Gln Val Ala Asn Lys Ser Gly Ser

355 360 365

Thr Gln Pro Leu Pro Ser Phe His Leu Pro Val Thr Leu Glu His Ser

370 375 380

Glu Asn Lys Asp Gln Tyr Tyr Leu Lys Thr Glu Gln Gly Tyr Ile Thr

385 390 395 400

Val Asp Ser Ser Gly Gln Ser Asn Trp Lys Asn Ala Leu Val Ile Asn

405 410 415

Gly Thr Lys Asp Lys Gly Leu Leu Leu Thr Phe Cys Ser Asp Ser Ser

420 425 430

Gly Thr Pro Thr Asn Pro Asp Asp Val Ile Pro Pro Ala Ile Asn Asp

435 440 445

Ile Pro Ser Pro Pro Ala Arg Glu Thr Leu Ser Leu Thr Pro Val Ser

450 455 460

Tyr Gln Leu Met Thr Asn Pro Ala Pro Thr Glu Asp Asp Ile Thr Asn

465 470 475 480

His Tyr Gly Phe Asn Gly Ala Ser Leu Arg Ala Ser Pro Leu Ser Thr

485 490 W4 »495

Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr Phe Cys Glu Lys

500 505 510

Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr Ala Gln Gln Ser

515 520 525

Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser Arg Tyr Val Arg

530 535 540

Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr Gly Ala Ala Tyr

545 550 555 560

Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln Tyr Leu Trp Ile

565 570 575

Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp Thr Val Val Ala

580 585 590

Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser Ser Gln Thr Gly

595 600 605

Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn Ala Ser Arg Ser

610 615 620

Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys Pro Val Leu Glu

625 630 635 640

Ala Leu Ala Glu Tyr Val Ser Leu Lys Gln Arg Tyr Gly Leu Asp Ala

645 650 655

Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro Tyr Thr Pro Asp

660 665 670

Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser Ala Asp Gly Asn

675 680 685

His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala Glu Asn Glu Gln

690 695 700

Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly Val Thr Ser Asp

705 710 715 720

Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn Ala Gly Ser Phe

725 730 735

Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg Phe Gly Ala Ile

740 745 750

Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu Ile Leu Trp Arg

755 760 765

Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln Leu Gly Gln Ala

770 775 780

Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr Glu Gln Val Leu

785 790 795 800

Asp Trp Met Ser Ser Val Asn Leu Ser Leu Thr Tyr Leu Gln Gly Met

805 810 815

Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu Met Phe Asn Phe

820 825 830

Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala Ala Thr Lys Glu

835 840 845

Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg Ala Leu Ser Ala

850 855 860

Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val Thr Phe Trp Leu

865 870 875 880

Glu Lys Ile Thr Ile Gly Ser Asp Asn Pro Phe Thr Leu Ala Asn Tyr

885 890 895

Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn Ala Thr Leu Glu

900 905 910

Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr Gln Gln Leu Ser

915 920 925

Gln Leu Val Leu Ile Val Lys Trp Leu Ser Leu Thr Glu Gln Asp Leu

930 935 940

Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn Gly Ile Thr Asn

945 950 955 960

Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu Ser Arg Phe Lys

965 970 975

Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu Ala Met Arg Cys

980 985 990

Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu Asn Ala Gly Ser

995 1000 1005

Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr Gly Ala Gln Val

1010 1015 1020

Asn Thr Leu Leu Leu Gly Glu Asn Asn Trp Pro Lys Ser Phe Thr Ser

1025 1030 1035 1040

Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln Arg Leu Asn Val

1045 1050 1055

Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met Gln Ala Asp Pro

1060 1065 1070

Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala Gln Asn Leu Ser

1075 1080 1085

Ala Ala Ile Ser Asn Arg Gln

1090 1095

(2) INFORMATION FOR SEQ ID NO: 35

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 603 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35 (TcaAiii protein):

Pro Leu Ser Thr Ser Glu Leu Thr Ser Lys Leu Asn Ser Ile Asp Thr

1 5 10 15

Phe Cys Glu Lys Thr Arg Leu Ser Phe Asn Gln Leu Met Asp Leu Thr

20 25 30

Ala Gln Gln Ser Tyr Ser Gln Ser Ser Ile Asp Ala Lys Ala Ala Ser

35 40 45

Arg Tyr Val Arg Phe Gly Glu Thr Thr Pro Thr Arg Val Asn Val Tyr

50 55 60

Gly Ala Ala Tyr Leu Asn Ser Thr Leu Ala Asp Ala Ala Asp Gly Gln

65 70 75 80

Tyr Leu Trp Ile Gln Thr Asp Gly Lys Ser Leu Asn Phe Thr Asp Asp

85 90 95

Thr Val Val Ala Leu Ala Gly Arg Ala Glu Lys Leu Val Arg Leu Ser

100 105 110

Ser Gln Thr Gly Leu Ser Phe Glu Glu Leu Asp Trp Leu Ile Ala Asn

115 120 125

Ala Ser Arg Ser Val Pro Asp His His Asp Lys Ile Val Leu Asp Lys

130 135 140

Pro Val Leu Glu Ala Leu Ala Glu Tyr Val Ser Le Le Lys Gln Arg Tyr

145 150 155 160

Gly Leu Asp Ala Asn Thr Phe Ala Thr Phe Ile Ser Ala Val Asn Pro

165 170 175

Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe Arg Ser

180 185 190

Ala Asp Gly Asn His Val Ile Ala Leu Gly Thr Glu Val Lys Tyr Ala

195 200 205

Glu Asn Glu Gln Asp Glu Leu Ala Ala Ile Cys Cys Lys Ala Leu Gly

210 215 220

Val Thr Ser Asp Glu Leu Leu Arg Ile Gly Arg Tyr Cys Phe Gly Asn

225 230 235 240

Ala Gly Arg Phe Thr Leu Asp Glu Tyr Thr Ala Ser Gln Leu Tyr Arg

245 250 255

Phe Gly Ala Ile Pro Arg Leu Phe Gly Leu Thr Phe Ala Gln Ala Glu

260 265 270

Ile Leu Trp Arg Leu Met Glu Gly Gly Lys Asp Ile Leu Leu Gln Gln

275 280 285

Xxx Gly Gln Ala Lys Ser Leu Gln Pro Leu Ala Ile Leu Arg Arg Thr

290 295 300

Glu Gln Val Leu Asp Trp Met Ser Pro Val Asn Leu Ser Leu Thr Tyr

305 310 315 320

Leu Gln Gly Met Val Ser Thr Gln Trp Ser Gly Thr Ala Thr Ala Glu

325 330 335

Met Phe Asn Phe Leu Glu Asn Val Cys Asp Ser Val Asn Ser Gln Ala

340 345 350

Xxx Thr Lys Glu Thr Met Asp Ser Ala Leu Gln Gln Lys Val Leu Arg

355 360 365

Ala Leu Ser Ala Gly Phe Gly Ile Lys Ser Asn Val Met Gly Ile Val

370 375 380

Thr Phe Trp Leu Glu Lys Ile Thr Ile Gly Arg Asp Asn Pro Phe Thr

385 390 395 400

Leu Ala Asn Tyr Trp His Asp Ile Gln Thr Leu Phe Ser His Asp Asn

405 410 415

Ala Thr Leu Glu Ser Leu Gln Thr Asp Thr Ser Leu Val Ile Ala Thr

420 425 430

Gln Gln Leu Ser Gln Leu Val Leu Ile Val Lys Trp Val Ser Leu Thr

435 440 445

Glu Gln Asp Leu Gln Leu Leu Thr Thr Tyr Pro Glu Arg Leu Ile Asn

450 455 460

Gly Ile Thr Asn Val Pro Val Pro Asn Pro Glu Leu Leu Leu Thr Leu

465 470 475 480

Ser Arg Phe Lys Gln Trp Glu Thr Gln Val Thr Val Ser Arg Asp Glu

485 490 495

Ala Met Arg Cys Phe Asp Gln Leu Asn Ala Asn Asp Met Thr Thr Glu

500 505 510

Asn Ala Gly Ser Leu Ile Ala Thr Leu Tyr Glu Met Asp Lys Gly Thr

515 520 525

Gly Ala Gln Val Asn Thr Leu Leu Gly Glu Asn Asn Trp Pro Lys

530 535 540

Ser Phe Thr Ser Leu Trp Gln Leu Leu Thr Trp Leu Arg Val Gly Gln

545 550 555 560

Arg Leu Asn Val Gly Ser Thr Thr Leu Gly Asn Leu Leu Ser Met Met

565 570 575

Gln Ala Asp Pro Ala Ala Glu Ser Ser Ala Leu Leu Ala Ser Val Ala

580 585 590

Gln Asn Leu Ser Ala Ala Ile Ser Asn Arg Gln *

595 600

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2557 base pairs

(B) TYPE: nucleic acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36 (tcdA internal

fragment):

GAATTCGGCT TGCGTTTAAT ATTGATGATG TCTCGCTCTT CCGCCTGCTT AAAATTACCG 60

ACCATGATAA TAAAGATGGA AAAATTAAAA ATAACCTAAA GAATCTTTCC AATTTATATA 120

TTGGAAAATT ACTGGCAGAT ATTCATCAAT TAACCATTGA TGAACTGGAT TTATTACTGA 180

TTGCCGTAGG TGAAGGAAAA ACTAATTTAT CCGCTATCAG TGATAAGCAA TTGGCTACCC 240

TGATCAGAAA ACTCAATACT ATTACCAGCT GGCTACATAC ACAGAAGTGG AGTGTATTCC 300

AGCTATTTAT CATGACCTCC ACCAGCTATA ACAAAACGCT AACGCCTGAA ATTAAGAATT 360

TGCTGGATAC CGTCTACCAC GGTTTACAAG GTTTTGATAA AGACAAAGCA GATTTGCTAC 420

ATGTCATGGC GCCCTATATT GCGGCCACCT TGCAATTATC ATCGGAAAAT GTCGCCCACT 480

CGGTACTCCT TTGGGCAGAT AAGTTACAGC CCGGCGACGG CGCAATGACA GCAGAGGGAN 540

TCTGGGACTG GTTGAATACT AAGTATACGC CGGGTTCATC GGAAGCCGTA GAAACGCAGG 600

AACATATCGT TCAGTATTGT CAGGCTCTGG CACAATTGGA AATGGTTTAC CATTCCACCG 660

GCATCAACGA AAACGCCTTC CGTCTATTTG TGACAAAACC AGAGATGTTT GGCGCTGCAA 720

CTGGAGCAGC GCCCGCGCAT GATGCCCTTT CACTGATTAT GCTGACACGT TTTGCGGATT 780

GGGTGAACGC ACTAGGCGAA AAAGCGTCCT CGGTGCTAGC GGCATTTGAA GCTAACTCGT 840

TAACGGCAGA ACAACTGGCT GATGCCATGA ATCTTGATGC TAATTTGCTG TTGCAAGCCA 900

GTATTCAAGC ACAAAATCAT CAACATCTTC CCCCAGTAAC TCCAGAAAAT GCGTTCTCCT 960

GTTGGACATC TATCAATACT ATCCTGCAAT GGGTTAATGT CGCACAACAA TTGAAATGTC 1020

GCCCCACAGG GCGTTTCCGC TTTGGTCGGG CTGGATTATA TTCAATCAAT GAAAGAGACA 1080

CCGACCTATG CCCAGTGGGA AAACGCGGCA GGCGTATTAA CCGCCGGGTT GAATTCAACA 1140

ACAGGCTAAT ACATTACAAC GCTTTTCTGG ATGAATCTCG CAGTGCCGCA TTAAGCACCT 1200

ACTATATCCG TCAAGTCGCC AAGGCAGCGG CGGCTATTAA AAGCCGTGAT GACTTGTATC 1260

AATACTTACT GATTGATAAT CAGGTTTCTG CGGCAATAAA AACCACCCGG ATCGCCGAAG 1320

CCATTGCCAG TATTCAACTG TACGTCAACC GGGCATTGGA AAATGTGGAA GAAAATGCCA 1380

ATTCGGGGGT TATCAGCCGC CAATTCTTTA TCGACTGGGA CAAATACAAT AAACGCTACA 1440

GCACTTGGGC GGGTGTTTCT CAATTAGTTT ACTACCCGGA AAACTATATT GATCCGACCA 1500

TGCGTATCGG ACAAACCAAA ATGATGGACG CATTACTGCA ATCCGTCAGC CAAAGCCAAT 1560

TAAACGCCGA TACCGTCGAA GATGCCTTTA TGTCTTATCT GACATCGTTT GAACAAGTGG 1620

CTAATCTTAA AGTTATTAGC GCATATCACG ATAATATTAA TAACGATCAA GGGCTGACCT 1680

ATTTTATCGG ACTCAGTGAA ACTGATGCCG GTGAATATTA TTGGCGCAGT GTCGATCACA 1740

GTAAATTCAA CGACGGTAAA TTCGCGGCTA ATGCCTGGAG TGAATGGCAT AAAATTGATT 1800

GTCCAATTAA CCCTTATAAA AGCACTATCC GTCCAGTGAT ATATAAATCC CGCCTGTATC 1860

TGCTCTGGTT GGAACAAAAG GAGATCACCA AACAGACAGG AAATAGTAAA GATGGCTATC 1920

AAACTGAAAC GGATTATCGT TATGAACTAA AATTGGCGCA TATCCGCTAT GATGGCACTT 1980

GGAATACGCC AATCACCTTT GATGTCAATA AAAAAATATC CGAGCTAAAA CTGGAAAAAA 2040

ATAGAGCGCC CGGACTCTAT TGTGCCGGTT ATCAAGGTGA AGATACGTTG CTGGTGATGT 2100

TTTATAACCA ACAAGACACA CTAGATAGTT ATAAAAACGC TTCAATGCAA GGACTATATA 2160

TCTTTGCTGA TATGGCATCC AAAGATATGA CCCCAGAACA GAGCAATGTT TATCGGGATA 2220

ATAGCTATCA ACAATTTGAT ACCAATAATG TCAGAAGAGT GAATAACCGC TATGCAGAGG 2280

ATTATGAGAT TCCTTCTTCG GTAAGTAGCC GTAAAGACTA TGGTTGGGGA GATTATTACC 2340

TCAGCATGGT ATATAACGGA GATATTCCAA CTATCAATTA CAAAGCCGCA TCAAGTGATT 2400

TAAAAATTTA TATTTCACCA AAATTAAGAA TTATTCATAA TGGATATGAA GGACAGAAGC 2460

GCAATCAATG CAATTTGATG AATAAATATG GCAAACTAGG TGATAAATTT ATTGTGTATA 2520

CCAGCCTGGG CGTTAATCCG AATAATAAGC CGAATTC 2557

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 845 amino acids

(B) TYPE: amino acids

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (partial)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37 (TcdA internal

peptide):

Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile Thr

1 5 10 15

Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn Leu

20 25 30

Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu Thr

35 40 45

Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys Thr

50 55 60

Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg Lys

65 70 75 80

Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val Phe

85 90 95

Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr Pro

100 105 110

Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly Phe

115 120 125

Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile Ala

130 135 140

Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu Leu

145 150 155 160

Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu Gly

165 170 175

Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu Ala

180 185 190

Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala Gln

195 200 205

Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe Arg

210 215 220

Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala Ala

225 230 235 240

Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala Asp

245 250 255

Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala Phe

260 265 270

Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn Leu

275 280 285

Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His Gln

290 295 300

His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr Ser

305 310 315 320

Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Lys Cys

325 330 335

Arg Pro Thr Gly Arg Phe Arg Phe Gly Arg Ala Gly Leu Tyr Ser Ile

340 345 350

Asn Glu Arg Asp Thr Asp Leu Cys Pro Val Gly Lys Arg Gly Arg Arg

355 360 365

Ile Asn Arg Arg Val Glu Phe Asn Asn Arg Leu Ile His Tyr Asn Ala

370 375 380

Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg

385 390 395 400

Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr

405 410 415

Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr

420 425 430

Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala

435 440 445

Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln

450 455 460

Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala

465 470 475 480

Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr

485 490 495

Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val

500 505 510

Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser

515 520 525

Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala

530 535 540

Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly

545 550 555 560

Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His

565 570 575

Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp

580 585 590

His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro

595 600 605

Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu

610 615 620

Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr

625 630 635 640

Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr

645 650 655

Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu

660 665 670

Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln

675 680 685

Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu

690 695 700

Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp

705 710 715 720

Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp

725 730 735

Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn

740 745 750

Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys

755 760 765

Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp

770 775 780

Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr

785 790 795 800

Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys

805 810 815

Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys

820 825 830

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn

835 840 845

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38 (TcdAii- pk71

internal peptide):

Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly

1 5 10 15

Lys

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39 (TcdAii- pK44

internal peptide):

Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala

1 5 10 15

Ile Ser Pro Ala Lys

20

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40 (TcbAiii N-terminus):

Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln

1 5 10

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41 (TcdAiii N-terminus):

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln

1 5 10

(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42 (TcdA-pk57 internal

peptide):

Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr

1 5 10 15

Ala Gly Leu Glu

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43 (TcdAiii-pK20

internal peptide):

Ile Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

Asp Asp Ser Gly Asp Asp Asp Lys Val Thr Asn Thr Asp Ile His Arg

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

Asp Val Xaa Gly Ser Glu Lys Ala Asn Glu Lys Leu Lys

1 5 10

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7551 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46 (tcdA):

ATG AAC GAG TCT GTA AAA GAG ATA CCT GAT GTA TTA AAA AGC CAG TGT 48

Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys

1 5 10 15

GGT TTT AAT TGT CTG ACA GAT ATT AGC CAC AGC TCT TTT AAT GAA TTT 96

Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe

20 25 30

CGC CAG CAA GTA TCT GAG CAC CTC TCC TGG TCC GAA ACA CAC GAC TTA 144

Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu

35 40 45

TAT CAT GAT GCA CAA CAG GCA CAA AAG GAT AAT CGC CTG TAT GAA GCG 192

Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala

50 55 60

CGT ATT CTC AAA CGC GCC AAT CCC CAA TTA CAA AAT GCG GTG CAT CTT 240

Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu

65 70 75 80

GCC ATT CTC GCT CCC AAT GCT GAA CTG ATA GGC TAT AAC AAT CAA TTT 288

Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe

85 90 95

AGC GGT AGA GCC AGT CAA TAT GTT GCG CCG GGT ACC GTT TCT TCC ATG 336

Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met

100 105 110

TTC TCC CCC GCC GCT TAT TTG ACT GAA CTT TAT CGT GAA GCA CGC AAT 384

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn

115 120 125

TTA CAC GCA AGT GAC TCC GTT TAT TAT CTG GAT ACC CGC CGC CCA GAT 432

Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp

130 135 140

CTC AAA TCA ATG GCG CTC AGT CAG CAA AAT ATG GAT ATA GAA TTA TCC 480

Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser

145 150 155 160

ACA CTC TCT TTG TCC AAT GAG CTG TTA TTG GAA AGC ATT AAA ACT GAA 528

Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu

165 170 175

TCT AAA CTG GAA AAC TAT ACT AAA GTG ATG GAA ATG CTC TCC ACT TTC 576

Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe

180 185 190

CGT CCT TCC GGC GCA ACG CCT TAT CAT GAT GCT TAT GAA AAT GTG CGT 624

Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg

195 200 205

GAA GTT ATC CAG CTA CAA GAT CCT GGA CTT GAG CAA CTC AAT GCA TCA 672

Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser

210 215 220

CCG GCA ATT GCC GGG TTG ATG CAT CAA GCC TCC CTA TTG GGT ATT AAC 720

Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn

225 230 235 240

GCT TCA ATC TCG CCT GAG CTA TTT AAT ATT CTG ACG GAG GAG ATT ACC 768

Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr

245 250 255

GAA GGT AAT GCT GAG GAA CTT TAT AAG AAA AAT TTT GGT AAT ATC GAA 816

Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu

260 265 270

CCG GCC TCA TTG GCT ATG CCG GAA TAC CTT AAA CGT TAT TAT AAT TTA 864

Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu

275 280 285

AGC GAT GAA GAA CTT AGT CAG TTT ATT GGT AAA GCC AGC AAT TTT GGT 912

Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly

290 295 300

CAA CAG GAA TAT AGT AAT AAC CAA CTT ATT ACT CCG GTA GTC AAC AGC 960

Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser

305 310 315 320

AGT GAT GGC ACG GTT AAG GTA TAT CGG ATC ACC CGC GAA TAT ACA ACC 1008

Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr

325 330 335

AAT GCT TAT CAA ATG GAT GTG GAG CTA TTT CCC TTC GGT GGT GAG AAT 1056

Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Glu Glu Asn

340 345 350

TAT CGG TTA GAT TAT AAA TTC AAA AAT TTT TAT AAT GCC TCT TAT TTA 1104

Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu

355 360 365

TCC ATC AAG TTA AAT GAT AAA AGA GAA CTT GTT CGA ACT GAA GGC GCT 1152

Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala

370 375 380

CCT CAA GTC AAT ATA GAA TAC TCC GCA AAT ATC ACA TTA AAT ACC GCT 1200

Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala

385 390 395 400

GAT ATC AGT CAA CCT TTT GAA ATT GGC CTG ACA CGA GTA CTT CCT TCC 1248

Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser

405 410 415

GGT TCT TGG GCA TAT GCC GCC GCA AAA TTT ACC GTT GAA GAG TAT AAC 1296

Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn

420 425 430

CAA TAC TCT TTT CTG CTA AAA CTT AAC AAG GCT ATT CGT CTA TCA CGT 1344

Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg

435 440 445

GCG ACA GAA TTG TCA CCC ACG ATT CTG GAA GGC ATT GTG CGC AGT GTT 1392

Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val

450 455 460

AAT CTA CAA CTG GAT ATC AAC ACA GAC GTA TTA GGT AAA GTT TTT CTG 1440

Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu

465 470 475 480

ACT AAA TAT TAT ATG CAG CGT TAT GCT ATT CAT GCT GAA ACT GCC CTG 1488

Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu

485 490 495

ATA CTA TGC AAC GCG CCT ATT TCA CAA CGT TCA TAT GAT AAT CAA CCT 1536

Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro

500 505 510

AGC CAA TTT GAT CGC CTG TTT AAT ACG CCA TTA CTG AAC GGA CAA TAT 1584

Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr

515 520 525

TTT TCT ACC GGC GAT GAG GAG ATT GAT TTA AAT TCA GGT AGC ACC GGC 1632

Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly

530 535 540

GAT TGG CGA AAA ACC ATA CTT AAG CGT GCA TTT AAT ATT GAT GAT GTC 1680

Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val

545 550 555 560

TCG CTC TTC CGC CTG CTT AAA ATT ACC GAC CAT GAT AAT AAA GAT GGA 1728

Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly

565 570 575

AAA ATT AAA AAT AAC CTA AAG AAT CTT TCC AAT TTA TAT ATT GGA AAA 1776

Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys

580 585 590

TTA CTG GCA GAT ATT CAT CAA TTA ACC ATT GAT GAA CTG GAT TTA TTA 1824

Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu

595 600 605

CTG ATT GCC GTA GGT GAA GGA AAA ACT AAT TTA TCC GCT ATC AGT GAT 1872

Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp

610 615 620

AAG CAA TTG GCT ACC CTG ATC AGA AAA CTC AAT ACT ATT ACC AGC TGG 1920

Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp

625 630 635 640

CTA CAT ACA CAG AAG TGG AGT GTA TTC CAG CTA TTT ATC ATG ACC TCC 1968

Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser

645 650 655

ACC AGC TAT AAC AAA ACG CTA ACG CCT GAA ATT AAG AAT TTG CTG GAT 2016

Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp

660 665 670

ACC GTC TAC CAC GGT TTA CAA GGT TTT GAT AAA GAC AAA GCA GAT TTG 2064

Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu

675 680 685

CTA CAT GTC ATG GCG CCC TAT ATT GCG GCC ACC TTG CAA TTA TCA TCG 2112

Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser

690 695 700

GAA AAT GTC GCC CAC TCG GTA CTC CTT TGG GCA GAT AAG TTA CAG CCC 2160

Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro

705 710 715 720

GGC GAC GGC GCA ATG ACA GCA GAA AAA TTC TGG GAC TGG TTG AAT ACT 2208

Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr

725 730 735

AAG TAT ACG CCG GGT TCA TCG GAA GCC GTA GAA ACG CAG GAA CAT ATC 2256

Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile

740 745 750

GTT CAG TAT TGT CAG GCT CTG GCA CAA TTG GAA ATG GTT TAC CAT TCC 2304

Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser

755 760 765

ACC GGC ATC AAC GAA AAC GCC TTC CGT CTA TTT GTG ACA AAA CCA GAG 2352

Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu

770 775 780

ATG TTT GGC GCT GCA ACT GGA GCA GCG CCC GCG CAT GAT GCC CTT TCA 2400

Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser

785 790 795 800

CTG ATT ATG CTG ACA CGT TTT GCG GAT TGG GTG AAC GCA CTA GGC GAA 2448

Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu

805 810 815

AAA GCG TCC TCG GTG CTA GCG GCA TTT GAA GCT AAC TCG TTA ACG GCA 2496

Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala

820 825 830

GAA CAA CTG GCT GAT GCC ATG AAT CTT GAT GCT AAT TTG CTG TTG CAA 2544

Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln

835 840 845

GCC AGT ATT CAA GCA CAA AAT CAT CAA CAT CTT CCC CCA GTA ACT CCA 2592

Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro

850 855 860

GAA AAT GCG TTC TCC TGT TGG ACA TCT ATC AAT ACT ATC CTG CAA TGG 2640

Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp

865 870 875 880

GTT AAT GTC GCA CAA CAA TTG AAT GTC GCC CCA CAG GGC GTT TCC GCT 2688

Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala

885 890 895

TTG GTC GGG CTG GAT TAT ATT CAA TCA ATG AAA GAG ACA CCG ACC TAT 2736

Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr

900 905 910

GCC CAG TGG GAA AAC GCG GCA GGC GTA TTA ACC GCC GGG TTG AAT TCA 2784

Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser

915 920 925

CAA CAG GCT AAT ACA TTA CAC GCT TTT CTG GAT GAA TCT CGC AGT GCC 2832

Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala

930 935 940

GCA TTA AGC ACC TAC TAT ATC CGT CAA GTC GCC AAG GCA GCG GCG GCT 2880

Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala

945 950 955 960

ATT AAA AGC CGT GAT GAC TTG TAT CAA TAC TTA CTG ATT GAT AAT CAG 2928

Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln

965 970 975

GTT TCT GCG GCA ATA AAA ACC ACC CGG ATC GCC GAA GCC ATT GCC AGT 2976

Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser

980 985 990

ATT CAA CTG TAC GTC AAC CGG GCA TTG GAA AAT GTG GAA GAA AAT GCC 3024

Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala

995 1000 1005

AAT TCG GGG GTT ATC AGC CGC CAA TTC TTT ATC GAC TGG GAC AAA TAC 3072

Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr

1010 1015 1020

AAT AAA CGC TAC AGC ACT TGG GCG GGT GTT TCT CAA TTA GTT TAC TAC 3120

Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr

1025 1030 1035 1040

CCG GAA AAC TAT ATT GAT CCG ACC ATG CGT ATC GGA CAA ACC AAA ATG 3168

Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met

1045 1050 1055

ATG GAC GCA TTA CTG CAA TCC GTC AGC CAA AGC CAA TTA AAC GCC GAT 3216

Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp

1060 1065 1070

ACC GTC GAA GAT GCC TTT ATG TCT TAT CTG ACA TCG TTT GAA CAA GTG 3264

Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val

1075 1080 1085

GCT AAT CTT AAA GTT ATT AGC GCA TAT CAC GAT AAT ATT AAT AAC GAT 3312

Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp

1090 1095 1100

CAA GGG CTG ACC TAT TTT ATC GGA CTC AGT GAA ACT GAT GCC GGT GAA 3360

Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu

1105 1110 1115 1120

TAT TAT TGG CGC AGT GTC GAT CAC AGT AAA TTC AAC GAC GGT AAA TTC 3408

Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe

1125 1130 1135

GCG GCT AAT GCC TGG AGT GAA TGG CAT AAA ATT GAT TGT CCA ATT AAC 3456

Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn

1140 1145 1150

CCT TAT AAA AGC ACT ATC CGT CCA GTG ATA TAT AAA TCC CGC CTG TAT 3504

Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr

1155 1160 1165

CTG CTC TGG TTG GAA CAA AAG GAG ATC ACC AAA CAG ACA GGA AAT AGT 3552

Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser

1170 1175 1180

AAA GAT GGC TAT CAA ACT GAA ACG GAT TAT CGT TAT GAA CTA AAA TTG 3600

Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu

1185 1190 1195 1200

GCG CAT ATC CGC TAT GAT GGC ACT TGG AAT ACG CCA ATC ACC TTT GAT 3648

Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp

1205 1210 1215

GTC AAT AAA AAA ATA TCC GAG CTA AAA CTG GAA AAA AAT AGA GCG CCC 3696

Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro

1220 1225 1230

GGA CTC TAT TGT GCC GGT TAT CAA GGT GAA GAT ACG TTG CTG GTG ATG 3744

Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met

1235 1240 1245

TTT TAT AAC CAA CAA GAC ACA CTA GAT AGT TAT AAA AAC GCT TCA ATG 3792

Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met

1250 1255 1260

CAA GGA CTA TAT ATC TTT GCT GAT ATG GCA TCC AAA GAT ATG ACC CCA 3840

Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro

1265 1270 1275 1280

GAA CAG AGC AAT GTT TAT CGG GAT AAT AGC TAT CAA CAA TTT GAT ACC 3888

Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr

1285 1290 1295

AAT AAT GTC AGA AGA GTG AAT AAC CGC TAT GCA GAG GAT TAT GAG ATT 3936

Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile

1300 1305 1310

CCT TCC TCG GTA AGT AGC CGT AAA GAC TAT GGT TGG GGA GAT TAT TAC 3984

Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr

1315 1320 1325

CTC AGC ATG GTA TAT AAC GGA GAT ATT CCA ACT ATC AAT TAC AAA GCC 4032

Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala

1330 1335 1340

GCA TCA AGT GAT TTA AAA ATC TAT ATC TCA CCA AAA TTA AGA ATT ATT 4080

Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile

1345 1350 1355 1360

CAT AAT GGA TAT GAA GGA CAG AAG CGC AAT CAA TGC AAT CTG ATG AAT 4 128

His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn

1365 1370 1375

AAA TAT GGC AAA CTA GGT GAT AAA TTT ATT GTT TAT ACT AGC TTG GGG 4176

Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly

1380 1385 1390

GTC AAT CCA AAT AAC TCG TCA AAT AAG CTC ATG TTT TAC CCC GTC TAT 4224

Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr

1395 1400 1405

CAA TAT AGC GGA AAC ACC AGT GGA CTC AAT CAA GGG AGA CTA CTA TTC 4272

Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe

1410 1415 1420

CAC CGT GAC ACC ACT TAT CCA TCT AAA GTA GAA GCT TGG ATT CCT GGA 4320

His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly

1425 1430 1435 1440

GCA AAA CGT TCT CTA ACC AAC CAA AAT GCC GCC ATT GGT GAT GAT TAT 4368

Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr

1445 1450 1455

GCT ACA GAC TCT CTG AAT AAA CCG GAT GAT CTT AAG CAA TAT ATC TTT 4416

Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe

1460 1465 1470

ATG ACT GAC AGT AAA GGG ACT GCT ACT GAT GTC TCA GGC CCA GTA GAG 4464

Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu

1475 1480 1485

ATT AAT ACT GCA ATT TCT CCA GCA AAA GTT CAG ATA ATA GTC AAA GCG 4512

Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala

1490 1495 1500

GGT GGC AAG GAG CAA ACT TTT ACC GCA GAT AAA GAT GTC TCC ATT CAG 4560

Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln

1505 1510 1515 1520

CCA TCA CCT AGC TTT GAT GAA ATG AAT TAT CAA TTT AAT GCC CTT GAA 4608

Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu

1525 1530 1535

ATA GAC GGT TCT GGT CTG AAT TTT ATT AAC AAC TCA GCC AGT ATT GAT 4656

Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp

1540 1545 1550

GTT ACT TTT ACC GCA TTT GCG GAG GAT GGC CGC AAA CTG GGT TAT GAA 4704

Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu

1555 1560 1565

AGT TTC AGT ATT CCT GTT ACC CTC AAG GTA AGT ACC GAT AAT GCC CTG 4752

Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu

1570 1575 1580

ACC CTG CAC CAT AAT GAA AAT GGT GCG CAA TAT ATG CAA TGG CAA TCC 4800

Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser

1585 1590 1595 1600

TAT CGT ACC CGC CTG AAT ACT CTA TTT GCC CGC CAG TTG GTT GCA CGC 4848

Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg

1605 1610 1615

GCC ACC ACC GGA ATC GAT ACA ATT CTG AGT ATG GAA ACT CAG AAT ATT 4896

Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile

1620 1625 1630

CAG GAA CCG CAG TTA GGC AAA GGT TTC TAT GCT ACG TTC GTG ATA CCT 4944

Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro

1635 1640 1645

CCC TAT AAC CTA TCA ACT CAT GGT GAT GAA CGT TGG TTT AAG CTT TAT 4992

Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr

1650 1655 1660

ATC AAA CAT GTT GTT GAT AAT AAT TCA CAT ATT ATC TAT TCA GGC CAG 5040

Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln

1665 1670 1675 1680

CTA ACA GAT ACA AAT ATA AAC ATC ACA TTA TTT ATT CCT CTT GAT GAT 5088

Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp

1685 1690 1695

GTC CCA TTG AAT CAA GAT TAT CAC GCC AAG GTT TAT ATG ACC TTC AAG 5136

Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys

1700 1705 1710

AAA TCA CCA TCA GAT GGT ACC TGG TGG GGC CCT CAC TTT GTT AGA GAT 5184

Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp

1715 1720 1725

GAT AAA GGA ATA GTA ACA ATA AAC CCT AAA TCC ATT TTG ACC CAT TTT 5232

Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe

1730 1735 1740

GAG AGC GTC AAT GTC CTG AAT AAT ATT AGT AGC GAA CCA ATG GAT TTC 5280

Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe

1745 1750 1755 1760

AGC GGC GCT AAC AGC CTC TAT TTC TGG GAA CTG TTC TAC TAT ACC CCG 5328

Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro

1765 1770 1775

ATG CTG GTT GCT CAA CGT TTG CTG CAT GAA CAG AAC TTC GAT GAA GCC 5376

Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala

1780 1785 1790

AAC CGT TGG CTG AAA TAT GTC TGG AGT CCA TCC GGT TAT ATT GTC CAC 5424

Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His

1795 1800 1805

GGC CAG ATT CAG AAC TAC CAG TGG AAC GTC CGC CCG TTA CTG GAA GAC 5472

Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp

1810 1815 1820

ACC AGT TGG AAC AGT GAT CCT TTG GAT TCC GTC GAT CCT GAC GCG GTA 5520

Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val

1825 1830 1835 1840

GCA CAG CAC GAT CCA ATG CAC TAC AAA GTT TCA ACT TTT ATG CGT ACC 5568

Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr

1845 1850 1855

TTG GAT CTA TTG ATA GCA CGC GGC GAC CAT GCT TAT CGC CAA CTG GAA 5616

Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu

1860 1865 1870

CGA GAT ACA CTC AAC GAA GCG AAG ATG TGG TAT ATG CAA GCG CTG CAT 5664

Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His

1875 1880 1885

CTA TTA GGT GAC AAA CCT TAT CTA CCG CTG AGT ACG ACA TGG AGT GAT 5712

Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp

1890 1895 1900

CCA CGA CTA GAC AGA GCC GCG GAT ATC ACT ACC CAA AAT GCT CAC GAC 5760

Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp

1905 1910 1915 1920

AGC GCA ATA GTC GCT CTG CGG CAG AAT ATA CCT ACA CCG GCA CCT TTA 5808

Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu

1925 1930 1935

TCA TTG CGC AGC GCT AAT ACC CTG ACT GAT CTC TTC CTG CCG CAA ATC 5856

Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile

1940 1945 1950

AAT GAA GTG ATG ATG AAT TAC TGG CAG ACA TTA GCT CAG AGA GTA TAC 5904

Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr

1955 1960 1965

AAT CTG CGT CAT AAC CTC TCT ATC GAC GGC CAG CCG TTA TAT CTG CCA 5952

Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro

1970 1975 1980

ATC TAT GCC ACA CCG GCC GAT CCG AAA GCG TTA CTC AGC GCC GCC GTT 6000

Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val

1985 1990 1995 2000

GCC ACT TCT CAA GGT GGA GGC AAG CTA CCG GAA TCA TTT ATG TCC CTG 6048

Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu

2005 2010 2015

TGG CGT TTC CCG CAC ATG CTG GAA AAT GCG CGC GGC ATG GTT AGC CAG 6096

Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln

2020 2025 2030

CTC ACC CAG TTC GGC TCC ACG TTA CAA AAT ATT ATC GAA CGT CAG GAC 6144

Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Glu Arg Gln Asp

2035 2040 2045

GCG GAA GCG CTC AAT GCG TTA TTA CAA AAT CAG GCC GCC GAG CTG ATA 6192

Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile

2050 2055 2060

TTG ACT AAC CTG AGC ATT CAG GAC AAA ACC ATT GAA GAA TTG GAT GCC 6240

Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala

2065 2070 2075 2080

GAG AAA ACG GTG TTG GAA AAA TCC AAA GCG GGA GCA CAA TCG CGC TTT 6288

Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe

2085 2090 2095

GAT AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC 6336

Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn

2100 2105 2110

CAA GCC ATG ACG CTA CGA GCG TCC GCC GCC GGG CTT ACC ACG GCA GTT 6384

Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val

2115 2120 2125

CAG GCA TCC CGT CTG GCC GGT GCG GCG GCT GAT CTG GTG CCT AAC ATC 6432

Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile

2130 2135 2140

TTC GGC TTT GCC GGT GGC GGC AGC CGT TGG GGG GCT ATC GCT GAG GCG 6 480

Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala

2145 2150 2155 2160

ACA GGT TAT GTG ATG GAA TTC TCC GCG AAT GTT ATG AAC ACC GAA GCG 6528

Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala

2165 2170 2175

GAT AAA ATT AGC CAA TCT GAA ACC TAC CGT CGT CGC CGT CAG GAG TGG 6576

Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp

2180 2185 2190

GAG ATC CAG CGG AAT AAT GCC GAA GCG GAA TTG AAG CAA ATC GAT GCT 6624

Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala

2195 2200 2205

CAG CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA 6672

Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys

2210 2215 2220

ACC AGT CTG AAA ACC CAA CAA GAA CAG ACC CAA TCT CAA TTG GCC TTC 6720

Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe

2225 2230 2235 2240

CTG CAA CGT AAG TTC AGC AAT CAG GCG TTA TAC AAC TGG CTG CGT GGT 6768

Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly

2245 2250 2255

CGA CTG GCG GCG ATT TAC TTC CAG TTC TAC GAT TTG GCC GTC GCG CGT 6816

Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg

2260 2265 2270

TGC CTG ATG GCA GAA CAA GCT TAC CGT TGG GAA CTC AAT GAT GAC TCT 6864

Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser

2275 2280 2285

GCC CGC TTC ATT AAA CCG GGC GCC TGG CAG GGA ACC TAT GCC GGT CTG 6912

Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu

2290 2295 2300

CTT GCA GGT GAA ACC TTG ATG CTG AGT CTG GCA CAA ATG GAA GAC GCT 6960

Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala

2305 2310 2315 2320

CAT CTG AAA CGC GAT AAA CGC GCA TTA GAG GTT GAA CGC ACA GTA TCG 7008

His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser

2325 2330 2335

CTG GCC GAA GTT TAT GCA GGA TTA CCA AAA GAT AAC GGT CCA TTT TCC 7056

Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser

2340 2345 2350

CTG GCT CAG GAA ATT GAC AAG CTG GTG AGT CAA GGT TCA GGC AGT GCC 7104

Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala

2355 2360 2365

GGC AGT GGT AAT AAT AAT TTG GCG TTC GGC GCC GGC ACG GAC ACT AAA 7 152

Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys

2370 2375 2380

ACC TCT TTG CAG GCA TCA GTT TCA TTC GCT GAT TTG AAA ATT CGT GAA 7200

Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu

2385 2390 2395 2400

GAT TAC CCG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC 7248

Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser

2405 2410 2415

GTC ACT TTG CCC GCG CTA CTG GGA CCG TAT CAG GAT GTA CAG GCA ATA 7296

Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile

2420 2425 2430

TTG TCT TAC GGC GAT AAA GCC GGA TTA GCT AAC GGC TGT GAA GCG CTG 7344

Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu

2435 2440 2445

GCA GTT TCT CAC GGT ATG AAT GAC AGC GGC CAA TTC CAG CTC GAT TTC 7392

Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe

2450 2455 2460

AAC GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC 7440

Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly

2465 2470 2475 2480

ACG CTG ACA CTG AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA 7488

Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys

2485 2490 2495

CAA GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC 7536

Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg

2500 2505 2510

TAC ACC ATT AAA TAA 7551

Tyr Thr Ile Lys

2516

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2516 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47 (TcdA):

Features From To Description

Peptide 1 2516 TcdA proteins

Peptide 89 1937 TcdAii peptide

Fragment 89 100 TcdAii N-terminus (SEQ

ID NO: 13)

Fragment 284 299 (SEQ ID NO: 38)

Fragment 554 563 (SEQ ID NO: 17)

Fragment 1080 1092 (SEQ ID NO: 23;

12/13)

Fragment 1385 1400 (SEQ ID NO: 18)

Fragment 1478 1497 (SEQ ID NO: 39)

Fragment 1620 1642 (SEQ ID NO: 21;

19/23)

Fragment 1938 1948 (SEQ ID NO: 41)

Peptide 1938 2516 TcdAiii peptide

Fragment 2327 2345 (SEQ ID NO: 42)

Fragment 2398 2408 (SEQ ID NO: 43)

Met Asn Glu Ser Val Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys

1 5 10 15

Gly Phe Asn Cys Leu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe

20 25 30

Arg Gln Gln Val Ser Glu His Leu Ser Trp Ser Glu Thr His Asp Leu

35 40 45

Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn Arg Leu Tyr Glu Ala

50 55 60

Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn Ala Val His Leu

65 70 75 80

Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn Gln Phe

85 90 95

Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser Ser Met

100 105 110

Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala Arg Asn

115 120 125

Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg Pro Asp

130 135 140

Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu Leu Ser

145 150 155 160

Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys Thr Glu

165 170 175

Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met Leu Ser Thr Phe

180 185 190

Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr Glu Asn Val Arg

195 200 205

Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln Leu Asn Ala Ser

210 215 220

Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu Leu Gly Ile Asn

225 230 235 240

Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr Glu Glu Ile Thr

245 250 255

Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn Phe Gly Asn Ile Glu

260 265 270

Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu

275 280 285

Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys Ala Ser Asn Phe Gly

290 295 300

Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr Pro Val Val Asn Ser

305 310 315 320

Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr

325 330 335

Asn Ala Tyr Gln Met Asp Val Glu Leu Phe Pro Phe Gly Glu Glu Asn

340 345 350

Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu

355 360 365

Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala

370 375 380

Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala

385 390 395 400

Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser

405 410 415

Gly Ser Trp Ala Tyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn

420 425 430

Gln Tyr Ser Phe Leu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg

435 440 445

Ala Thr Glu Leu Ser Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val

450 455 460

Asn Leu Gln Leu Asp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu

465 470 475 480

Thr Lys Tyr Tyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu

485 490 495

Ile Leu Cys Asn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro

500 505 510

Ser Gln Phe Asp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr

515 520 525

Phe Ser Thr Gly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly

530 535 540

Asp Trp Arg Lys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val

545 550 555 560

Ser Leu Phe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly

565 570 575

Lys Ile Lys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys

580 585 590

Leu Leu Ala Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu

595 600 605

Leu Ile Ala Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp

610 615 620

Lys Gln Leu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp

625 630 635 640

Leu His Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser

645 650 655

Thr Ser Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp

660 665 670

Thr Val Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu

675 680 685

Leu His Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser

690 695 700

Glu Asn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro

705 710 715 720

Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr

725 730 735

Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile

740 745 750

Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser

755 760 765

Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu

770 775 780

Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser

785 790 795 800

Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu

805 810 815

Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala

820 825 830

Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln

835 840 845

Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro

850 855 860

Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp

865 870 875 880

Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala

885 890 895

Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr

900 905 910

Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser

915 920 925

Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala

930 935 940

Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala

945 950 955 960

Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp Asn Gln

965 970 975

Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile Ala Ser

980 985 990

Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu Asn Ala

995 1000 1005

Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp Lys Tyr

1010 1015 1020

Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val Tyr Tyr

1025 1030 1035 1040

Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr Lys Met

1045 1050 1055

Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn Ala Asp

1060 1065 1070

Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser Phe Glu Gln Val

1075 1080 1085

Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp Asn Ile Asn Asn Asp

1090 1095 1100

Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser Glu Thr Asp Ala Gly Glu

1105 1110 1115 1120

Tyr Tyr Trp Arg Ser Val Asp His Ser Lys Phe Asn Asp Gly Lys Phe

1125 1130 1135

Ala Ala Asn Ala Trp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn

1140 1145 1150

Pro Tyr Lys Ser Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr

1155 1160 1165

Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser

1170 1175 1180

Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu

1185 1190 1195 1200

Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp

1205 1210 1215

Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg Ala Pro

1220 1225 1230

Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met

1235 1240 1245

Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala Ser Met

1250 1255 1260

Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met Thr Pro

1265 1270 1275 1280

Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe Asp Thr

1285 1290 1295

Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile

1300 1305 1310

Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr Gly Trp Gly Asp Tyr Tyr

1315 1320 1325

Leu Ser Met Val Tyr Asn Gly Asp Ile Pro Thr Ile Asn Tyr Lys Ala

1330 1335 1340

Ala Ser Ser Asp Leu Lys Ile Tyr Ile Ser Pro Lys Leu Arg Ile Ile

1345 1350 1355 1360

His Asn Gly Tyr Glu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn

1365 1370 1375

Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly

1380 1385 1390

Val Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr

1395 1400 1405

Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe

1410 1415 1420

His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly

1425 1430 1435 1440

Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp Asp Tyr

1445 1450 1455

Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr Ile Phe

1460 1465 1470

Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro Val Glu

1475 1480 1485

Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val Lys Ala

1490 1495 1500

Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp Val Ser Ile Gln

1505 1510 1515 1520

Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln Phe Asn Ala Leu Glu

1525 1530 1535

Ile Asp Gly Ser Gly Leu Asn Phe Ile Asn Asn Ser Ala Ser Ile Asp

1540 1545 1550

Val Thr Phe Thr Ala Phe Ala Glu Asp Gly Arg Lys Leu Gly Tyr Glu

1555 1560 1565

Ser Phe Ser Ile Pro Val Thr Leu Lys Val Ser Thr Asp Asn Ala Leu

1570 1575 1580

Thr Leu His His Asn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser

1585 1590 1595 1600

Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg

1605 1610 1615

Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile

1620 1625 1630

Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro

1635 1640 1645

Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys Leu Tyr

1650 1655 1660

Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser Gly Gln

1665 1670 1675 1680

Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu Asp Asp

1685 1690 1695

Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr Phe Lys

1700 1705 1710

Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His Phe Val Arg Asp

1715 1720 1725

Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser Ile Leu Thr His Phe

1730 1735 1740

Glu Ser Val Asn Val Leu Asn Asn Ile Ser Ser Glu Pro Met Asp Phe

1745 1750 1755 1760

Ser Gly Ala Asn Ser Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro

1765 1770 1775

Met Leu Val Ala Gln Arg Leu Leu His Glu Gln Asn Phe Asp Glu Ala

1780 1785 1790

Asn Arg Trp Leu Lys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His

1795 1800 1805

Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp

1810 1815 1820

Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val

1825 1830 1835 1840

Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr

1845 1850 1855

Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln Leu Glu

1860 1865 1870

Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala Leu His

1875 1880 1885

Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp Ser Asp

1890 1895 1900

Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala His Asp

1905 1910 1915 1920

Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala Pro Leu

1925 1930 1935

Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile

1940 1945 1950

Asn Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr

1955 1960 1965

Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro

1970 1975 1980

Ile Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val

1985 1990 1995 2000

Ala Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu

2005 2010 2015

Trp Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln

2020 2025 2030

Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Glu Arg Gln Asp

2035 2040 2045

Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile

2050 2055 2060

Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala

2065 2070 2075 2080

Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe

2085 2090 2095

Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn

2100 2105 2110

Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val

2115 2120 2125

Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile

2130 2135 2140

Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala

2145 2150 2155 2160

Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala

2165 2170 2175

Asp Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp

2180 2185 2190

Glu Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala

2195 2200 2205

Gln Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys

2210 2215 2220

Thr Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe

2225 2230 2235 2240

Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly

2245 2250 2255

Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg

2260 2265 2270

Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser

2275 2280 2285

Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu

2290 2295 2300

Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala

2305 2310 2315 2320

His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser

2325 2330 2335

Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser

2340 2345 2350

Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala

2355 2360 2365

Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys

2370 2375 2380

Thr Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu

2385 2390 2395 2400

Asp Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser

2405 2410 2415

Val Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile

2420 2425 2430

Leu Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu

2435 2440 2445

Ala Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe

2450 2455 2460

Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly

2465 2470 2475 2480

Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys

2485 2490 2495

Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg

2500 2505 2510

Tyr Thr Ile Lys

2516

(2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5547 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48 (tcdAii coding

region):

CTG ATA GGC TAT AAC AAT CAA TTT AGC GGT AGA GCC AGT CAA TAT GTT 48

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val

1 5 10 15

GCG CCG GGT ACC GTT TCT TCC ATG TTC TCC CCC GCC GCT TAT TTG ACT 96

Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr

20 25 30

GAA CTT TAT CGT GAA GCA CGC AAT TTA CAC GCA AGT GAC TCC GTT TAT 144

Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr

35 40 45

TAT CTG GAT ACC CGC CGC CCA GAT CTC AAA TCA ATG GCG CTC AGT CAG 192

Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln

50 55 60

CAA AAT ATG GAT ATA GAA TTA TCC ACA CTC TCT TTG TCC AAT GAG CTG 240

Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu

65 70 75 80

TTA TTG GAA AGC ATT AAA ACT GAA TCT AAA CTG GAA AAC TAT ACT AAA 288

Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys

85 90 95

GTG ATG GAA ATG CTC TCC ACT TTC CGT CCT TCC GGC GCA ACG CCT TAT 336

Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr

100 105 110

CAT GAT GCT TAT GAA AAT GTG CGT GAA GTT ATC CAG CTA CAA GAT CCT 384

His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro

115 120 125

GGA CTT GAG CAA CTC AAT GCA TCA CCG GCA ATT GCC GGG TTG ATG CAT 432

Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His

130 135 140

CAA GCC TCC CTA TTG GGT ATT AAC GCT TCA ATC TCG CCT GAG CTA TTT 480

Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe

145 150 155 160

AAT ATT CTG ACG GAG GAG ATT ACC GAA GGT AAT GCT GAG GAA CTT TAT 528

Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr

165 170 175

AAG AAA AAT TTT GGT AAT ATC GAA CCG GCC TCA TTG GCT ATG CCG GAA 576

Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu

180 185 190

TAC CTT AAA CGT TAT TAT AAT TTA AGC GAT GAA GAA CTT AGT CAG TTT 624

Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe

195 200 205

ATT GGT AAA GCC AGC AAT TTT GGT CAA CAG GAA TAT AGT AAT AAC CAA 672

Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln

210 215 220

CTT ATT ACT CCG GTA GTC AAC AGC AGT GAT GGC ACG GTT AAG GTA TAT 720

Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr

225 230 235 240

CGG ATC ACC CGC GAA TAT ACA ACC AAT GCT TAT CAA ATG GAT GTG GAG 768

Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu

245 250 255

CTA TTT CCC TTC GGT GGT GAG AAT TAT CGG TTA GAT TAT AAA TTC AAA 816

Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys

260 265 270

AAT TTT TAT AAT GCC TCT TAT TTA TCC ATC AAG TTA AAT GAT AAA AGA 864

Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg

275 280 285

GAA CTT GTT CGA ACT GAA GGC GCT CCT CAA GTC AAT ATA GAA TAC TCC 912

Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser

290 295 300

GCA AAT ATC ACA TTA AAT ACC GCT GAT ATC AGT CAA CCT TTT GAA ATT 960

Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile

305 310 315 320

GGC CTG ACA CGA GTA CTT CCT TCC GGT TCT TGG GCA TAT GCC GCC GCA 1008

Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala

325 330 335

AAA TTT ACC GTT GAA GAG TAT AAC CAA TAC TCT TTT CTG CTA AAA CTT 1056

Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu

340 345 350

AAC AAG GCT ATT CGT CTA TCA CGT GCG ACA GAA TTG TCA CCC ACG ATT 1104

Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile

355 360 365

CTG GAA GGC ATT GTG CGC AGT GTT AAT CTA CAA CTG GAT ATC AAC ACA 1152

Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr

370 375 380

GAC GTA TTA GGT AAA GTT TTT CTG ACT AAA TAT TAT ATG CAG CGT TAT 1200

Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr

385 390 395 400

GCT ATT CAT GCT GAA ACT GCC CTG ATA CTA TGC AAC GCG CCT ATT TCA 1248

Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser

405 410 415

CAA CGT TCA TAT GAT AAT CAA CCT AGC CAA TTT GAT CGC CTG TTT AAT 1296

Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn

420 425 430

ACG CCA TTA CTG AAC GGA CAA TAT TTT TCT ACC GGC GAT GAG GAG ATT 1344

Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile

435 440 445

GAT TTA AAT TCA GGT AGC ACC GGC GAT TGG CGA AAA ACC ATA CTT AAG 1392

Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys

450 455 460

CGT GCA TTT AAT ATT GAT GAT GTC TCG CTC TTC CGC CTG CTT AAA ATT 1440

Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile

465 470 475 480

ACC GAC CAT GAT AAT AAA GAT GGA AAA ATT AAA AAT AAC CTA AAG AAT 1488

Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn

485 490 495

CTT TCC AAT TTA TAT ATT GGA AAA TTA CTG GCA GAT ATT CAT CAA TTA 1536

Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu

500 505 510

ACC ATT GAT GAA CTG GAT TTA TTA CTG ATT GCC GTA GGT GAA GGA AAA 1584

Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys

515 520 525

ACT AAT TTA TCC GCT ATC AGT GAT AAG CAA TTG GCT ACC CTG ATC AGA 1632

Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg

530 535 540

AAA CTC AAT ACT ATT ACC AGC TGG CTA CAT ACA CAG AAG TGG AGT GTA 1680

Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val

545 550 555 560

TTC CAG CTA TTT ATC ATG ACC TCC ACC AGC TAT AAC AAA ACG CTA ACG 1728

Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr

565 570 575

CCT GAA ATT AAG AAT TTG CTG GAT ACC GTC TAC CAC GGT TTA CAA GGT 1776

Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly

580 585 590

TTT GAT AAA GAC AAA GCA GAT TTG CTA CAT GTC ATG GCG CCC TAT ATT 1824

Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile

595 600 605

GCG GCC ACC TTG CAA TTA TCA TCG GAA AAT GTC GCC CAC TCG GTA CTC 1872

Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu

610 615 620

CTT TGG GCA GAT AAG TTA CAG CCC GGC GAC GGC GCA ATG ACA GCA GAA 1920

Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu

625 630 635 640

AAA TTC TGG GAC TGG TTG AAT ACT AAG TAT ACG CCG GGT TCA TCG GAA 1968

Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu

645 650 655

GCC GTA GAA ACG CAG GAA CAT ATC GTT CAG TAT TGT CAG GCT CTG GCA 2016

Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala

660 665 670

CAA TTG GAA ATG GTT TAC CAT TCC ACC GGC ATC AAC GAA AAC GCC TTC 2064

Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe

675 680 685

CGT CTA TTT GTG ACA AAA CCA GAG ATG TTT GGC GCT GCA ACT GGA GCA 2112

Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala

690 695 700

GCG CCC GCG CAT GAT GCC CTT TCA CTG ATT ATG CTG ACA CGT TTT GCG 2160

Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala

705 710 715 720

GAT TGG GTG AAC GCA CTA GGC GAA AAA GCG TCC TCG GTG CTA GCG GCA 2208

Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala

725 730 735

TTT GAA GCT AAC TCG TTA ACG GCA GAA CAA CTG GCT GAT GCC ATG AAT 2256

Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn

740 745 750

CTT GAT GCT AAT TTG CTG TTG CAA GCC AGT ATT CAA GCA CAA AAT CAT 2304

Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His

755 760 765

CAA CAT CTT CCC CCA GTA ACT CCA GAA AAT GCG TTC TCC TGT TGG ACA 2352

Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr

770 775 780

TCT ATC AAT ACT ATC CTG CAA TGG GTT AAT GTC GCA CAA CAA TTG AAT 2400

Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn

785 790 795 800

GTC GCC CCA CAG GGC GTT TCC GCT TTG GTC GGG CTG GAT TAT ATT CAA 2448

Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln

805 810 815

TCA ATG AAA GAG ACA CCG ACC TAT GCC CAG TGG GAA AAC GCG GCA GGC 2496

Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly

820 825 830

GTA TTA ACC GCC GGG TTG AAT TCA CAA CAG GCT AAT ACA TTA CAC GCT 2544

Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala

835 840 845

TTT CTG GAT GAA TCT CGC AGT GCC GCA TTA AGC ACC TAC TAT ATC CGT 2592

Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg

850 855 860

CAA GTC GCC AAG GCA GCG GCG GCT ATT AAA AGC CGT GAT GAC TTG TAT 2640

Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr

865 870 875 880

CAA TAC TTA CTG ATT GAT AAT CAG GTT TCT GCG GCA ATA AAA ACC ACC 2688

Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr

885 890 895

CGG ATC GCC GAA GCC ATT GCC AGT ATT CAA CTG TAC GTC AAC CGG GCA 2736

Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala

900 905 910

TTG GAA AAT GTG GAA GAA AAT GCC AAT TCG GGG GTT ATC AGC CGC CAA 2784

Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln

915 920 925

TTC TTT ATC GAC TGG GAC AAA TAC AAT AAA CGC TAC AGC ACT TGG GCG 2832

Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala

930 935 940

GGT GTT TCT CAA TTA GTT TAC TAC CCG GAA AAC TAT ATT GAT CCG ACC 2880

Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr

945 950 955 960

ATG CGT ATC GGA CAA ACC AAA ATG ATG GAC GCA TTA CTG CAA TCC GTC 2928

Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val

965 970 975

AGC CAA AGC CAA TTA AAC GCC GAT ACC GTC GAA GAT GCC TTT ATG TCT 2976

Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser

980 985 990

TAT CTG ACA TCG TTT GAA CAA GTG GCT AAT CTT AAA GTT ATT AGC GCA 3024

Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala

995 1000 1005

TAT CAC GAT AAT ATT AAT AAC GAT CAA GGG CTG ACC TAT TTT ATC GGA 3072

Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly

1010 1015 1020

CTC AGT GAA ACT GAT GCC GGT GAA TAT TAT TGG CGC AGT GTC GAT CAC 3120

Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His

1025 1030 1035 1040

AGT AAA TTC AAC GAC GGT AAA TTC GCG GCT AAT GCC TGG AGT GAA TGG 3168

Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp

1045 1050 1055

CAT AAA ATT GAT TGT CCA ATT AAC CCT TAT AAA AGC ACT ATC CGT CCA 3216

His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro

1060 1065 1070

GTG ATA TAT AAA TCC CGC CTG TAT CTG CTC TGG TTG GAA CAA AAG GAG 3264

Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu

1075 1080 1085

ATC ACC AAA CAG ACA GGA AAT AGT AAA GAT GGC TAT CAA ACT GAA ACG 3312

Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr

1090 1095 1100

GAT TAT CGT TAT GAA CTA AAA TTG GCG CAT ATC CGC TAT GAT GGC ACT 3360

Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr

1105 1110 1115 1120

TGG AAT ACG CCA ATC ACC TTT GAT GTC AAT AAA AAA ATA TCC GAG CTA 3408

Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu

1125 1130 1135

AAA CTG GAA AAA AAT AGA GCG CCC GGA CTC TAT TGT GCC GGT TAT CAA 3456

Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln

1140 1145 1150

GGT GAA GAT ACG TTG CTG GTG ATG TTT TAT AAC CAA CAA GAC ACA CTA 3504

Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu

1155 1160 1165

GAT AGT TAT AAA AAC GCT TCA ATG CAA GGA CTA TAT ATC TTT GCT GAT 3552

Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp

1170 1175 1180

ATG GCA TCC AAA GAT ATG ACC CCA GAA CAG AGC AAT GTT TAT CGG GAT 3600

Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp

1185 1190 1195 1200

AAT AGC TAT CAA CAA TTT GAT ACC AAT AAT GTC AGA AGA GTG AAT AAC 3648

Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn

1205 1210 1215

CGC TAT GCA GAG GAT TAT GAG ATT CCT TCC TCG GTA AGT AGC CGT AAA 3696

Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys

1220 1225 1230

GAC TAT GGT TGG GGA GAT TAT TAC CTC AGC ATG GTA TAT AAC GGA GAT 3744

Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp

1235 1240 1245

ATT CCA ACT ATC AAT TAC AAA GCC GCA TCA AGT GAT TTA AAA ATC TAT 3792

Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr

1250 1255 1260

ATC TCA CCA AAA TTA AGA ATT ATT CAT AAT GGA TAT GAA GGA CAG AAG 3840

Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys

1265 1270 1275 1280

CGC AAT CAA TGC AAT CTG ATG AAT AAA TAT GGC AAA CTA GGT GAT AAA 3888

Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys

1285 1290 1295

TTT ATT GTT TAT ACT AGC TTG GGG GTC AAT CCA AAT AAC TCG TCA AAT 3936

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn

1300 1305 1310

AAG CTC ATG TTT TAC CCC GTC TAT CAA TAT AGC GGA AAC ACC AGT GGA 3984

Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly

1315 1320 1325

CTC AAT CAA GGG AGA CTA CTA TTC CAC CGT GAC ACC ACT TAT CCA TCT 4032

Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser

1330 1335 1340

AAA GTA GAA GCT TGG ATT CCT GGA GCA AAA CGT TCT CTA ACC AAC CAA 4080

Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln

1345 1350 1355 1360

AAT GCC GCC ATT GGT GAT GAT TAT GCT ACA GAC TCT CTG AAT AAA CCG 4128

Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro

1365 1370 1375

GAT GAT CTT AAG CAA TAT ATC TTT ATG ACT GAC AGT AAA GGG ACT GCT 4176

Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala

1380 1385 1390

ACT GAT GTC TCA GGC CCA GTA GAG ATT AAT ACT GCA ATT TCT CCA GCA 4224

Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala

1395 1400 1405

AAA GTT CAG ATA ATA GTC AAA GCG GGT GGC AAG GAG CAA ACT TTT ACC 4272

Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr

1410 1415 1420

GCA GAT AAA GAT GTC TCC ATT CAG CCA TCA CCT AGC TTT GAT GAA ATG 4320

Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met

1425 1430 1435 1440

AAT TAT CAA TTT AAT GCC CTT GAA ATA GAC GGT TCT GGT CTG AAT TTT 4368

Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe

1445 1450 1455

ATT AAC AAC TCA GCC AGT ATT GAT GTT ACT TTT ACC GCA TTT GCG GAG 4416

Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu

1460 1465 1470

GAT GGC CGC AAA CTG GGT TAT GAA AGT TTC AGT ATT CCT GTT ACC CTC 4464

Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu

1475 1480 1485

AAG GTA AGT ACC GAT AAT GCC CTG ACC CTG CAC CAT AAT GAA AAT GGT 4512

Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly

1490 1495 1500

GCG CAA TAT ATG CAA TGG CAA TCC TAT CGT ACC CGC CTG AAT ACT CTA 4560

Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu

1505 1510 1515 1520

TTT GCC CGC CAG TTG GTT GCA CGC GCC ACC ACC GGA ATC GAT ACA ATT 4608

Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile

1525 1530 1535

CTG AGT ATG GAA ACT CAG AAT ATT CAG GAA CCG CAG TTA GGC AAA GGT 4656

Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly

1540 1545 1550

TTC TAT GCT ACG TTC GTG ATA CCT CCC TAT AAC CTA TCA ACT CAT GGT 4704

Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly

1555 1560 1565

GAT GAA CGT TGG TTT AAG CTT TAT ATC AAA CAT GTT GTT GAT AAT AAT 4752

Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn

1570 1575 1580

TCA CAT ATT ATC TAT TCA GGC CAG CTA ACA GAT ACA AAT ATA AAC ATC 4800

Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile

1585 1590 1595 1600

ACA TTA TTT ATT CCT CTT GAT GAT GTC CCA TTG AAT CAA GAT TAT CAC 4848

Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His

1605 1610 1615

GCC AAG GTT TAT ATG ACC TTC AAG AAA TCA CCA TCA GAT GGT ACC TGG 4896

Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp

1620 1625 1630

TGG GGC CCT CAC TTT GTT AGA GAT GAT AAA GGA ATA GTA ACA ATA AAC 4944

Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn

1635 1640 1645

CCT AAA TCC ATT TTG ACC CAT TTT GAG AGC GTC AAT GTC CTG AAT AAT 4992

Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn

1650 1655 1660

ATT AGT AGC GAA CCA ATG GAT TTC AGC GGC GCT AAC AGC CTC TAT TTC 5040

Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe

1665 1670 1675 1680

TGG GAA CTG TTC TAC TAT ACC CCG ATG CTG GTT GCT CAA CGT TTG CTG 5088

Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu

1685 1690 1695

CAT GAA CAG AAC TTC GAT GAA GCC AAC CGT TGG CTG AAA TAT GTC TGG 5136

His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp

1700 1705 1710

AGT CCA TCC GGT TAT ATT GTC CAC GGC CAG ATT CAG AAC TAC CAG TGG 5184

Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp

1715 1720 1725

AAC GTC CGC CCG TTA CTG GAA GAC ACC AGT TGG AAC AGT GAT CCT TTG 5232

Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu

1730 1735 1740

GAT TCC GTC GAT CCT GAC GCG GTA GCA CAG CAC GAT CCA ATG CAC TAC 5280

Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr

1745 1750 1755 1760

AAA GTT TCA ACT TTT ATG CGT ACC TTG GAT CTA TTG ATA GCA CGC GGC 5328

Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly

1765 1770 1775

GAC CAT GCT TAT CGC CAA CTG GAA CGA GAT ACA CTC AAC GAA GCG AAG 5376

Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys

1780 1785 1790

ATG TGG TAT ATG CAA GCG CTG CAT CTA TTA GGT GAC AAA CCT TAT CTA 5424

Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu

1795 1800 1805

CCG CTG AGT ACG ACA TGG AGT GAT CCA CGA CTA GAC AGA GCC GCG GAT 5472

Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp

1810 1815 1820

ATC ACT ACC CAA AAT GCT CAC GAC AGC GCA ATA GTC GCT CTG CGG CAG 5520

Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln

1825 1830 1835 1840

AAT ATA CCT ACA CCG GCA CCT TTA TCA 5547

Asn Ile Pro Thr Pro Ala Pro Leu Ser

1845 1849

(2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1849 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49 (TcdAii):

Features From To Description

Peptide 1 1849 TcdAii peptide

Fragment 1 12 TcdAii N-terminus (SEQ ID NO: 13)

Fragment 196 211 (SEQ ID NO: 38)

Fragment 466 475 (SEQ ID NO: 17)

Fragment 993 1004 (SEQ ID NO: 23; 12/13)

Fragment 1297 1312 (SEQ ID NO: 18)

Fragment 1390 1409 (SEQ ID NO: 39)

Fragment 1532 1554 (SEQ ID NO: 21; 19/23)

Leu Ile Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val

1 5 10 15

Ala Pro Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr

20 25 30

Glu Leu Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val Tyr

35 40 45

Tyr Leu Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser Gln

50 55 60

Gln Asn Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu

65 70 75 80

Leu Leu Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys

85 90 95

Val Met Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr

100 105 110

His Asp Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln Asp Pro

115 120 125

Gly Leu Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu Met His

130 135 140

Gln Ala Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe

145 150 155 160

Asn Ile Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr

165 170 175

Lys Lys Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala Met Pro Glu

180 185 190

Tyr Leu Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe

195 200 205

Ile Gly Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln

210 215 220

Leu Ile Thr Pro Val Val Asn Ser Ser Asp Gly Thr Val Lys Val Tyr

225 230 235 240

Arg Ile Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln Met Asp Val Glu

245 250 255

Leu Phe Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys

260 265 270

Asn Phe Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg

275 280 285

Glu Leu Val Arg Thr Glu Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser

290 295 300

Ala Asn Ile Thr Leu Asn Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile

305 310 315 320

Gly Leu Thr Arg Val Leu Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala

325 330 335

Lys Phe Thr Val Glu Glu Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu

340 345 350

Asn Lys Ala Ile Arg Leu Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile

355 360 365

Leu Glu Gly Ile Val Arg Ser Val Asn Leu Gln Leu Asp Ile Asn Thr

370 375 380

Asp Val Leu Gly Lys Val Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr

385 390 395 400

Ala Ile His Ala Glu Thr Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser

405 410 415

Gln Arg Ser Tyr Asp Asn Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn

420 425 430

Thr Pro Leu Leu Asn Gly Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile

435 440 445

Asp Leu Asn Ser Gly Ser Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys

450 455 460

Arg Ala Phe Asn Ile Asp Asp Val Ser Leu Phe Arg Leu Leu Lys Ile

465 470 475 480

Thr Asp His Asp Asn Lys Asp Gly Lys Ile Lys Asn Asn Leu Lys Asn

485 490 495

Leu Ser Asn Leu Tyr Ile Gly Lys Leu Leu Ala Asp Ile His Gln Leu

500 505 510

Thr Ile Asp Glu Leu Asp Leu Leu Leu Ile Ala Val Gly Glu Gly Lys

515 520 525

Thr Asn Leu Ser Ala Ile Ser Asp Lys Gln Leu Ala Thr Leu Ile Arg

530 535 540

Lys Leu Asn Thr Ile Thr Ser Trp Leu His Thr Gln Lys Trp Ser Val

545 550 555 560

Phe Gln Leu Phe Ile Met Thr Ser Thr Ser Tyr Asn Lys Thr Leu Thr

565 570 575

Pro Glu Ile Lys Asn Leu Leu Asp Thr Val Tyr His Gly Leu Gln Gly

580 585 590

Phe Asp Lys Asp Lys Ala Asp Leu Leu His Val Met Ala Pro Tyr Ile

595 600 605

Ala Ala Thr Leu Gln Leu Ser Ser Glu Asn Val Ala His Ser Val Leu

610 615 620

Leu Trp Ala Asp Lys Leu Gln Pro Gly Asp Gly Ala Met Thr Ala Glu

625 630 635 640

Lys Phe Trp Asp Trp Leu Asn Thr Lys Tyr Thr Pro Gly Ser Ser Glu

645 650 655

Ala Val Glu Thr Gln Glu His Ile Val Gln Tyr Cys Gln Ala Leu Ala

660 665 670

Gln Leu Glu Met Val Tyr His Ser Thr Gly Ile Asn Glu Asn Ala Phe

675 680 685

Arg Leu Phe Val Thr Lys Pro Glu Met Phe Gly Ala Ala Thr Gly Ala

690 695 700

Ala Pro Ala His Asp Ala Leu Ser Leu Ile Met Leu Thr Arg Phe Ala

705 710 715 720

Asp Trp Val Asn Ala Leu Gly Glu Lys Ala Ser Ser Val Leu Ala Ala

725 730 735

Phe Glu Ala Asn Ser Leu Thr Ala Glu Gln Leu Ala Asp Ala Met Asn

740 745 750

Leu Asp Ala Asn Leu Leu Leu Gln Ala Ser Ile Gln Ala Gln Asn His

755 760 765

Gln His Leu Pro Pro Val Thr Pro Glu Asn Ala Phe Ser Cys Trp Thr

770 775 780

Ser Ile Asn Thr Ile Leu Gln Trp Val Asn Val Ala Gln Gln Leu Asn

785 790 795 800

Val Ala Pro Gln Gly Val Ser Ala Leu Val Gly Leu Asp Tyr Ile Gln

805 810 815

Ser Met Lys Glu Thr Pro Thr Tyr Ala Gln Trp Glu Asn Ala Ala Gly

820 825 830

Val Leu Thr Ala Gly Leu Asn Ser Gln Gln Ala Asn Thr Leu His Ala

835 840 845

Phe Leu Asp Glu Ser Arg Ser Ala Ala Leu Ser Thr Tyr Tyr Ile Arg

850 855 860

Gln Val Ala Lys Ala Ala Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr

865 870 875 880

Gln Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr

885 890 895

Arg Ile Ala Glu Ala Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala

900 905 910

Leu Glu Asn Val Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln

915 920 925

Phe Phe Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala

930 935 940

Gly Val Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro Thr

945 950 955 960

Met Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln Ser Val

965 970 975

Ser Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe Met Ser

980 985 990

Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala

995 1000 1005

Tyr His Asp Asn Ile Asn Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly

1010 1015 1020

Leu Ser Glu Thr Asp Ala Gly Glu Tyr Tyr Trp Arg Ser Val Asp His

1025 1030 1035 1040

Ser Lys Phe Asn Asp Gly Lys Phe Ala Ala Asn Ala Trp Ser Glu Trp

1045 1050 1055

His Lys Ile Asp Cys Pro Ile Asn Pro Tyr Lys Ser Thr Ile Arg Pro

1060 1065 1070

Val Ile Tyr Lys Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln Lys Glu

1075 1080 1085

Ile Thr Lys Gln Thr Gly Asn Ser Lys Asp Gly Tyr Gln Thr Glu Thr

1090 1095 1100

Asp Tyr Arg Tyr Glu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr

1105 1110 1115 1120

Trp Asn Thr Pro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu

1125 1130 1135

Lys Leu Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln

1140 1145 1150

Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu

1155 1160 1165

Asp Ser Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala Asp

1170 1175 1180

Met Ala Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp

1185 1190 1195 1200

Asn Ser Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg Arg Val Asn Asn

1205 1210 1215

Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser Ser Val Ser Ser Arg Lys

1220 1225 1230

Asp Tyr Gly Trp Gly Asp Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp

1235 1240 1245

Ile Pro Thr Ile Asn Tyr Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr

1250 1255 1260

Ile Ser Pro Lys Leu Arg Ile Ile His Asn Gly Tyr Glu Gly Gln Lys

1265 1270 1275 1280

Arg Asn Gln Cys Asn Leu Met Asn Lys Tyr Gly Lys Leu Gly Asp Lys

1285 1290 1295

Phe Ile Val Tyr Thr Ser Leu Gly Val Asn Pro Asn Asn Ser Ser Asn

1300 1305 1310

Lys Leu Met Phe Tyr Pro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly

1315 1320 1325

Leu Asn Gln Gly Arg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser

1330 1335 1340

Lys Val Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln

1345 1350 1355 1360

Asn Ala Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro

1365 1370 1375

Asp Asp Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr Ala

1380 1385 1390

Thr Asp Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser Pro Ala

1395 1400 1405

Lys Val Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr

1410 1415 1420

Ala Asp Lys Asp Val Ser Ile Gln Pro Ser Pro Ser Phe Asp Glu Met

1425 1430 1435 1440

Asn Tyr Gln Phe Asn Ala Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe

1445 1450 1455

Ile Asn Asn Ser Ala Ser Ile Asp Val Thr Phe Thr Ala Phe Ala Glu

1460 1465 1470

Asp Gly Arg Lys Leu Gly Tyr Glu Ser Phe Ser Ile Pro Val Thr Leu

1475 1480 1485

Lys Val Ser Thr Asp Asn Ala Leu Thr Leu His His Asn Glu Asn Gly

1490 1495 1500

Ala Gln Tyr Met Gln Trp Gln Ser Tyr Arg Thr Arg Leu Asn Thr Leu

1505 1510 1515 1520

Phe Ala Arg Gln Leu Val Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile

1525 1530 1535

Leu Ser Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly

1540 1545 1550

Phe Tyr Ala Thr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly

1555 1560 1565

Asp Glu Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn

1570 1575 1580

Ser His Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn Ile

1585 1590 1595 1600

Thr Leu Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp Tyr His

1605 1610 1615

Ala Lys Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp

1620 1625 1630

Trp Gly Pro His Phe Val Arg Asp Asp Lys Gly Ile Val Thr Ile Asn

1635 1640 1645

Pro Lys Ser Ile Leu Thr His Phe Glu Ser Val Asn Val Leu Asn Asn

1650 1655 1660

Ile Ser Ser Glu Pro Met Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe

1665 1670 1675 1680

Trp Glu Leu Phe Tyr Tyr Thr Pro Met Leu Val Ala Gln Arg Leu Leu

1685 1690 1695

His Glu Gln Asn Phe Asp Glu Ala Asn Arg Trp Leu Lys Tyr Val Trp

1700 1705 1710

Ser Pro Ser Gly Tyr Ile Val His Gly Gln Ile Gln Asn Tyr Gln Trp

1715 1720 1725

Asn Val Arg Pro Leu Leu Glu Asp Thr Ser Trp Asn Ser Asp Pro Leu

1730 1735 1740

Asp Ser Val Asp Pro Asp Ala Val Ala Gln His Asp Pro Met His Tyr

1745 1750 1755 1760

Lys Val Ser Thr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly

1765 1770 1775

Asp His Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys

1780 1785 1790

Met Trp Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu

1795 1800 1805

Pro Leu Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala Asp

1810 1815 1820

Ile Thr Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala Leu Arg Gln

1825 1830 1835 1840

Asn Ile Pro Thr Pro Ala Pro Leu Ser

1845 1849

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1740 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50 (tcdAiii coding

region):

TTG CGC AGC GCT AAT ACC CTG ACT GAT CTC TTC CTG CCG CAA ATC AAT 48

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn

1 5 10 15

GAA GTG ATG ATG AAT TAC TGG CAG ACA TTA GCT CAG AGA GTA TAC AAT 96

Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn

20 25 30

CTG CGT CAT AAC CTC TCT ATC GAC GGC CAG CCG TTA TAT CTG CCA ATC 144

Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile

35 40 45

TAT GCC ACA CCG GCC GAT CCG AAA GCG TTA CTC AGC GCC GCC GTT GCC 192

Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala

50 55 60

ACT TCT CAA GGT GGA GGC AAG CTA CCG GAA TCA TTT ATG TCC CTG TGG 240

Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp

65 70 75 80

CGT TTC CCG CAC ATG CTG GAA AAT GCG CGC GGC ATG GTT AGC CAG CTC 288

Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu

85 90 95

ACC CAG TTC GGC TCC ACG TTA CAA AAT ATT ATC GAA CGT CAG GAC GCG 336

Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Glu Arg Gln Asp Ala

100 105 110

GAA GCG CTC AAT GCG TTA TTA CAA AAT CAG GCC GCC GAG CTG ATA TTG 384

Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu

115 120 125

ACT AAC CTG AGC ATT CAG GAC AAA ACC ATT GAA GAA TTG GAT GCC GAG 432

Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu

130 135 140

AAA ACG GTG TTG GAA AAA TCC AAA GCG GGA GCA CAA TCG CGC TTT GAT 480

Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp

145 150 155 160

AGC TAC GGC AAA CTG TAC GAT GAG AAT ATC AAC GCC GGT GAA AAC CAA 528

Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln

165 170 175

GCC ATG ACG CTA CGA GCG TCC GCC GCC GGG CTT ACC ACG GCA GTT CAG 576

Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln

180 185 190

GCA TCC CGT CTG GCC GGT GCG GCG GCT GAT CTG GTG CCT AAC ATC TTC 624

Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe

195 200 205

GGC TTT GCC GGT GGC GGC AGC CGT TGG GGG GCT ATC GCT GAG GCG ACA 672

Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr

210 215 220

GGT TAT GTG ATG GAA TTC TCC GCG AAT GTT ATG AAC ACC GAA GCG GAT 720

Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp

225 230 235 240

AAA ATT AGC CAA TCT GAA ACC TAC CGT CGT CGC CGT CAG GAG TGG GAG 768

Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu

245 250 255

ATC CAG CGG AAT AAT GCC GAA GCG GAA TTG AAG CAA ATC GAT GCT CAG 816

Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln

260 265 270

CTC AAA TCA CTC GCT GTA CGC CGC GAA GCC GCC GTA TTG CAG AAA ACC 864

Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr

275 280 285

AGT CTG AAA ACC CAA CAA GAA CAG ACC CAA TCT CAA TTG GCC TTC CTG 912

Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu

290 295 300

CAA CGT AAG TTC AGC AAT CAG GCG TTA TAC AAC TGG CTG CGT GGT CGA 960

Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg

305 310 315 320

CTG GCG GCG ATT TAC TTC CAG TTC TAC GAT TTG GCC GTC GCG CGT TGC 1008

Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys

325 330 335

CTG ATG GCA GAA CAA GCT TAC CGT TGG GAA CTC AAT GAT GAC TCT GCC 1056

Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala

340 345 350

CGC TTC ATT AAA CCG GGC GCC TGG CAG GGA ACC TAT GCC GGT CTG CTT 1104

Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu

355 360 365

GCA GGT GAA ACC TTG ATG CTG AGT CTG GCA CAA ATG GAA GAC GCT CAT 1152

Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His

370 375 380

CTG AAA CGC GAT AAA CGC GCA TTA GAG GTT GAA CGC ACA GTA TCG CTG 1200

Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu

385 390 395 400

GCC GAA GTT TAT GCA GGA TTA CCA AAA GAT AAC GGT CCA TTT TCC CTG 1248

Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu

405 410 415

GCT CAG GAA ATT GAC AAG CTG GTG AGT CAA GGT TCA GGC AGT GCC GGC 1296

Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly

420 425 430

AGT GGT AAT AAT AAT TTG GCG TTC GGC GCC GGC ACG GAC ACT AAA ACC 1344

Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr

435 440 445

TCT TTG CAG GCA TCA GTT TCA TTC GCT GAT TTG AAA ATT CGT GAA GAT 1392

Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp

450 455 460

TAC CCG GCA TCG CTT GGC AAA ATT CGA CGT ATC AAA CAG ATC AGC GTC 1440

Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val

465 470 475 480

ACT TTG CCC GCG CTA CTG GGA CCG TAT CAG GAT GTA CAG GCA ATA TTG 1488

Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu

485 490 495

TCT TAC GGC GAT AAA GCC GGA TTA GCT AAC GGC TGT GAA GCG CTG GCA 1536

Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala

500 505 510

GTT TCT CAC GGT ATG AAT GAC AGC GGC CAA TTC CAG CTC GAT TTC AAC 1584

Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn

515 520 525

GAT GGC AAA TTC CTG CCA TTC GAA GGC ATC GCC ATT GAT CAA GGC ACG 1632

Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr

530 535 540

CTG ACA CTG AGC TTC CCA AAT GCA TCT ATG CCG GAG AAA GGT AAA CAA 1680

Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln

545 550 555 560

GCC ACT ATG TTA AAA ACC CTG AAC GAT ATC ATT TTG CAT ATT CGC TAC 1728

Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr

565 570 575

ACC ATT AAA TAA 1740

Thr Ile Lys

579

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 579 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51 (TcdAiii):

Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu Phe Leu Pro Gln Ile Asn

1 5 10 15

Glu Val Met Met Asn Tyr Trp Gln Thr Leu Ala Gln Arg Val Tyr Asn

20 25 30

Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Tyr Leu Pro Ile

35 40 45

Tyr Ala Thr Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ala

50 55 60

Thr Ser Gln Gly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu Trp

65 70 75 80

Arg Phe Pro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln Leu

85 90 95

Thr Gln Phe Gly Ser Thr Leu Gln Asn Ile Glu Arg Gln Asp Ala

100 105 110

Glu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile Leu

115 120 125

Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala Glu

130 135 140

Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser Arg Phe Asp

145 150 155 160

Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly Glu Asn Gln

165 170 175

Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr Ala Val Gln

180 185 190

Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro Asn Ile Phe

195 200 205

Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala Ile Ala Glu Ala Thr

210 215 220

Gly Tyr Val Met Glu Phe Ser Ala Asn Val Met Asn Thr Glu Ala Asp

225 230 235 240

Lys Ile Ser Gln Ser Glu Thr Tyr Arg Arg Arg Arg Gln Glu Trp Glu

245 250 255

Ile Gln Arg Asn Asn Ala Glu Ala Glu Leu Lys Gln Ile Asp Ala Gln

260 265 270

Leu Lys Ser Leu Ala Val Arg Arg Glu Ala Ala Val Leu Gln Lys Thr

275 280 285

Ser Leu Lys Thr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe Leu

290 295 300

Gln Arg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly Arg

305 310 315 320

Leu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg Cys

325 330 335

Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser Ala

340 345 350

Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu

355 360 365

Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu Asp Ala His

370 375 380

Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu

385 390 395 400

Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro Phe Ser Leu

405 410 415

Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly Ser Gly Ser Ala Gly

420 425 430

Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala Gly Thr Asp Thr Lys Thr

435 440 445

Ser Leu Gln Ala Ser Val Ser Phe Ala Asp Leu Lys Ile Arg Glu Asp

450 455 460

Tyr Pro Ala Ser Leu Gly Lys Ile Arg Arg Ile Lys Gln Ile Ser Val

465 470 475 480

Thr Leu Pro Ala Leu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile Leu

485 490 495

Ser Tyr Gly Asp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu Ala

500 505 510

Val Ser His Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn

515 520 525

Asp Gly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly Thr

530 535 540

Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys Gln

545 550 555 560

Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His Ile Arg Tyr

565 570 575

Thr Ile Lys

579

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5532 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52 (tcbAii coding

region):

TTT ATA CAA GGT TAT AGT GAT CTG TTT GGT AAT CGT GCT GAT AAC TAT 48

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr

1 5 10 15

GCC GCG CCG GGC TCG GTT GCA TCG ATG TTC TCA CCG GCG GCT TAT TTG 96

Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu

20 25 30

ACG GAA TTG TAC CGT GAA GCC AAA AAC TTG CAT GAC AGC AGC TCA ATT 144

Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile

35 40 45

TAT TAC CTA GAT AAA CGT CGC CCG GAT TTA GCA AGC TTA ATG CTC AGC 192

Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser

50 55 60

CAG AAA AAT ATG GAT GAG GAA ATT TCA ACG CTG GCT CTC TCT AAT GAA 240

Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu

65 70 75 80

TTG TGC CTT GCC GGG ATC GAA ACA AAA ACA GGA AAA TCA CAA GAT GAA 288

Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu

85 90 95

GTG ATG GAT ATG TTG TCA ACT TAT CGT TTA AGT GGA GAG ACA CCT TAT 336

Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr

100 105 110

CAT CAC GCT TAT GAA ACT GTT CGT GAA ATC GTT CAT GAA CGT GAT CCA 384

His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro

115 120 125

GGA TTT CGT CAT TTG TCA CAG GCA CCC ATT GTT GCT GCT AAG CTC GAT 432

Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp

130 135 140

CCT GTG ACT TTG TTG GGT ATT AGC TCC CAT ATT TCG CCA GAA CTG TAT 480

Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr

145 150 155 160

AAC TTG CTG ATT GAG GAG ATC CCG GAA AAA GAT GAA GCC GCG CTT GAT 528

Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp

165 170 175

ACG CTT TAT AAA ACA AAC TTT GGC GAT ATT ACT ACT GCT CAG TTA ATG 576

Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met

180 185 190

TCC CCA AGT TAT CTG GCC CGG TAT TAT GGC GTC TCA CCG GAA GAT ATT 624

Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile

195 200 205

GCC TAC GTG ACG ACT TCA TTA TCA CAT GTT GGA TAT AGC AGT GAT ATT 672

Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile

210 215 220

CTG GTT ATT CCG TTG GTC GAT GGT GTG GGT AAG ATG GAA GTA GTT CGT 720

Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg

225 230 235 240

GTT ACC CGA ACA CCA TCG GAT AAT TAT ACC AGT CAG ACG AAT TAT ATT 768

Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile

245 250 255

GAG CTG TAT CCA CAG GGT GGC GAC AAT TAT TTG ATC AAA TAC AAT CTA 816

Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu

260 265 270

AGC AAT AGT TTT GGT TTG GAT GAT TTT TAT CTG CAA TAT AAA GAT GGT 864

Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly

275 280 285

TCC GCT GAT TGG ACT GAG ATT GCC CAT AAT CCC TAT CCT GAT ATG GTC 912

Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val

290 295 300

ATA AAT CAA AAG TAT GAA TCA CAG GCG ACA ATC AAA CGT AGT GAC TCT 960

Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser

305 310 315 320

GAC AAT ATA CTC AGT ATA GGG TTA CAA AGA TGG CAT AGC GGT AGT TAT 1008

Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr

325 330 335

AAT TTT GCC GCC GCC AAT TTT AAA ATT GAC CAA TAC TCC CCG AAA GCT 1056

Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala

340 345 350

TTC CTG CTT AAA ATG AAT AAG GCT ATT CGG TTG CTC AAA GCT ACC GGC 1104

Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly

355 360 365

CTC TCT TTT GCT ACG TTG GAG CGT ATT GTT GAT AGT GTT AAT AGC ACC 1152

Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr

370 375 380

AAA TCC ATC ACG GTT GAG GTA TTA AAC AAG GTT TAT CGG GTA AAA TTC 1200

Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe

385 390 395 400

TAT ATT GAT CGT TAT GGC ATC AGT GAA GAG ACA GCC GCT ATT TTG GCT 1248

Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala

405 410 415

AAT ATT AAT ATC TCT CAG CAA GCT GTT GGC AAT CAG CTT AGC CAG TTT 1296

Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe

420 425 430

GAG CAA CTA TTT AAT CAC CCG CCG CTC AAT GGT ATT CGC TAT GAA ATC 1344

Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile

435 440 445

AGT GAG GAC AAC TCC AAA CAT CTT CCT AAT CCT GAT CTG AAC CTT AAA 1392

Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys

450 455 460

CCA GAC AGT ACC GGT GAT GAT CAA CGC AAG GCG GTT TTA AAA CGC GCG 1440

Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala

465 470 475 480

TTT CAG GTT AAC GCC AGT GAG TTG TAT CAG ATG TTA TTG ATC ACT GAT 1488

Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp

485 490 495

CGT AAA GAA GAC GGT GTT ATC AAA AAT AAC TTA GAG AAT TTG TCT GAT 1536

Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp

500 505 510

CTG TAT TTG GTT AGT TTG CTG GCC CAG ATT CAT AAC CTG ACT ATT GCT 1584

Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala

515 520 525

GAA TTG AAC ATT TTG TTG GTG ATT TGT GGC TAT GGC GAC ACC AAC ATT 1632

Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile

530 535 540

TAT CAG ATT ACC GAC GAT AAT TTA GCC AAA ATA GTG GAA ACA TTG TTG 1680

Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu

545 550 555 560

TGG ATC ACT CAA TGG TTG AAG ACC CAA AAA TGG ACA GTT ACC GAC CTG 1728

Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu

565 570 575

TTT CTG ATG ACC ACG GCC ACT TAC AGC ACC ACT TTA ACG CCA GAA ATT 1776

Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile

580 585 590

AGC AAT CTG ACG GCT ACG TTG TCT TCA ACT TTG CAT GGC AAA GAG AGT 1824

Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser

595 600 605

CTG ATT GGG GAA GAT CTG AAA AGA GCA ATG GCG CCT TGC TTC ACT TCG 1872

Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser

610 615 620

GCT TTG CAT TTG ACT TCT CAA GAA GTT GCG TAT GAC CTG CTG TTG TGG 1920

Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp

625 630 635 640

ATA GAC CAG ATT CAA CCG GCA CAA ATA ACT GTT GAT GGG TTT TGG GAA 1968

Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu

645 650 655

GAA GTG CAA ACA ACA CCA ACC AGC TTG AAG GTG ATT ACC TTT GCT CAG 2016

Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln

660 665 670

GTG CTG GCA CAA TTG AGC CTG ATC TAT CGT CGT ATT GGG TTA AGT GAA 2064

Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu

675 680 685

ACG GAA CTG TCA CTG ATC GTG ACT CAA TCT TCT CTG CTA GTG GCA GGC 2112

Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly

690 695 700

AAA AGC ATA CTG GAT CAC GGT CTG TTA ACC CTG ATG GCC TTG GAA GGT 2160

Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly

705 710 715 720

TTT CAT ACC TGG GTT AAT GGC TTG GGG CAA CAT GCC TCC TTG ATA TTG 2208

Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu

725 730 735

GCG GCG TTG AAA GAC GGA GCC TTG ACA GTT ACC GAT GTA GCA CAA GCT 2256

Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala

740 745 750

ATG AAT AAG GAG GAA TCT CTC CTA CAA ATG GCA GCT AAT CAG GTG GAG 2304

Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu

755 760 765

AAG GAT CTA ACA AAA CTG ACC AGT TGG ACA CAG ATT GAC GCT ATT CTG 2352

Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu

770 775 780

CAA TGG TTA CAG ATG TCT TCG GCC TTG GCG GTT TCT CCA CTG GAT CTG 2400

Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu

785 790 795 800

GCA GGG ATG ATG GCC CTG AAA TAT GGG ATA GAT CAT AAC TAT GCT GCC 2448

Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala

805 810 815

TGG CAA GCT GCG GCG GCT GCG CTG ATG GCT GAT CAT GCT AAT CAG GCA 2496

Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala

820 825 830

CAG AAA AAA CTG GAT GAG ACG TTC AGT AAG GCA TTA TGT AAC TAT TAT 2544

Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr

835 840 845

ATT AAT GCT GTT GTC GAT AGT GCT GCT GGA GTA CGT GAT CGT AAC GGT 2592

Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly

850 855 860

TTA TAT ACC TAT TTG CTG ATT GAT AAT CAG GTT TCT GCC GAT GTG ATC 2640

Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile

865 870 875 880

ACT TCA CGT ATT GCA GAA GCT ATC GCC GGT ATT CAA CTG TAC GTT AAC 2688

Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn

885 890 895

CGG GCT TTA AAC CGA GAT GAA GGT CAG CTT GCA TCG GAC GTT AGT ACC 2736

Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr

900 905 910

CGT CAG TTC TTC ACT GAC TGG GAA CGT TAC AAT AAA CGT TAC AGT ACT 2784

Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr

915 920 925

TGG GCT GGT GTC TCT GAA CTG GTC TAT TAT CCA GAA AAC TAT GTT GAT 2832

Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp

930 935 940

CCC ACT CAG CGC ATT GGG CAA ACC AAA ATG ATG GAT GCG CTG TTG CAA 2880

Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln

945 950 955 960

TCC ATC AAC CAG AGC CAG CTA AAT GCG GAT ACG GTG GAA GAT GCT TTC 2928

Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe

965 970 975

AAA ACT TAT TTG ACC AGC TTT GAG CAG GTA GCA AAT CTG AAA GTA ATT 2976

Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile

980 985 990

AGT GCT TAC CAC GAT AAT GTG AAT GTG GAT CAA GGA TTA ACT TAT TTT 3024

Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe

995 1000 1005

ATC GGT ATC GAC CAA GCA GCT CCG GGT ACG TAT TAC TGG CGT AGT GTT 3072

Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val

1010 1015 1020

GAT CAC AGC AAA TGT GAA AAT GGC AAG TTT GCC GCT AAT GCT TGG GGT 3120

Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly

1025 1030 1035 1040

GAG TGG AAT AAA ATT ACC TGT GCT GTC AAT CCT TGG AAA AAT ATC ATC 3168

Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile

1045 1050 1055

CGT CCG GTT GTT TAT ATG TCC CGC TTA TAT CTG CTA TGG CTG GAG CAG 3216

Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln

1060 1065 1070

CAA TCA AAG AAA AGT GAT GAT GGT AAA ACC ACG ATT TAT CAA TAT AAC 3264

Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn

1075 1080 1085

TTA AAA CTG GCT CAT ATT CGT TAC GAC GGT AGT TGG AAT ACA CCA TTT 3312

Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe

1090 1095 1100

ACT TTT GAT GTG ACA GAA AAG GTA AAA AAT TAC ACG TCG AGT ACT GAT 3360

Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp

1105 1110 1115 1120

GCT GCT GAA TCT TTA GGG TTG TAT TGT ACT GGT TAT CAA GGG GAA GAC 3408

Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp

1125 1130 1135

ACT CTA TTA GTT ATG TTC TAT TCG ATG CAG AGT AGT TAT AGC TCC TAT 3456

Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr

1140 1145 1150

ACC GAT AAT AAT GCG CCG GTC ACT GGG CTA TAT ATT TTC GCT GAT ATG 3504

Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met

1155 1160 1165

TCA TCA GAC AAT ATG ACG AAT GCA CAA GCA ACT AAC TAT TGG AAT AAC 3552

Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn

1170 1175 1180

AGT TAT CCG CAA TTT GAT ACT GTG ATG GCA GAT CCG GAT AGC GAC AAT 3600

Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn

1185 1190 1195 1200

AAA AAA GTC ATA ACC AGA AGA GTT AAT AAC CGT TAT GCG GAG GAT TAT 3648

Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr

1205 1210 1215

GAA ATT CCT TCC TCT GTG ACA AGT AAC AGT AAT TAT TCT TGG GGT GAT 3696

Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp

1220 1225 1230

CAC AGT TTA ACC ATG CTT TAT GGT GGT AGT GTT CCT AAT ATT ACT TTT 3744

His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe

1235 1240 1245

GAA TCG GCG GCA GAA GAT TTA AGG CTA TCT ACC AAT ATG GCA TTG AGT 3792

Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser

1250 1255 1260

ATT ATT CAT AAT GGA TAT GCG GGA ACC CGC CGT ATA CAA TGT AAT CTT 3840

Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu

1265 1270 1275 1280

ATG AAA CAA TAC GCT TCA TTA GGT GAT AAA TTT ATA ATT TAT GAT TCA 3888

Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser

1285 1290 1295

TCA TTT GAT GAT GCA AAC CGT TTT AAT CTG GTG CCA TTG TTT AAA TTC 3936

Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe

1300 1305 1310

GGA AAA GAC GAG AAC TCA GAT GAT AGT ATT TGT ATA TAT AAT GAA AAC 3984

Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn

1315 1320 1325

CCT TCC TCT GAA GAT AAG AAG TGG TAT TTT TCT TCG AAA GAT GAC AAT 4032

Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn

1330 1335 1340

AAA ACA GCG GAT TAT AAT GGT GGA ACT CAA TGT ATA GAT GCT GGA ACC 4080

Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr

1345 1350 1355 1360

AGT AAC AAA GAT TTT TAT TAT AAT CTC CAG GAG ATT GAA GTA ATT AGT 4128

Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser

1365 1370 1375

GTT ACT GGT GGG TAT TGG TCG AGT TAT AAA ATA TCC AAC CCG ATT AAT 4176

Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn

1380 1385 1390

ATC AAT ACG GGC ATT GAT AGT GCT AAA GTA AAA GTC ACC GTA AAA GCG 4224

Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala

1395 1400 1405

GGT GGT GAC GAT CAA ATC TTT ACT GCT GAT AAT AGT ACC TAT GTT CCT 4272

Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro

1410 1415 1420

CAG CAA CCG GCA CCC AGT TTT GAG GAG ATG ATT TAT CAG TTC AAT AAC 4320

Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn

1425 1430 1435 1440

CTG ACA ATA GAT TGT AAG AAT TTA AAT TTC ATC GAC AAT CAG GCA CAT 4368

Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His

1445 1450 1455

ATT GAG ATT GAT TTC ACC GCT ACG GCA CAA GAT GGC CGA TTC TTG GGT 4416

Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly

1460 1465 1470

GCA GAA ACT TTT ATT ATC CCG GTA ACT AAA AAA GTT CTC GGT ACT GAG 4464

Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu

1475 1480 1485

AAC GTG ATT GCG TTA TAT AGC GAA AAT AAC GGT GTT CAA TAT ATG CAA 4512

Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln

1490 1495 1500

ATT GGC GCA TAT CGT ACC CGT TTG AAT ACG TTA TTC GCT CAA CAG TTG 4560

Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu

1505 1510 1515 1520

GTT AGC CGT GCT AAT CGT GGC ATT GAT GCA GTG CTC AGT ATG GAA ACT 4608

Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr

1525 1530 1535

CAG AAT ATT CAG GAA CCG CAA TTA GGA GCG GGC ACA TAT GTG CAG CTT 4656

Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu

1540 1545 1550

GTG TTG GAT AAA TAT GAT GAG TCT ATT CAT GGC ACT AAT AAA AGC TTT 4704

Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe

1555 1560 1565

GCT ATT GAA TAT GTT GAT ATA TTT AAA GAG AAC GAT AGT TTT GTG ATT 4752

Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile

1570 1575 1580

TAT CAA GGA GAA CTT AGC GAA ACA AGT CAA ACT GTT GTG AAA GTT TTC 4800

Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe

1585 1590 1595 1600

TTA TCC TAT TTT ATA GAG GCG ACT GGA AAT AAG AAC CAC TTA TGG GTA 4848

Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val

1605 1610 1615

CGT GCT AAA TAC CAA AAG GAA ACG ACT GAT AAG ATC TTG TTC GAC CGT 4896

Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg

1620 1625 1630

ACT GAT GAG AAA GAT CCG CAC GGT TGG TTT CTC AGC GAC GAT CAC AAG 4944

Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys

1635 1640 1645

ACC TTT AGT GGT CTC TCT TCC GCA CAG GCA TTA AAG AAC GAC AGT GAA 4992

Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu

1650 1655 1660

CCG ATG GAT TTC TCT GGC GCC AAT GCT CTC TAT TTC TGG GAA CTG TTC 5040

Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe

1665 1670 1675 1680

TAT TAC ACG CCG ATG ATG ATG GCT CAT CGT TTG TTG CAG GAA CAG AAT 5088

Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn

1685 1690 1695

TTT GAT GCG GCG AAC CAT TGG TTC CGT TAT GTC TGG AGT CCA TCC GGT 5136

Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly

1700 1705 1710

TAT ATC GTT GAT GGT AAA ATT GCT ATC TAC CAC TGG AAC GTG CGA CCG 5184

Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro

1715 1720 1725

CTG GAA GAA GAC ACC AGT TGG AAT GCA CAA CAA CTG GAC TCC ACC GAT 5232

Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp

1730 1735 1740

CCA GAT GCT GTA GCC CAA GAT GAT CCG ATG CAC TAC AAG GTG GCT ACC 5280

Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr

1745 1750 1755 1760

TTT ATG GCG ACG TTG GAT CTG CTA ATG GCC CGT GGT GAT GCT GCT TAC 5328

Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr

1765 1770 1775

CGC CAG TTA GAG CGT GAT ACG TTG GCT GAA GCT AAA ATG TGG TAT ACA 5376

Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr

1780 1785 1790

CAG GCG CTT AAT CTG TTG GGT GAT GAG CCA CAA GTG ATG CTG AGT ACG 5424

Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr

1795 1800 1805

ACT TGG GCT AAT CCA ACA TTG GGT AAT GCT GCT TCA AAA ACC ACA CAG 5472

Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln

1810 1815 1820

CAG GTT CGT CAG CAA GTG CTT ACC CAG TTG CGT CTC AAT AGC AGG GTA 5520

Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val

1825 1830 1835 1840

AAA ACC CCG TTG 5532

Lys Thr Pro Leu

1844

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1844 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53 (TcbAii):

Features From To Description

Peptide 1 1844 TcbAii peptide

Fragment 1 11 (SEQ ID NO: 1)

Fragment 978 990 (SEQ ID NO: 23)

Fragment 1387 1401 (SEQ ID NO: 22)

Fragment 1484 1505 (SEQ ID NO: 24)

Fragment 1527 1552 (SEQ ID NO: 21)

Phe Ile Gln Gly Tyr Ser Asp Leu Phe Gly Asn Arg Ala Asp Asn Tyr

1 5 10 15

Ala Ala Pro Gly Ser Val Ala Ser Met Phe Ser Pro Ala Ala Tyr Leu

20 25 30

Thr Glu Leu Tyr Arg Glu Ala Lys Asn Leu His Asp Ser Ser Ser Ile

35 40 45

Tyr Tyr Leu Asp Lys Arg Arg Pro Asp Leu Ala Ser Leu Met Leu Ser

50 55 60

Gln Lys Asn Met Asp Glu Glu Ile Ser Thr Leu Ala Leu Ser Asn Glu

65 70 75 80

Leu Cys Leu Ala Gly Ile Glu Thr Lys Thr Gly Lys Ser Gln Asp Glu

85 90 95

Val Met Asp Met Leu Ser Thr Tyr Arg Leu Ser Gly Glu Thr Pro Tyr

100 105 110

His His Ala Tyr Glu Thr Val Arg Glu Ile Val His Glu Arg Asp Pro

115 120 125

Gly Phe Arg His Leu Ser Gln Ala Pro Ile Val Ala Ala Lys Leu Asp

130 135 140

Pro Val Thr Leu Leu Gly Ile Ser Ser His Ile Ser Pro Glu Leu Tyr

145 150 155 160

Asn Leu Leu Ile Glu Glu Ile Pro Glu Lys Asp Glu Ala Ala Leu Asp

165 170 175

Thr Leu Tyr Lys Thr Asn Phe Gly Asp Ile Thr Thr Ala Gln Leu Met

180 185 190

Ser Pro Ser Tyr Leu Ala Arg Tyr Tyr Gly Val Ser Pro Glu Asp Ile

195 200 205

Ala Tyr Val Thr Thr Ser Leu Ser His Val Gly Tyr Ser Ser Asp Ile

210 215 220

Leu Val Ile Pro Leu Val Asp Gly Val Gly Lys Met Glu Val Val Arg

225 230 235 240

Val Thr Arg Thr Pro Ser Asp Asn Tyr Thr Ser Gln Thr Asn Tyr Ile

245 250 255

Glu Leu Tyr Pro Gln Gly Gly Asp Asn Tyr Leu Ile Lys Tyr Asn Leu

260 265 270

Ser Asn Ser Phe Gly Leu Asp Asp Phe Tyr Leu Gln Tyr Lys Asp Gly

275 280 285

Ser Ala Asp Trp Thr Glu Ile Ala His Asn Pro Tyr Pro Asp Met Val

290 295 300

Ile Asn Gln Lys Tyr Glu Ser Gln Ala Thr Ile Lys Arg Ser Asp Ser

305 310 315 320

Asp Asn Ile Leu Ser Ile Gly Leu Gln Arg Trp His Ser Gly Ser Tyr

325 330 335

Asn Phe Ala Ala Ala Asn Phe Lys Ile Asp Gln Tyr Ser Pro Lys Ala

340 345 350

Phe Leu Leu Lys Met Asn Lys Ala Ile Arg Leu Leu Lys Ala Thr Gly

355 360 365

Leu Ser Phe Ala Thr Leu Glu Arg Ile Val Asp Ser Val Asn Ser Thr

370 375 380

Lys Ser Ile Thr Val Glu Val Leu Asn Lys Val Tyr Arg Val Lys Phe

385 390 395 400

Tyr Ile Asp Arg Tyr Gly Ile Ser Glu Glu Thr Ala Ala Ile Leu Ala

405 410 415

Asn Ile Asn Ile Ser Gln Gln Ala Val Gly Asn Gln Leu Ser Gln Phe

420 425 430

Glu Gln Leu Phe Asn His Pro Pro Leu Asn Gly Ile Arg Tyr Glu Ile

435 440 445

Ser Glu Asp Asn Ser Lys His Leu Pro Asn Pro Asp Leu Asn Leu Lys

450 455 460

Pro Asp Ser Thr Gly Asp Asp Gln Arg Lys Ala Val Leu Lys Arg Ala

465 470 475 480

Phe Gln Val Asn Ala Ser Glu Leu Tyr Gln Met Leu Leu Ile Thr Asp

485 490 495

Arg Lys Glu Asp Gly Val Ile Lys Asn Asn Leu Glu Asn Leu Ser Asp

500 505 510

Leu Tyr Leu Val Ser Leu Leu Ala Gln Ile His Asn Leu Thr Ile Ala

515 520 525

Glu Leu Asn Ile Leu Leu Val Ile Cys Gly Tyr Gly Asp Thr Asn Ile

530 535 540

Tyr Gln Ile Thr Asp Asp Asn Leu Ala Lys Ile Val Glu Thr Leu Leu

545 550 555 560

Trp Ile Thr Gln Trp Leu Lys Thr Gln Lys Trp Thr Val Thr Asp Leu

565 570 575

Phe Leu Met Thr Thr Ala Thr Tyr Ser Thr Thr Leu Thr Pro Glu Ile

580 585 590

Ser Asn Leu Thr Ala Thr Leu Ser Ser Thr Leu His Gly Lys Glu Ser

595 600 605

Leu Ile Gly Glu Asp Leu Lys Arg Ala Met Ala Pro Cys Phe Thr Ser

610 615 620

Ala Leu His Leu Thr Ser Gln Glu Val Ala Tyr Asp Leu Leu Leu Trp

625 630 635 640

Ile Asp Gln Ile Gln Pro Ala Gln Ile Thr Val Asp Gly Phe Trp Glu

645 650 655

Glu Val Gln Thr Thr Pro Thr Ser Leu Lys Val Ile Thr Phe Ala Gln

660 665 670

Val Leu Ala Gln Leu Ser Leu Ile Tyr Arg Arg Ile Gly Leu Ser Glu

675 680 685

Thr Glu Leu Ser Leu Ile Val Thr Gln Ser Ser Leu Leu Val Ala Gly

690 695 700

Lys Ser Ile Leu Asp His Gly Leu Leu Thr Leu Met Ala Leu Glu Gly

705 710 715 720

Phe His Thr Trp Val Asn Gly Leu Gly Gln His Ala Ser Leu Ile Leu

725 730 735

Ala Ala Leu Lys Asp Gly Ala Leu Thr Val Thr Asp Val Ala Gln Ala

740 745 750

Met Asn Lys Glu Glu Ser Leu Leu Gln Met Ala Ala Asn Gln Val Glu

755 760 765

Lys Asp Leu Thr Lys Leu Thr Ser Trp Thr Gln Ile Asp Ala Ile Leu

770 775 780

Gln Trp Leu Gln Met Ser Ser Ala Leu Ala Val Ser Pro Leu Asp Leu

785 790 795 800

Ala Gly Met Met Ala Leu Lys Tyr Gly Ile Asp His Asn Tyr Ala Ala

805 810 815

Trp Gln Ala Ala Ala Ala Ala Leu Met Ala Asp His Ala Asn Gln Ala

820 825 830

Gln Lys Lys Leu Asp Glu Thr Phe Ser Lys Ala Leu Cys Asn Tyr Tyr

835 840 845

Ile Asn Ala Val Val Asp Ser Ala Ala Gly Val Arg Asp Arg Asn Gly

850 855 860

Leu Tyr Thr Tyr Leu Leu Ile Asp Asn Gln Val Ser Ala Asp Val Ile

865 870 875 880

Thr Ser Arg Ile Ala Glu Ala Ile Ala Gly Ile Gln Leu Tyr Val Asn

885 890 895

Arg Ala Leu Asn Arg Asp Glu Gly Gln Leu Ala Ser Asp Val Ser Thr

900 905 910

Arg Gln Phe Phe Thr Asp Trp Glu Arg Tyr Asn Lys Arg Tyr Ser Thr

915 920 925

Trp Ala Gly Val Ser Glu Leu Val Tyr Tyr Pro Glu Asn Tyr Val Asp

930 935 940

Pro Thr Gln Arg Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln

945 950 955 960

Ser Ile Asn Gln Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala Phe

965 970 975

Lys Thr Tyr Leu Thr Ser Phe Glu Gln Val Ala Asn Leu Lys Val Ile

980 985 990

Ser Ala Tyr His Asp Asn Val Asn Val Asp Gln Gly Leu Thr Tyr Phe

995 1000 1005

Ile Gly Ile Asp Gln Ala Ala Pro Gly Thr Tyr Tyr Trp Arg Ser Val

1010 1015 1020

Asp His Ser Lys Cys Glu Asn Gly Lys Phe Ala Ala Asn Ala Trp Gly

1025 1030 1035 1040

Glu Trp Asn Lys Ile Thr Cys Ala Val Asn Pro Trp Lys Asn Ile Ile

1045 1050 1055

Arg Pro Val Val Tyr Met Ser Arg Leu Tyr Leu Leu Trp Leu Glu Gln

1060 1065 1070

Gln Ser Lys Lys Ser Asp Asp Gly Lys Thr Thr Ile Tyr Gln Tyr Asn

1075 1080 1085

Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Ser Trp Asn Thr Pro Phe

1090 1095 1100

Thr Phe Asp Val Thr Glu Lys Val Lys Asn Tyr Thr Ser Ser Thr Asp

1105 1110 1115 1120

Ala Ala Glu Ser Leu Gly Leu Tyr Cys Thr Gly Tyr Gln Gly Glu Asp

1125 1130 1135

Thr Leu Leu Val Met Phe Tyr Ser Met Gln Ser Ser Tyr Ser Ser Tyr

1140 1145 1150

Thr Asp Asn Asn Ala Pro Val Thr Gly Leu Tyr Ile Phe Ala Asp Met

1155 1160 1165

Ser Ser Asp Asn Met Thr Asn Ala Gln Ala Thr Asn Tyr Trp Asn Asn

1170 1175 1180

Ser Tyr Pro Gln Phe Asp Thr Val Met Ala Asp Pro Asp Ser Asp Asn

1185 1190 1195 1200

Lys Lys Val Ile Thr Arg Arg Val Asn Asn Arg Tyr Ala Glu Asp Tyr

1205 1210 1215

Glu Ile Pro Ser Ser Val Thr Ser Asn Ser Asn Tyr Ser Trp Gly Asp

1220 1225 1230

His Ser Leu Thr Met Leu Tyr Gly Gly Ser Val Pro Asn Ile Thr Phe

1235 1240 1245

Glu Ser Ala Ala Glu Asp Leu Arg Leu Ser Thr Asn Met Ala Leu Ser

1250 1255 1260

Ile Ile His Asn Gly Tyr Ala Gly Thr Arg Arg Ile Gln Cys Asn Leu

1265 1270 1275 1280

Met Lys Gln Tyr Ala Ser Leu Gly Asp Lys Phe Ile Ile Tyr Asp Ser

1285 1290 1295

Ser Phe Asp Asp Ala Asn Arg Phe Asn Leu Val Pro Leu Phe Lys Phe

1300 1305 1310

Gly Lys Asp Glu Asn Ser Asp Asp Ser Ile Cys Ile Tyr Asn Glu Asn

1315 1320 1325

Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe Ser Ser Lys Asp Asp Asn

1330 1335 1340

Lys Thr Ala Asp Tyr Asn Gly Gly Thr Gln Cys Ile Asp Ala Gly Thr

1345 1350 1355 1360

Ser Asn Lys Asp Phe Tyr Tyr Asn Leu Gln Glu Ile Glu Val Ile Ser

1365 1370 1375

Val Thr Gly Gly Tyr Trp Ser Ser Tyr Lys Ile Ser Asn Pro Ile Asn

1380 1385 1390

Ile Asn Thr Gly Ile Asp Ser Ala Lys Val Lys Val Thr Val Lys Ala

1395 1400 1405

Gly Gly Asp Asp Gln Ile Phe Thr Ala Asp Asn Ser Thr Tyr Val Pro

1410 1415 1420

Gln Gln Pro Ala Pro Ser Phe Glu Glu Met Ile Tyr Gln Phe Asn Asn

1425 1430 1435 1440

Leu Thr Ile Asp Cys Lys Asn Leu Asn Phe Ile Asp Asn Gln Ala His

1445 1450 1455

Ile Glu Ile Asp Phe Thr Ala Thr Ala Gln Asp Gly Arg Phe Leu Gly

1460 1465 1470

Ala Glu Thr Phe Ile Ile Pro Val Thr Lys Lys Val Leu Gly Thr Glu

1475 1480 1485

Asn Val Ile Ala Leu Tyr Ser Glu Asn Asn Gly Val Gln Tyr Met Gln

1490 1495 1500

Ile Gly Ala Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala Gln Gln Leu

1505 1510 1515 1520

Val Ser Arg Ala Asn Arg Gly Ile Asp Ala Val Leu Ser Met Glu Thr

1525 1530 1535

Gln Asn Ile Gln Glu Pro Gln Leu Gly Ala Gly Thr Tyr Val Gln Leu

1540 1545 1550

Val Leu Asp Lys Tyr Asp Glu Ser Ile His Gly Thr Asn Lys Ser Phe

1555 1560 1565

Ala Ile Glu Tyr Val Asp Ile Phe Lys Glu Asn Asp Ser Phe Val Ile

1570 1575 1580

Tyr Gln Gly Glu Leu Ser Glu Thr Ser Gln Thr Val Val Lys Val Phe

1585 1590 1595 1600

Leu Ser Tyr Phe Ile Glu Ala Thr Gly Asn Lys Asn His Leu Trp Val

1605 1610 1615

Arg Ala Lys Tyr Gln Lys Glu Thr Thr Asp Lys Ile Leu Phe Asp Arg

1620 1625 1630

Thr Asp Glu Lys Asp Pro His Gly Trp Phe Leu Ser Asp Asp His Lys

1635 1640 1645

Thr Phe Ser Gly Leu Ser Ser Ala Gln Ala Leu Lys Asn Asp Ser Glu

1650 1655 1660

Pro Met Asp Phe Ser Gly Ala Asn Ala Leu Tyr Phe Trp Glu Leu Phe

1665 1670 1675 1680

Tyr Tyr Thr Pro Met Met Met Ala His Arg Leu Leu Gln Glu Gln Asn

1685 1690 1695

Phe Asp Ala Ala Asn His Trp Phe Arg Tyr Val Trp Ser Pro Ser Gly

1700 1705 1710

Tyr Ile Val Asp Gly Lys Ile Ala Ile Tyr His Trp Asn Val Arg Pro

1715 1720 1725

Leu Glu Glu Asp Thr Ser Trp Asn Ala Gln Gln Leu Asp Ser Thr Asp

1730 1735 1740

Pro Asp Ala Val Ala Gln Asp Asp Pro Met His Tyr Lys Val Ala Thr

1745 1750 1755 1760

Phe Met Ala Thr Leu Asp Leu Leu Met Ala Arg Gly Asp Ala Ala Tyr

1765 1770 1775

Arg Gln Leu Glu Arg Asp Thr Leu Ala Glu Ala Lys Met Trp Tyr Thr

1780 1785 1790

Gln Ala Leu Asn Leu Leu Gly Asp Glu Pro Gln Val Met Leu Ser Thr

1795 1800 1805

Thr Trp Ala Asn Pro Thr Leu Gly Asn Ala Ala Ser Lys Thr Thr Gln

1810 1815 1820

Gln Val Arg Gln Gln Val Leu Thr Gln Leu Arg Leu Asn Ser Arg Val

1825 1830 1835 1840

Lys Thr Pro Leu

1844

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1722 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54 (tcbAiii coding

region):

CTA GGA ACA GCC AAT TCC CTG ACC GCT TTA TTC CTG CCG CAG GAA AAT 48

Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn

1 5 10 15

AGC AAG CTC AAA GGC TAC TGG CGG ACA CTG GCG CAG CGT ATG TTT AAT 96

Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn

20 25 30

TTA CGT CAT AAT CTG TCG ATT GAC GGC CAG CCG CTC TCC TTG CCG CTG 144

Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu

35 40 45

TAT GCT AAA CCG GCT GAT CCA AAA GCT TTA CTG AGT GCG GCG GTT TCA 192

Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser

50 55 60

GCT TCT CAA GGG GGA GCC GAC TTG CCG AAG GCG CCG CTG ACT ATT CAC 240

Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His

65 70 75 80

CGC TTC CCT CAA ATG CTA GAA GGG GCA CGG GGC TTG GTT AAC CAG CTT 288

Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu

85 90 95

ATA CAG TTC GGT AGT TCA CTA TTG GGG TAC AGT GAG CGT CAG GAT GCG 336

Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala

100 105 110

GAA GCT ATG AGT CAA CTA CTG CAA ACC CAA GCC AGC GAG TTA ATA CTG 384

Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu

115 120 125

ACC AGT ATT CGT ATG CAG GAT AAC CAA TTG GCA GAG CTG GAT TCG GAA 432

Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu

130 135 140

AAA ACC GCC TTG CAA GTC TCT TTA GCT GGA GTG CAA CAA CGG TTT GAC 480

Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp

145 150 155 160

AGC TAT AGC CAA CTG TAT GAG GAG AAC ATC AAC GCA GGT GAG CAG CGA 528

Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg

165 170 175

GCG CTG GCG TTA CGC TCA GAA TCT GCT ATT GAG TCT CAG GGA GCG CAG 576

Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln

180 185 190

ATT TCC CGT ATG GCA GGC GCG GGT GTT GAT ATG GCA CCA AAT ATC TTC 624

Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe

195 200 205

GGC CTG GCT GAT GGC GGC ATG CAT TAT GGT GCT ATT GCC TAT GCC ATC 672

Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile

210 215 220

GCT GAC GGT ATT GAG TTG AGT GCT TCT GCC AAG ATG GTT GAT GCG GAG 720

Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu

225 230 235 240

AAA GTT GCT CAG TCG GAA ATA TAT CGC CGT CGC CGT CAA GAA TGG AAA 768

Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys

245 250 255

ATT CAG CGT GAC AAC GCA CAA GCG GAG ATT AAC CAG TTA AAC GCG CAA 816

Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln

260 265 270

CTG GAA TCA CTG TCT ATT CGC CGT GAA GCC GCT GAA ATG CAA AAA GAG 864

Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu

275 280 285

TAC CTG AAA ACC CAG CAA GCT CAG GCG CAG GCA CAA CTT ACT TTC TTA 912

Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu

290 295 300

AGA AGC AAA TTC AGT AAT CAA GCG TTA TAT AGT TGG TTA CGA GGG CGT 960

Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg

305 310 315 320

TTG TCA GGT ATT TAT TTC CAG TTC TAT GAC TTG GCC GTA TCA CGT TGC 1008

Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys

325 330 335

CTG ATG GCA GAG CAA TCC TAT CAA TGG GAA GCT AAT GAT AAT TCC ATT 1056

Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile

340 345 350

AGC TTT GTC AAA CCG GGT GCA TGG CAA GGA ACT TAC GCC GGC TTA TTG 1104

Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu

355 360 365

TGT GGA GAA GCT TTG ATA CAA AAT CTG GCA CAA ATG GAA GAG GCA TAT 1152

Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr

370 375 380

CTG AAA TGG GAA TCT CGC GCT TTG GAA GTA GAA CGC ACG GTT TCA TTG 1200

Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu

385 390 395 400

GCA GTG GTT TAT GAT TCA CTG GAA GGT AAT GAT CGT TTT AAT TTA GCG 1248

Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala

405 410 415

GAA CAA ATA CCT GCA TTA TTG GAT AAG GGG GAG GGA ACA GCA GGA ACT 1296

Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr

420 425 430

AAA GAA AAT GGG TTA TCA TTG GCT AAT GCT ATC CTG TCA GCT TCG GTC 1344

Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val

435 440 445

AAA TTG TCC GAC TTG AAA CTG GGA ACG GAT TAT CCA GAC AGT ATC GTT 1392

Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val

450 455 460

GGT AGC AAC AAG GTT CGT CGT ATT AAG CAA ATC AGT GTT TCG CTA CCT 1440

Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro

465 470 475 480

GCA TTG GTT GGG CCT TAT CAG GAT GTT CAG GCT ATG CTC AGC TAT GGT 1488

Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly

485 490 495

GGC AGT ACT CAA TTG CCG AAA GGT TGT TCA GCG TTG GCT GTG TCT CAT 1536

Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His

500 505 510

GGT ACC AAT GAT AGT GGT CAG TTC CAG TTG GAT TTC AAT GAC GGC AAA 1584

Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys

515 520 525

TAC CTG CCA TTT GAA GGT ATT GCT CTT GAT GAT CAG GGT ACA CTG AAT 1632

Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn

530 535 540

CTT CAA TTT CCG AAT GCT ACC GAC AAG CAG AAA GCA ATA TTG CAA ACT 1680

Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr

545 550 555 560

ATG AGC GAT ATT ATT TTG CAT ATT CGT TAT ACC ATC CGT TAA 1722

Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg ⅴ?

565 570 573

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 573 amino acids

(B) TYPE: amino acids

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55 (TcbAiii):

Leu Gly Thr Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn

1 5 10 15

Ser Lys Leu Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe Asn

20 25 30

Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu Pro Leu

35 40 45

Tyr Ala Lys Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala Ala Val Ser

50 55 60

Ala Ser Gln Gly Gly Ala Asp Leu Pro Lys Ala Pro Leu Thr Ile His

65 70 75 80

Arg Phe Pro Gln Met Leu Glu Gly Ala Arg Gly Leu Val Asn Gln Leu

85 90 95

Ile Gln Phe Gly Ser Ser Leu Leu Gly Tyr Ser Glu Arg Gln Asp Ala

100 105 110

Glu Ala Met Ser Gln Leu Leu Gln Thr Gln Ala Ser Glu Leu Ile Leu

115 120 125

Thr Ser Ile Arg Met Gln Asp Asn Gln Leu Ala Glu Leu Asp Ser Glu

130 135 140

Lys Thr Ala Leu Gln Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp

145 150 155 160

Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg

165 170 175

Ala Leu Ala Leu Arg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln

180 185 190

Ile Ser Arg Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe

195 200 205

Gly Leu Ala Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile

210 215 220

Ala Asp Gly Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu

225 230 235 240

Lys Val Ala Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu Trp Lys

245 250 255

Ile Gln Arg Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu Asn Ala Gln

260 265 270

Leu Glu Ser Leu Ser Ile Arg Arg Glu Ala Ala Glu Met Gln Lys Glu

275 280 285

Tyr Leu Lys Thr Gln Gln Ala Gln Ala Gln Ala Gln Leu Thr Phe Leu

290 295 300

Arg Ser Lys Phe Ser Asn Gln Ala Leu Tyr Ser Trp Leu Arg Gly Arg

305 310 315 320

Leu Ser Gly Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ser Arg Cys

325 330 335

Leu Met Ala Glu Gln Ser Tyr Gln Trp Glu Ala Asn Asp Asn Ser Ile

340 345 350

Ser Phe Val Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala Gly Leu Leu

355 360 365

Cys Gly Glu Ala Leu Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr

370 375 380

Leu Lys Trp Glu Ser Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu

385 390 395 400

Ala Val Val Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala

405 410 415

Glu Gln Ile Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr

420 425 430

Lys Glu Asn Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val

435 440 445

Lys Leu Ser Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile Val

450 455 460

Gly Ser Asn Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser Leu Pro

465 470 475 480

Ala Leu Val Gly Pro Tyr Gln Asp Val Gln Ala Met Leu Ser Tyr Gly

485 490 495

Gly Ser Thr Gln Leu Pro Lys Gly Cys Ser Ala Leu Ala Val Ser His

500 505 510

Gly Thr Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe Asn Asp Gly Lys

515 520 525

Tyr Leu Pro Phe Glu Gly Ile Ala Leu Asp Asp Gln Gly Thr Leu Asn

530 535 540

Leu Gln Phe Pro Asn Ala Thr Asp Lys Gln Lys Ala Ile Leu Gln Thr

545 550 555 560

Met Ser Asp Ile Ile Leu His Ile Arg Tyr Thr Ile Arg ⅴ?

565 570 573

(2) INFORMATION FOR SEQ ID NO: 56

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2994 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56 (tccA)

1 ATG AAT CAA CTC GCC AGT CCC CTG ATT TCC CGC ACC GAA GAG ATC CAC 48

1 Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Glu Ile His 16

49 AAC TTA CCC GGT AAA TTG ACC GAT CTT GGT TAT ACC TCA GTG TTT GAT 96

17 Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp 32

97 GTG GTA CGT ATG CCG CGT GAG CGT TTT ATT CGT GAG CAT CGT GCT GAT 144

33 Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp 48

145 CTC GGG CGC AGT GCT GAA AAA ATG TAT GAC CTG GCA GTG GGC TAT GCT 192

49 Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala 64

193 CAT CAG GTG TTA CAC CAT TTT CGC CGT AAT TCT CTT AGT GAA GCT GTT 240

65 His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val 80

241 CAG TTT GGC TTG AGA AGT CCG TTC TCC GTA TCA GGC CCG GAT TAC GCC 288

81 Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala 96

289 AAT CAG TTT CTT GAT GCA AAC ACG GGT TGG AAA GAT AAA GCA CCA AGT 336

97 Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser 112

337 GGA TCA CCG GAA GCC AAT GAT GCG CCG GTA GCC TAT CTG ACT CAT ATT 384

113 Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile 128

385 TAT CAA TTG GCC CTT GAA CAG GAA AAG AAT GGC GCC ACT ACC ATT ATG 432

129 Tyr Gln Leu Ala Leu Glu Gln Glu Lys Asn Gly Ala Thr Thr Ile Met 144

433 AAT ACG CTG GCG GAG CGT CGC CCC GAT CTG GGT GCT TTG TTA ATT AAT 480

145 Asn Thr Leu Ala Glu Arg Arg Pro Asp Leu Gly Ala Leu Leu Ile Asn 160

481 GAT AAA GCA ATC AAT GAG GTG ATA CCG CAA TTG CAG TTG GTC AAT GAA 528

161 Asp Lys Ala Ile Asn Glu Val Ile Pro Gln Leu Gln Leu Val Asn Glu 176

529 ATT CTG TCC AAA GCT ATT CAG AAG AAA CTG AGT TTG ACT GAT CTG GAA 576

177 Ile Leu Ser Lys Ala Ile Gln Lys Lys Leu Ser Leu Thr Asp Leu Glu 192

577 GCG GTA AAC GCC AGA CTT TCC ACT ACC CGT TAC CCG AAT AAT CTG CCG 624

193 Ala Val Asn Ala Arg Leu Ser Thr Thr Arg Tyr Pro Asn Asn Leu Pro 208

625 TAT CAT TAT GGT CAT CAG CAG ATT CAG ACA GCT CAA TCG GTA TTG GGT 672

209 Tyr His Tyr Gly His Gln Gln Ile Gln Thr Ala Gln Ser Val Leu Gly 224

673 ACT ACG TTG CAA GAT ATC ACT TTG CCA CAG ACG CTG GAT CTG CCG CAA 720

225 Thr Thr Leu Gln Asp Ile Thr Leu Pro Gln Thr Leu Asp Leu Pro Gln 240

721 AAC TTC TGG GCA ACA GCA AAA GGA AAA CTG AGC GAT ACG ACT GCC AGT 768

241 Asn Phe Trp Ala Thr Ala Lys Gly Lys Leu Ser Asp Thr Thr Ala Ser 256

769 GCT TTG ACC CGA CTG CAA ATC ATG GCG AGT CAG TTT TCG CCA GAG CAG 816

257 Ala Leu Thr Arg Leu Gln Ile Met Ala Ser Gln Phe Ser Pro Glu Gln 272

817 CAG AAA ATC ATT ACG GAG ACT GTC GGT CAG GAT TTC TAT CAG CTT AAC 864

273 Gln Lys Ile Ile Thr Glu Thr Val Gly Gln Asp Phe Tyr Gln Leu Asn 288

865 TAT GGT GAC AGT TCG CTT ACT GTG AAT AGT TTC AGC GAC ATG ACC ATA 912

289 Tyr Gly Asp Ser Ser Leu Thr Val Asn Ser Phe Ser Asp Met Thr Ile 304

913 ATG ACT GAT CGA ACA AGT TTG ACT GTA CCC CAG GTA GAA CTG ATG TTG 960

305 Met Thr Asp Arg Thr Ser Leu Thr Val Pro Gln Val Glu Leu Met Leu 320

961 TGT TCA ACT GTC GGA GGT TCT ACG GTT GTT AAG TCT GAT AAT GTG AGT

1008

321 Cys Ser Thr Val Gly Gly Ser Thr Val Val Lys Ser Asp Asn Val Ser 336

1009 TCT GGT GAC ACG ACA GCG ACG CCA TTT GCG TAT GGC GCC CGC TTT ATT

1056

337 Ser Gly Asp Thr Thr Ala Thr Pro Phe Ala Tyr Gly Ala Arg Phe Ile 352

1057 CAT GCC GGT AAG CCG GAG GCG ATT ACC CTG AGT CGC AGT GGT GCG GAG

1104

353 His Ala Gly Lys Pro Glu Ala Ile Thr Leu Ser Arg Ser Gly Ala Glu 368

1105 GCG CAT TTT GCT CTG ACG GTT AAC AAT CTG ACA GAT GAC AAG TTG GAC

1152

369 Ala His Phe Ala Leu Thr Val Asn Asn Leu Thr Asp Asp Lys Leu Asp 384

1153 CGT ATT AAC CGC ACA GTG CGC CTG CAA AAA TGG CTG AAT CTG CCT TAT

1200

385 Arg Ile Asn Arg Thr Val Arg Leu Gln Lys Trp Leu Asn Leu Pro Tyr 400

1201 GAG GAT ATT GAC CTG TTA GTG ACT TCT GCT ATG GAT GCG GAA ACA GGA

1248

401 Glu Asp Ile Asp Leu Leu Val Thr Ser Ala Met Asp Ala Glu Thr Gly 416

1249 AAT ACC GCG CTG TCG ATG AAC GAC AAT ACG CTG CGT ATG TTG GGA GTG

1296

417 Asn Thr Ala Leu Ser Met Asn Asp Asn Thr Leu Arg Met Leu Gly Val 432

1297 TTC AAA CAT TAT CAG GCG AAG TAT GGT GTT AGC GCT AAA CAA TTT GCT

1344

433 Phe Lys His Tyr Gln Ala Lys Tyr Gly Val Ser Ala Lys Gln Phe Ala 448

1345 GGC TGG CTG CGC GTA GTG GCC CCG TTT GCC ATT ACA CCG GCA ACG CCG

1392

449 Gly Trp Leu Arg Val Val Ala Pro Phe Ala Ile Thr Pro Ala Thr Pro 464

1393 TTT TTA GAC CAA GTG TTT AAC TCC GTC GGC ACC TTT GAT ACA CCG TTT

1440

465 Phe Leu Asp Gln Val Phe Asn Ser Val Gly Thr Phe Asp Thr Pro Phe 480

1441 GTG ATA GAT AAT CAG GAT TTT GTC TAT ACA TTG ACC ACC GGG GGC GAT

1488

481 Val Ile Asp Asn Gln Asp Phe Val Tyr Thr Leu Thr Thr Gly Gly Asp 496

1489 GGG GCG CGT GTT AAG CAT ATC AGC ACG GCA CTG GGC CTC AAT CAT CGT

1536

497 Gly Ala Arg Val Lys His Ile Ser Thr Ala Leu Gly Leu Asn His Arg 512

1537 CAG TTC CTG TTA TTG GCG GAT AAT ATT GCC CGT CAA CAG GGG AAT GTC

1584

513 Gln Phe Leu Leu Leu Ala Asp Asn Ile Ala Arg Gln Gln Gly Asn Val 528

1585 ACG CAA AGC ACA CTC AAC TGT AAT CTG TTT GTG GTG TCA GCT TTC TAC

1632

529 Thr Gln Ser Thr Leu Asn Cys Asn Leu Phe Val Val Ser Ala Phe Tyr 544

1633 CGT CTG GCT AAT TTG GCG CGC ACA TTG GGG ATA AAT CCA GAG TCT TTC

1680

545 Arg Leu Ala Asn Leu Ala Arg Thr Leu Gly Ile Asn Pro Glu Ser Phe 560

1681 TGT GCC TTG GTT GAT CGA TTA GAT GCA GGT ACA GGC ATC GTC TGG CAG

1728

561 Cys Ala Leu Val Asp Arg Leu Asp Ala Gly Thr Gly Ile Val Trp Gln 576

1729 CAA TTG GCA GGG AAA CCC ACA ATC ACG GTA CCA CAA AAA GAT TCC CCG

1776

577 Gln Leu Ala Gly Lys Pro Thr Ile Thr Val Pro Gln Lys Asp Ser Pro 592

1777 CTG GCG GCG GAT ATT CTG AGT TTG CTG CAA GCG CTA AGT GCG ATT GCT

1824

593 Leu Ala Ala Asp Ile Leu Ser Leu Leu Gln Ala Leu Ser Ala Ile Ala 608

1825 CAA TGG CAA CAA CAG CAC GAT TTA GAA TTT TCA GCA CTG CTT TTG CTG

1872

609 Gln Trp Gln Gln Gln His Asp Leu Glu Phe Ser Ala Leu Leu Leu Leu 624

1873 TTG AGT GAC AAC CCT ATT TCT ACC TCG CAG GGC ACT GAC GAT CAA TTG

1920

625 Leu Ser Asp Asn Pro Ile Ser Thr Ser Gln Gly Thr Asp Asp Gln Leu 640

1921 AAC TTT ATC CGT CAA GTG TGG CAG AAC CTA GGC AGT ACG TTT GTG GGT

1968

641 Asn Phe Ile Arg Gln Val Trp Gln Asn Leu Gly Ser Thr Phe Val Gly 656

1969 GCA ACA TTG TTG TCC CGC AGT GGG GCA CCA TTA GTC GAT ACC AAC GGC

2016

657 Ala Thr Leu Leu Ser Arg Ser Gly Ala Pro Leu Val Asp Thr Asn Gly 672

2017 CAC GCT ATT GAC TGG TTT GCT CTG CTC TCA GCA GGT AAT AGT CCG CTT

2064

673 His Ala Ile Asp Trp Phe Ala Leu Leu Ser Ala Gly Asn Ser Pro Leu 688

2065 ATC GAT AAG GTT GGT CTG GTG ACT GAT GCT GGC ATA CAA AGT GTT ATA

2112

689 Ile Asp Lys Val Gly Leu Val Thr Asp Ala Gly Ile Gln Ser Val Ile 704

2113 GCA ACG GTG GTC AAT ACA CAA AGC TTA TCT GAT GAA GAT AAG AAG CTG

2160

705 Ala Thr Val Val Asn Thr Gln Ser Leu Ser Asp Glu Asp Lys Lys Leu 720

2161 GCA ATC ACT ACT CTG ACT AAT ACG TTG AAT CAG GTA CAG AAA ACT CAA

2208

721 Ala Ile Thr Thr Leu Thr Asn Thr Leu Asn Gln Val Gln Lys Thr Gln 736

2209 CAG GGC GTG GCC GTC AGT CTG TTG GCG CAG ACT CTG AAC GTG AGT CAG

2256

737 Gln Gly Val Ala Val Ser Leu Leu Ala Gln Thr Leu Asn Val Ser Gln 752

2257 TCA CTG CCT GCG TTA TTG TTG CGC TGG AGT GGA CAA ACA ACC TAC CAG

2304

753 Ser Leu Pro Ala Leu Leu Leu Arg Trp Ser Gly Gln Thr Thr Tyr Gln 768

2305 TGG TTG AGT GCG ACT TGG GCA TTG AAG GAT GCC GTT AAG ACT GCC GCC

2352

769 Trp Leu Ser Ala Thr Trp Ala Leu Lys Asp Ala Val Lys Thr Ala Ala 784

2353 GAT ATT CCC GCT GAC TAT CTG CGT CAA TTA CGT GAA GTG GTA CGC CGC

2400

785 Asp Ile Pro Ala Asp Tyr Leu Arg Gln Leu Arg Glu Val Val Arg Arg 800

2401 TCC TTG TTG ACC CAA CAA TTC ACG CTG AGT CCT GCA ATG GTG CAA ACC

2448

801 Ser Leu Leu Thr Gln Gln Phe Thr Leu Ser Pro Ala Met Val Gln Thr 816

2449 TTG CTG GAC TAT CCA GCC TAT TTT GGC GCT TCC GCA GAA ACA GTG ACC

2496

817 Leu Leu Asp Tyr Pro Ala Tyr Phe Gly Ala Ser Ala Glu Thr Val Thr 832

2497 GAT ATC AGT TTG TGG ATG CTT TAT ACC CTG AGC TGT TAT AGC GAT TTA

2544

833 Asp Ile Ser Leu Trp Met Leu Tyr Thr Leu Ser Cys Tyr Ser Asp Leu 848

2545 TTG CTC CAA ATG GGT GAA GCT GGT GGT ACC GAA GAT GAT GTA CTG GCC

2592

849 Leu Leu Gln Met Gly Glu Ala Gly Gly Thr Glu Asp Asp Val Leu Ala 864

2593 TAC TTA CGC ACA GCT AAT GCT ACC ACA CCG TTG AGC CAA TCT GAT GCT

2640

865 Tyr Leu Arg Thr Ala Asn Ala Thr Thr Pro Leu Ser Gln Ser Asp Ala 880

2641 GCA CAG ACG TTG GCA ACG CTA TTG GGT TGG GAG GTT AAC GAG TTG CAA

2688

881 Ala Gln Thr Leu Ala Thr Leu Leu Gly Trp Glu Val Asn Glu Leu Gln 896

2689 GCC GCT TGG TCG GTA TTG GGC GGG ATT GCC AAA ACC ACA CCG CAA CTG

2736

897 Ala Ala Trp Ser Val Leu Gly Gly Ile Ala Lys Thr Thr Pro Gln Leu 912

2737 GAT GCG CTT CTG CGT TTG CAA CAG GCA CAG AAC CAA ACT GGT CTT GGC

2784

913 Asp Ala Leu Leu Arg Leu Gln Gln Ala Gln Asn Gln Thr Gly Leu Gly 928

2785 GTT ACA CAG CAA CAG CAA GGC TAT CTC CTG AGT CGT GAC AGT GAT TAT

2832

929 Val Thr Gln Gln Gln Gln Gly Tyr Leu Leu Ser Arg Asp Ser Asp Tyr 944

2833 ACC CTT TGG CAA AGC ACC GGT CAG GCG CTG GTG GCT GGC GTA TCC CAT

2880

945 Thr Leu Trp Gln Ser Thr Gly Gln Ala Leu Val Ala Gly Val Ser His 960

2881 GTC AAG GGC AGT AAC TGA GCATGGCAGA GCTCACTACC TGAGTGGATT TGATTT

2934

961 Val Lys Gly Ser Asn End 965

2935 TTCCGTATGG CCTAATGAGG CTATTTCTAA ACCGCCATTT AAGTAAGGCA GATAATTATG

2994

(2) INFORMATION FOR SEQ ID NO: 57

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 965 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57 (TccA peptide)

Features From To Description

1 10 SEQ ID NO: 8

1 Met Asn Gln Leu Ala Ser Pro Leu Ile Ser Arg Thr Glu Glu Ile His 16

17 Asn Leu Pro Gly Lys Leu Thr Asp Leu Gly Tyr Thr Ser Val Phe Asp 32

33 Val Val Arg Met Pro Arg Glu Arg Phe Ile Arg Glu His Arg Ala Asp 48

49 Leu Gly Arg Ser Ala Glu Lys Met Tyr Asp Leu Ala Val Gly Tyr Ala 64

65 His Gln Val Leu His His Phe Arg Arg Asn Ser Leu Ser Glu Ala Val 80

81 Gln Phe Gly Leu Arg Ser Pro Phe Ser Val Ser Gly Pro Asp Tyr Ala 96

97 Asn Gln Phe Leu Asp Ala Asn Thr Gly Trp Lys Asp Lys Ala Pro Ser 112

113 Gly Ser Pro Glu Ala Asn Asp Ala Pro Val Ala Tyr Leu Thr His Ile 128

129 Tyr Gln Leu Ala Leu Glu Gln Glu Lys Asn Gly Ala Thr Thr Ile Met 144

145 Asn Thr Leu Ala Glu Arg Arg Pro Asp Leu Gly Ala Leu Leu Ile Asn 160

161 Asp Lys Ala Ile Asn Glu Val Ile Pro Gln Leu Gln Leu Val Asn Glu 176

177 Ile Leu Ser Lys Ala Ile Gln Lys Lys Leu Ser Leu Thr Asp Leu Glu 192

193 Ala Val Asn Ala Arg Leu Ser Thr Thr Arg Tyr Pro Asn Asn Leu Pro 208

209 Tyr His Tyr Gly His Gln Gln Ile Gln Thr Ala Gln Ser Val Leu Gly 224

225 Thr Thr Leu Gln Asp Ile Thr Leu Pro Gln Thr Leu Asp Leu Pro Gln 240

241 Asn Phe Trp Ala Thr Ala Lys Gly Lys Leu Ser Asp Thr Thr Ala Ser 256

257 Ala Leu Thr Arg Leu Gln Ile Met Ala Ser Gln Phe Ser Pro Glu Gln 272

273 Gln Lys Ile Ile Thr Glu Thr Val Gly Gln Asp Phe Tyr Gln Leu Asn 288

289 Tyr Gly Asp Ser Ser Leu Thr Val Asn Ser Phe Ser Asp Met Thr Ile 304

305 Met Thr Asp Arg Thr Ser Leu Thr Val Pro Gln Val Glu Leu Met Leu 320

321 Cys Ser Thr Val Gly Gly Ser Thr Val Val Lys Ser Asp Asn Val Ser 336

337 Ser Gly Asp Thr Thr Ala Thr Pro Phe Ala Tyr Gly Ala Arg Phe Ile 352

353 His Ala Gly Lys Pro Glu Ala Ile Thr Leu Ser Arg Ser Gly Ala Glu 368

369 Ala His Phe Ala Leu Thr Val Asn Asn Leu Thr Asp Asp Lys Leu Asp 384

385 Arg Ile Asn Arg Thr Val Arg Leu Gln Lys Trp Leu Asn Leu Pro Tyr 400

401 Glu Asp Ile Asp Leu Leu Val Thr Ser Ala Met Asp Ala Glu Thr Gly 416

417 Asn Thr Ala Leu Ser Met Asn Asp Asn Thr Leu Arg Met Leu Gly Val 432

433 Phe Lys His Tyr Gln Ala Lys Tyr Gly Val Ser Ala Lys Gln Phe Ala 448

449 Gly Trp Leu Arg Val Val Ala Pro Phe Ala Ile Thr Pro Ala Thr Pro 464

465 Phe Leu Asp Gln Val Phe Asn Ser Val Gly Thr Phe Asp Thr Pro Phe 480

481 Val Ile Asp Asn Gln Asp Phe Val Tyr Thr Leu Thr Thr Gly Gly Asp 496

497 Gly Ala Arg Val Lys His Ile Ser Thr Ala Leu Gly Leu Asn His Arg 512

513 Gln Phe Leu Leu Leu Ala Asp Asn Ile Ala Arg Gln Gln Gly Asn Val 528

529 Thr Gln Ser Thr Leu Asn Cys Asn Leu Phe Val Val Ser Ala Phe Tyr 544

545 Arg Leu Ala Asn Leu Ala Arg Thr Leu Gly Ile Asn Pro Glu Ser Phe 560

561 Cys Ala Leu Val Asp Arg Leu Asp Ala Gly Thr Gly Ile Val Trp Gln 576

577 Gln Leu Ala Gly Lys Pro Thr Ile Thr Val Pro Gln Lys Asp Ser Pro 592

593 Leu Ala Ala Asp Ile Leu Ser Leu Leu Gln Ala Leu Ser Ala Ile Ala 608

609 Gln Trp Gln Gln Gln His Asp Leu Glu Phe Ser Ala Leu Leu Leu Leu 624

625 Leu Ser Asp Asn Pro Ile Ser Thr Ser Gln Gly Thr Asp Asp Gln Leu 640

641 Asn Phe Ile Arg Gln Val Trp Gln Asn Leu Gly Ser Thr Phe Val Gly 656

657 Ala Thr Leu Leu Ser Arg Ser Gly Ala Pro Leu Val Asp Thr Asn Gly 672

673 His Ala Ile Asp Trp Phe Ala Leu Leu Ser Ala Gly Asn Ser Pro Leu 688

689 Ile Asp Lys Val Gly Leu Val Thr Asp Ala Gly Ile Gln Ser Val Ile 704

705 Ala Thr Val Val Asn Thr Gln Ser Leu Ser Asp Glu Asp Lys Lys Leu 720

721 Ala Ile Thr Thr Leu Thr Asn Thr Leu Asn Gln Val Gln Lys Thr Gln 736

737 Gln Gly Val Ala Val Ser Leu Leu Ala Gln Thr Leu Asn Val Ser Gln 752

753 Ser Leu Pro Ala Leu Leu Leu Arg Trp Ser Gly Gln Thr Thr Tyr Gln 768

769 Trp Leu Ser Ala Thr Trp Ala Leu Lys Asp Ala Val Lys Thr Ala Ala 784

785 Asp Ile Pro Ala Asp Tyr Leu Arg Gln Leu Arg Glu Val Val Arg Arg 800

801 Ser Leu Leu Thr Gln Gln Phe Thr Leu Ser Pro Ala Met Val Gln Thr 816

817 Leu Leu Asp Tyr Pro Ala Tyr Phe Gly Ala Ser Ala Glu Thr Val Thr 832

833 Asp Ile Ser Leu Trp Met Leu Tyr Thr Leu Ser Cys Tyr Ser Asp Leu 848

849 Leu Leu Gln Met Gly Glu Ala Gly Gly Thr Glu Asp Asp Val Leu Ala 864

865 Tyr Leu Arg Thr Ala Asn Ala Thr Thr Pro Leu Ser Gln Ser Asp Ala 880

881 Ala Gln Thr Leu Ala Thr Leu Leu Gly Trp Glu Val Asn Glu Leu Gln 896

897 Ala Ala Trp Ser Val Leu Gly Gly Ile Ala Lys Thr Thr Pro Gln Leu 912

913 Asp Ala Leu Leu Arg Leu Gln Gln Ala Gln Asn Gln Thr Gly Leu Gly 928

929 Val Thr Gln Gln Gln Gln Gly Tyr Leu Leu Ser Arg Asp Ser Asp Tyr 944

945 Thr Leu Trp Gln Ser Thr Gly Gln Ala Leu Val Ala Gly Val Ser His 960

961 Val Lys Gly Ser Asn 965

(2) INFORMATION FOR SEQ ID NO: 58

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4932 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58 (tccB)

1 ATG TTA TCG ACA ATG GAA AAA CAA CTG AAT GAA TCC CAG CGT GAT GCG 48

1 Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu Ser Gln Arg Asp Ala 16

49 TTG GTG ACT GGC TAT ATG AAT TTT GTG GCG CCG ACG TTG AAA GGC GTC 96

17 Leu Val Thr Gly Tyr Met Asn Phe Val Ala Pro Thr Leu Lys Gly Val 32

97 AGT GGT CAG CCG GTG ACG GTG GAA GAT TTA TAC GAA TAT TTG CTG ATT 144

33 Ser Gly Gln Pro Val Thr Val Glu Asp Leu Tyr Glu Tyr Leu Leu Ile 48

145 GAC CCG GAA GTG GCT GAT GAG GTT GAG ACG AGT CGG GTA GCA CAA GCG 192

49 Asp Pro Glu Val Ala Asp Glu Val Glu Thr Ser Arg Val Ala Gln Ala 64

193 ATT GCC AGC ATA CAG CAA TAT ATG ACT CGT CTG GTC AAC GGC TCT GAA 240

65 Ile Ala Ser Ile Gln Gln Tyr Met Thr Arg Leu Val Asn Gly Ser Glu 80

241 CCG GGG CGT CAG GCG ATG GAG CCT TCT ACA GCT AAC GAA TGG CGT GAT 288

81 Pro Gly Arg Gln Ala Met Glu Pro Ser Thr Ala Asn Glu Trp Arg Asp 96

289 AAT GAT AAC CAA TAT GCT ATC TGG GCT GCG GGG GCT GAG GTT CGA AAT 336

97 Asn Asp Asn Gln Tyr Ala Ile Trp Ala Ala Gly Ala Glu Val Arg Asn 112

337 TAC GCT GAA AAC TAT ATT TCA CCC ATC ACC CGG CAG GAA AAA AGC CAT 384

113 Tyr Ala Glu Asn Tyr Ile Ser Pro Ile Thr Arg Gln Glu Lys Ser His 128

385 TAT TTC TCG GAG CTG GAG ACG ACT TTA AAT CAG AAT CGA CTC GAT CCG 432

129 Tyr Phe Ser Glu Leu Glu Thr Thr Leu Asn Gln Asn Arg Leu Asp Pro 144

433 GAT CGT GTG CAG GAT GCT GTT TTG GCG TAT CTC AAT GAG TTT GAG GCA 480

145 Asp Arg Val Gln Asp Ala Val Leu Ala Tyr Leu Asn Glu Phe Glu Ala 160

481 GTG AGT AAT CTA TAT GTG CTC AGT GGT TAT ATT AAT CAG GAT AAA TTT 528

161 Val Ser Asn Leu Tyr Val Leu Ser Gly Tyr Ile Asn Gln Asp Lys Phe 176

529 GAC CAA GCT ATC TAC TAC TTT ATT GGT CGC ACT ACC ACT AAA CCG TAT 576

177 Asp Gln Ala Ile Tyr Tyr Phe Ile Gly Arg Thr Thr Thr Ly Lys Pro Tyr 192

577 CGC TAC TAC TGG CGT CAG ATG GAT TTG AGT AAG AAC CGT CAA GAT CCG 624

193 Arg Tyr Tyr Trp Arg Gln Met Asp Leu Ser Lys Asn Arg Gln Asp Pro 208

625 GCA GGG AAT CCG GTG ACG CCA AAT TGC TGG AAT GAT TGG CAG GAA ATC 672

209 Ala Gly Asn Pro Val Thr Pro Asn Cys Trp Asn Asp Trp Gln Glu Ile 224

673 ACT TTG CCG CTG TCT GGT GAT ACG GTG CTG GAG CAT ACA GTT CGC CCG 720

225 Thr Leu Pro Leu Ser Gly Asp Thr Val Leu Glu His Thr Val Arg Pro 240

721 GTA TTT TAT AAT GAT CGA CTA TAT GTG GCT TGG GTT GAG CGT GAC CCG 768

241 Val Phe Tyr Asn Asp Arg Leu Tyr Val Ala Trp Val Glu Arg Asp Pro 256

769 GCA GTA CAG AAG GAT GCT GAC GGT AAA AAC ATC GGT AAA ACC CAT GCC 816

257 Ala Val Gln Lys Asp Ala Asp Gly Lys Asn Ile Gly Lys Thr His Ala 272

817 TAC AAC ATA AAG TTT GGT TAT AAA CGT TAT GAT GAT ACT TGG ACA GCG 864

273 Tyr Asn Ile Lys Phe Gly Tyr Lys Arg Tyr Asp Asp Thr Trp Thr Ala 288

865 CCG AAT ACG ACC ACG TTA ATG ACA CAA CAA GCA GGG GAA AGT TCA GAA 912

289 Pro Asn Thr Thr Thr Leu Met Thr Gln Gln Ala Gly Glu Ser Ser Glu 304

913 ACA CAG CGA TCC AGC CTG CTG ATT GAT GAA TCT AGC ACC ACA TTG CGC 960

305 Thr Gln Arg Ser Ser Leu Leu Ile Asp Glu Ser Ser Thr Thr Leu Arg 320

961 CAA GTT AAT CTG TTG GCT ACC ACC GAT TTT AGT ATC GAT CCG ACG GAG

1008

321 Gln Val Asn Leu Leu Ala Thr Thr Asp Phe Ser Ile Asp Pro Thr Glu 336

1009 GAA ACG GAC AGT AAC CCG TAT GGC CGC CTA ATG TTG GGG GTG TTT GTC

1056

337 Glu Thr Asp Ser Asn Pro Tyr Gly Arg Leu Met Leu Gly Val Phe Val 352

1057 CGT CAA TTT GAA GGT GAT GGG GCC AAT AGA AAA AAT AAA CCC GTT GTT

1104

353 Arg Gln Phe Glu Gly Asp Gly Ala Asn Arg Lys Asn Lys Pro Val Val 368

1105 TAT GGT TAT CTC TAT TGT GAC TCA GCT TTC AAT CGT CAT GTT CTC AGG

1152

369 Tyr Gly Tyr Leu Tyr Cys Asp Ser Ala Phe Asn Arg His Val Leu Arg 384

1153 CCG TTA AGT AAG AAC TTT TTG TTC AGT ACT TAC CGT GAT GAA ACG GAT

1200

385 Pro Leu Ser Lys Asn Phe Leu Phe Ser Thr Tyr Arg Asp Glu Thr Asp 400

1201 GGT CAA AAC AGC TTG CAA TTT GCG GTA TAC GAT AAA AAG TAT GTA ATT

1248

401 Gly Gln Asn Ser Leu Gln Phe Ala Val Tyr Asp Lys Lys Tyr Val Ile 416

1249 ACT AAG GTT GTT ACA GGT GCA ACG GAA GAT CCC GAA AAT ACA GGA TGG

1296

417 Thr Lys Val Val Thr Gly Ala Thr Glu Asp Pro Glu Asn Thr Gly Trp 432

1297 GTA AGT AAA GTT GAT GAC TTG AAA CAA GGC ACT ACT GGG GCC TAT GTG

1344

433 Val Ser Lys Val Asp Asp Leu Lys Gln Gly Thr Thr Gly Ala Tyr Val 448

1345 TAT ATC GAT CAA GAT GGC CTG ACG CTT CAT ATA CAA ACC ACA ACT AAT

1392

449 Tyr Ile Asp Gln Asp Gly Leu Thr Leu His Ile Gln Thr Thr Thr Asn 464

1393 GGG GAT TTT ATT AAC CGT CAT ACG TTT GGA TAT AAC GAT CTT GTA TAT

1440

465 Gly Asp Phe Ile Asn Arg His Thr Phe Gly Tyr Asn Asp Leu Val Tyr 480

1441 GAT TCT AAG TCT GGT TAT GGT TTC ACG TGG TCA GGA AAT GAA GGT TTT

1488

481 Asp Ser Lys Ser Gly Tyr Gly Phe Thr Trp Ser Gly Asn Glu Gly Phe 496

1489 TAT CTG GAT TAC CAT GAT GGA AAT TAT TAC ACC TTT CAT AAT GCA ATA

1536

497 Tyr Leu Asp Tyr His Asp Gly Asn Tyr Tyr Thr Phe His Asn Ala Ile 512

1537 ATC AAC TAC TAT CCG TCT GGA TAT GGT GGT GGA TCT GTT CCT AAT GGA

1584

513 Ile Asn Tyr Tyr Pro Ser Gly Tyr Gly Gly Gly Ser Val Pro Asn Gly 528

1585 ACG TGG GCG TTA GAG CAA AGG ATT AAT GAG GGA TGG GCT ATT GCT CCC

1632

529 Thr Trp Ala Leu Glu Gln Arg Ile Asn Glu Gly Trp Ala Ile Ala Pro 544

1633 CTG CTT GAT ACT CTC CAT ACT GTT ACT GTG AAG GGC AGT TAT ATC GCT

1680

545 Leu Leu Asp Thr Leu His Thr Val Thr Val Lys Gly Ser Tyr Ile Ala 560

1681 TGG GAA GGG GAA ACA CCT ACC GGT TAT AAT CTG TAT ATT CCA GAT GGT

1728

561 Trp Glu Gly Glu Thr Pro Thr Gly Tyr Asn Leu Tyr Ile Pro Asp Gly 576

1729 ACC GTG TTG CTA GAT TGG TTT GAT AAA ATA AAT TTT GCT ATT GGT CTT

1776

577 Thr Val Leu Leu Asp Trp Phe Asp Lys Ile Asn Phe Ala Ile Gly Leu 592

1777 AAT AAG CTT GAG TCT GTA TTT ACG TCG CCA GAT TGG CCA ACA CTA ACC

1824

593 Asn Lys Leu Glu Ser Val Phe Thr Ser Pro Asp Trp Pro Thr Leu Thr 608

1825 ACT ATC AAA AAT TTC AGT AAA ATC GCC GAT AAC CGC AAA TTC TAT CAG

1872

609 Thr Ile Lys Asn Phe Ser Lys Ile Ala Asp Asn Arg Lys Phe Tyr Gln 624

1873 GAA ATC AAT GCT GAG ACG GCG GAT GGA CGC AAC CTG TTT AAA CGT TAC

1920

625 Glu Ile Asn Ala Glu Thr Ala Asp Gly Arg Asn Leu Phe Lys Arg Tyr 640

1921 AGT ACT CAA ACT TTC GGA CTT ACC AGC GGT GCG ACT TAT TCT ACA ACT

1968

641 Ser Thr Gln Thr Phe Gly Leu Thr Ser Gly Ala Thr Tyr Ser Thr Thr 656

1969 TAT ACT TTG TCT GAG GCG GAT TTC TCC ACT GAT CCG GAC AAA AAC TAC

2016

657 Tyr Thr Leu Ser Glu Ala Asp Phe Ser Thr Asp Pro Asp Lys Asn Tyr 672

2017 CTA CAG GTT TGT TTG AAT GTC GTG TGG GAT CAT TAT GAC CGC CCG TCA

2064

673 Leu Gln Val Cys Leu Asn Val Val Trp Asp His Tyr Asp Arg Pro Ser 688

2065 GGG AAA AAA GGG GCT TAT TCT TGG GTC AGT AAG TGG TTT AAC GTC TAT

2112

689 Gly Lys Lys Gly Ala Tyr Ser Trp Val Ser Lys Trp Phe Asn Val Tyr 704

2113 GTT GCG TTG CAA GAT AGC AAA GCT CCG GAT GCC ATT CCT CGA TTA GTT

2160

705 Val Ala Leu Gln Asp Ser Lys Ala Pro Asp Ala Ile Pro Arg Leu Val 720

2161 TCC CGT TAC GAT AGT AAA CGT GGT CTG GTG CAA TAT CTG GAC TTC TGG

2208

721 Ser Arg Tyr Asp Ser Lys Arg Gly Leu Val Gln Tyr Leu Asp Phe Trp 736

2209 ACC TCA TCA TTA CCC GCG AAA ACC CGT CTT AAC ACC ACC TTT GTG CGT

2256

737 Thr Ser Ser Leu Pro Ala Lys Thr Arg Leu Asn Thr Thr Phe Val Arg 752

2257 ACT TTG ATT GAG AAG GCT AAT CTG GGG CTG GAT AGT TTG CTG GAT TAC

2304

753 Thr Leu Ile Glu Lys Ala Asn Leu Gly Leu Asp Ser Leu Leu Asp Tyr 768

2305 ACC TTG CAG GCA GAT CCT TCT CTG GAA GCA GAT TTA GTG ACT GAC GGC

2352

769 Thr Leu Gln Ala Asp Pro Ser Leu Glu Ala Asp Leu Val Thr Asp Gly 784

2353 AAA AGC GAA CCA ATG GAC TTT AAT GGT TCA AAC GGT CTC TAT TTC TGG

2400

785 Lys Ser Glu Pro Met Asp Phe Asn Gly Ser Asn Gly Leu Tyr Phe Trp 800

2401 GAA TTG TTC TTT CAC CTG CCG TTT TTG GTT GCT ACA CGC TTT GCC AAC

2448

801 Glu Leu Phe Phe His Leu Pro Phe Leu Val Ala Thr Arg Phe Ala Asn 816

2449 GAA CAG CAA TTT TCG CCG GCA CAA AAG AGT TTG CAT TAC ATC TTT GAC

2496

817 Glu Gln Gln Phe Ser Pro Ala Gln Lys Ser Leu His Tyr Ile Phe Asp 832

2497 CCG GCG ATG AAA AAC AAG CCA CAC AAT GCC CCG GCT TAT TGG AAT GTA

2544

833 Pro Ala Met Lys Asn Lys Pro His Asn Ala Pro Ala Tyr Trp Asn Val 848

2545 CGT CCG TTG GTT GAA GGA AAC AGC GAT TTG TCA CGT CAT TTG GAC GAT

2592

849 Arg Pro Leu Val Glu Gly Asn Ser Asp Leu Ser Arg His Leu Asp Asp 864

2593 TCT ATA GAC CCA GAT ACT CAA GCT TAT GCT CAT CCG GTG ATA TAC CAG

2640

865 Ser Ile Asp Pro Asp Thr Gln Ala Tyr Ala His Pro Val Ile Tyr Gln 880

2641 AAA GCG GTG TTT ATT GCC TAT GTC AGT AAC CTG ATT GCT CAG GGA GAT

2688

881 Lys Ala Val Phe Ile Ala Tyr Val Ser Asn Leu Ile Ala Gln Gly Asp 896

2689 ATG TGG TAT CGC CAA TTG ACT CGT GAC GGT CTG ACT CAG GCC CGT GTC

2736

897 Met Trp Tyr Arg Gln Leu Thr Arg Asp Gly Leu Thr Gln Ala Arg Val 912

2737 TAT TAC AAT CTG GCC GCT GAA TTG CTA GGG CCT CGT CCG GAT GTA TCG

2784

913 Tyr Tyr Asn Leu Ala Ala Glu Leu Leu Gly Pro Arg Pro Asp Val Ser 928

2785 CTG AGT AGC ATT TGG ACG CCG CAA ACC CTG GAT ACC TTA GCA GCC GGG

2832

929 Leu Ser Ser Ile Trp Thr Pro Gln Thr Leu Asp Thr Leu Ala Ala Gly 944

2833 CAA AAA GCG GTT TTA CGT GAT TTT GAG CAC CAG TTG GCT AAT AGT GAT

2880

945 Gln Lys Ala Val Leu Arg Asp Phe Glu His Gln Leu Ala Asn Ser Asp 960

2881 ACC GCT TTA CCC GCA TTG CCG GGC CGC AAT GTC AGC TAC TTG AAA CTG

2928

961 Thr Ala Leu Pro Ala Leu Pro Gly Arg Asn Val Ser Tyr Leu Lys Leu 976

2929 GCA GAT AAT GGC TAC TTT AAT GAA CCG CTC AAT GTT CTG ATG TTG TCT

2976

977 Ala Asp Asn Gly Tyr Phe Asn Glu Pro Leu Asn Val Leu Met Leu Ser 992

2977 CAC TGG GAT ACG TTG GAT GCA CGG TTA TAC AAT CTG CGT CAT AAC CTG

3024

993 His Trp Asp Thr Leu Asp Ala Arg Leu Tyr Asn Leu Arg His Asn Leu

1008

3025 ACC GTT GAT GGC AAG CCG CTT TCG CTG CCG CTG TAT GCT GCG CCT GTT

3072

1009 Thr Val Asp Gly Lys Pro Leu Ser Leu Pro Leu Tyr Ala Ala Pro Val

1024

3073 GAT CCG GTA GCG TTG TTG GCT CAG CGT GCT CAG TCC GGC ACG TTG ACG

3120

1025 Asp Pro Val Ala Leu Leu Ala Gln Arg Ala Gln Ser Gly Thr Leu Thr

1040

3121 AAT GGC GTC AGT GGC GCC ATG TTG ACG GTG CCG CCA TAC CGT TTC AGC

3168

1041 Asn Gly Val Ser Gly Ala Met Leu Thr Val Pro Pro Tyr Arg Phe Ser

1056

3169 GCT ATG TTG CCG CGA GCT TAC AGC GCC GTG GGT ACG TTG ACC AGT TTT

3216

1057 Ala Met Leu Pro Arg Ala Tyr Ser Ala Val Gly Thr Leu Thr Ser Phe

1072

3217 GGT CAG AAC CTG CTT AGT TTG TTG GAA CGT AGC GAA CGA GCC TGT CAA

3264

1073 Gly Gln Asn Leu Leu Ser Leu Leu Glu Arg Ser Glu Arg Ala Cys Gln

1088

3265 GAA GAG TTG GCG CAA CAG CAA CTG TTG GAT ATG TCC AGC TAT GCC ATC

3312

1089 Glu Glu Leu Ala Gln Gln Gln Leu Leu Asp Met Ser Ser Tyr Ala Ile

1104

3313 ACG TTG CAA CAA CAG GCG CTG GAT GGA TTG GCG GCA GAT CGT CTG GCG

3360

1105 Thr Leu Gln Gln Gln Ala Leu Asp Gly Leu Ala Ala Asp Arg Leu Ala

1120

3361 CTG CTA GCT AGT CAG GCT ACG GCA CAA CGT CAT GAC CAT TAT TAC

3408

1121 Leu Leu Ala Ser Gln Ala Thr Ala Gln Gln Arg His Asp His Tyr Tyr

1136

3409 ACT CTG TAT CAG AAC AAC ATC TCC AGT GCG GAA CAA CTG GTG ATG GAC

3456

1137 Thr Leu Tyr Gln Asn Asn Ile Ser Ser Ala Glu Gln Leu Val Met Asp

1152

3457 ACC CAA ACG TCA GCA CAA TCC CTG ATT TCT TCT TCC ACT GGT GTA CAA

3504

1153 Thr Gln Thr Ser Ala Gln Ser Leu Ile Ser Ser Ser Thr Gly Val Gln

1168

3505 ACT GCC AGT GGG GCA CTG AAA GTG ATC CCG AAT ATC TTT GGT TTG GCT

3552

1169 Thr Ala Ser Gly Ala Leu Lys Val Ile Pro Asn Ile Phe Gly Leu Ala

1184

3553 GAT GGC GGC TCG CGC TAT GAA GGA GTA ACG GAA GCG ATT GCC ATC GGG

3600

1185 Asp Gly Gly Ser Arg Tyr Glu Gly Val Thr Glu Ala Ile Ala Ile Gly

1200

3601 TTA ATG GCT GCC GGA CAA GCC ACC AGC GTG GTG GCC GAG CGT CTG GCA

3648

1201 Leu Met Ala Ala Gly Gln Ala Thr Ser Val Val Ala Glu Arg Leu Ala

1216

3649 ACC ACG GAG AAT TAC CGC CGC CGC CGT GAA GAG TGG CAA ATC CAA TAC

3696

1217 Thr Thr Glu Asn Tyr Arg Arg Arg Arg Glu Glu Trp Gln Ile Gln Tyr

1232

3697 CAG CAG GCA CAG TCT GAG GTC GAC GCA TTA CAG AAA CAG TTG GAT GCG

3744

1233 Gln Gln Ala Gln Ser Glu Val Asp Ala Leu Gln Lys Gln Leu Asp Ala

1248

3745 CTG GCA GTG CGC GAG AAA GCA GCT CAA ACT TCC CTG CAA CAG GCG AAG

3792

1249 Leu Ala Val Arg Glu Lys Ala Ala Gln Thr Ser Leu Gln Gln Ala Lys

1264

3793 GCA CAG CAG GTA CAA ATT CGG ACC ATG CTG ACT TAC TTA ACT ACT CGT

3840

1265 Ala Gln Gln Val Gln Ile Arg Thr Met Leu Thr Tyr Leu Thr Thr Arg

1280

3841 TTC ACC CAG GCG ACT CTG TAC CAG TGG CTG AGT GGT CAA TTA TCC GCG

3888

1281 Phe Thr Gln Ala Thr Leu Tyr Gln Trp Leu Ser Gly Gln Leu Ser Ala

1296

3889 TTG TAT TAT CAA GCG TAT GAT GCC GTG GTT GCT CTC TGC CTC TCC GCC

3936

1297 Leu Tyr Tyr Gln Ala Tyr Asp Ala Val Val Ala Leu Cys Leu Ser Ala

1312

3937 CAA GCT TGC TGG CAG TAT GAA TTG GGT GAT TAC GCT ACC ACT TTT ATC

3984

1313 Gln Ala Cys Trp Gln Tyr Glu Leu Gly Asp Tyr Ala Thr Thr Phe Ile

1328

3985 CAG ACC GGT ACC TGG AAC GAC CAT TAC CGT GGT TTG CAA GTG GGG GAG

4032

1329 Gln Thr Gly Thr Trp Asn Asp His Tyr Arg Gly Leu Gln Val Gly Glu

1344

4033 ACA CTG CAA CTC AAT TTG CAT CAG ATG GAA GCG GCC TAT TTA GTT CGT

4080

1345 Thr Leu Gln Leu Asn Leu His Gln Met Glu Ala Ala Tyr Leu Val Arg

1360

4081 CAC GAA CGC CGT CTT AAT GTG ATC CGT ACT GTG TCG CTC AAA AGC CTA

4128

1361 His Glu Arg Arg Leu Asn Val Ile Arg Thr Val Ser Leu Lys Ser Leu

1376

4129 TTG GGT GAT GAT GGT TTT GGT AAG TTA AAA ACC GAA GGC AAA GTC GAC

4176

1377 Leu Gly Asp Asp Gly Phe Gly Lys Leu Lys Thr Glu Gly Lys Val Asp

1392

4177 TTT CCA TTA AGC GAA AAG CTG TTT GAC AAC GAC TAT CCG GGG CAC TAT

4224

1393 Phe Pro Leu Ser Glu Lys Leu Phe Asp Asn Asp Tyr Pro Gly His Tyr

1408

4225 TTG CGC CAG ATT AAA ACT GTG TCA GTG ACG TTG CCG ACG TTA GTC GGG

4272

1409 Leu Arg Gln Ile Lys Thr Val Ser Val Thr Leu Pro Thr Leu Val Gly

1424

4273 CCG TAT CAA AAC GTG AAG GCA ACG CTC ACT CAG ACC AGC AGC AGT ATA

4320

1425 Pro Tyr Gln Asn Val Lys Ala Thr Leu Thr Gln Thr Ser Ser Ser Ile

1440

4321 TTG TTA GCA GCA GAT ATC AAT GGT GTT AAA CGT CTC AAT GAT CCG ACA

4368

1441 Leu Leu Ala Ala Asp Ile Asn Gly Val Lys Arg Leu Asn Asp Pro Thr

1456

4369 GGT AAA GAG GGT GAT GCG ACG CAT ATT GTC ACC AAT CTG CGT GCC AGC

4416

1457 Gly Lys Glu Gly Asp Ala Thr His Ile Val Thr Asn Leu Arg Ala Ser

1472

4417 CAG CAG GTG GCG CTC TCT TCT GGC ATT AAT GAT GCC GGT AGC TTT GAG

4464

1473 Gln Gln Val Ala Leu Ser Ser Gly Ile Asn Asp Ala Gly Ser Phe Glu

1488

4465 TTG CGT TTG GAA GAT GAG CGC TAT CTA TCA TTT GAG GGG ACT GGA GCT

4512

1489 Leu Arg Leu Glu Asp Glu Arg Tyr Leu Ser Phe Glu Gly Thr Gly Ala

1504

4513 GTT TCC AAA TGG ACT CTT AAC TTC CCG CGT TCT GTG GAT GAG CAT ATT

4560

1505 Val Ser Lys Trp Thr Leu Asn Phe Pro Arg Ser Val Asp Glu His Ile

1520

4561 GAC GAT AAG ACA TTG AAA GCG GAT GAG ATG CAG GCC GCA CTG TTG GCG

4608

1521 Asp Asp Lys Thr Leu Lys Ala Asp Glu Met Gln Ala Ala Leu Leu Ala

1536

4609 AAT ATG GAT GAT GTG CTG GTG CAG GTG CAT TAT ACC GCC TGC GAC GGC

4656

1537 Asn Met Asp Asp Val Leu Val Gln Val His Tyr Thr Ala Cys Asp Gly

1552

4657 GGC GCC AGT TTC GCA AAC CAG GTC AAG AAA ACA CTC TCT TAA CATTAACTTT 4708

1553 Gly Ala Ser Phe Ala Asn Gln Val Lys Lys Thr Leu Ser End 1565

4709 TAACTAATCC CTCCCACTCT GTTCGCCAGA GTGGGAGAAG GTTTGTCATA TCTAAAATCA 4768

4770 ATCTTGCGAT CTTTCTCCAT TTCATTGGAA GGGAAGCTGT AAAACAAATA AGGAATATGA 4828

4829 TATG 4932

(2) INFORMATION FOR SEQ ID NO: 59

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1565 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59 (TccB peptide)

Features From To Description

1 Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu Ser Gln Arg Asp Ala

16

17 Leu Val Thr Gly Tyr Met Asn Phe Val Ala Pro Thr Leu Lys Gly Val

32

33 Ser Gly Gln Pro Val Thr Val Glu Asp Leu Tyr Glu Tyr Leu Leu Ile

48

49 Asp Pro Glu Val Ala Asp Glu Val Glu Thr Ser Arg Val Ala Gln Ala

64

65 Ile Ala Ser Ile Gln Gln Tyr Met Thr Arg Leu Val Asn Gly Ser Glu

80

81 Pro Gly Arg Gln Ala Met Glu Pro Ser Thr Ala Asn Glu Trp Arg Asp

96

97 Asn Asp Asn Gln Tyr Ala Ile Trp Ala Ala Gly Ala Glu Val Arg Asn

112

113 Tyr Ala Glu Asn Tyr Ile Ser Pro Ile Thr Arg Gln Glu Lys Ser His

128

129 Tyr Phe Ser Glu Leu Glu Thr Thr Leu Asn Gln Asn Arg Leu Asp Pro

144

145 Asp Arg Val Gln Asp Ala Val Leu Ala Tyr Leu Asn Glu Phe Glu Ala

160

161 Val Ser Asn Leu Tyr Val Leu Ser Gly Tyr Ile Asn Gln Asp Lys Phe

176

177 Asp Gln Ala Ile Tyr Tyr Phe Ile Gly Arg Thr Thr Thr Lys Pro Tyr

192

193 Arg Tyr Tyr Trp Arg Gln Met Asp Leu Ser Lys Asn Arg Gln Asp Pro

208

209 Ala Gly Asn Pro Val Thr Pro Asn Cys Trp Asn Asp Trp Gln Glu Ile

224

225 Thr Leu Pro Leu Ser Gly Asp Thr Val Leu Glu His Thr Val Arg Pro

240

241 Val Phe Tyr Asn Asp Arg Leu Tyr Val Ala Trp Val Glu Arg Asp Pro

256

257 Ala Val Gln Lys Asp Ala Asp Gly Lys Asn Ile Gly Lys Thr His Ala

272

273 Tyr Asn Ile Lys Phe Gly Tyr Lys Arg Tyr Asp Asp Thr Trp Thr Ala

288

289 Pro Asn Thr Thr Thr Leu Met Thr Gln Gln Ala Gly Glu Ser Ser Glu

304

305 Thr Gln Arg Ser Ser Leu Leu Ile Asp Glu Ser Ser Thr Thr Leu Arg

320

321 Gln Val Asn Leu Leu Ala Thr Thr Asp Phe Ser Ile Asp Pro Thr Glu

336

337 Glu Thr Asp Ser Asn Pro Tyr Gly Arg Leu Met Leu Gly Val Phe Val

352

353 Arg Gln Phe Glu Gly Asp Gly Ala Asn Arg Lys Asn Lys Pro Val Val

368

369 Tyr Gly Tyr Leu Tyr Cys Asp Ser Ala Phe Asn Arg His Val Leu Arg

384

385 Pro Leu Ser Lys Asn Phe Leu Phe Ser Thr Tyr Arg Asp Glu Thr Asp

400

401 Gly Gln Asn Ser Leu Gln Phe Ala Val Tyr Asp Lys Lys Tyr Val Ile

416

417 Thr Lys Val Val Thr Gly Ala Thr Glu Asp Pro Glu Asn Thr Gly Trp

432

433 Val Ser Lys Val Asp Asp Leu Lys Gln Gly Thr Thr Gly Ala Tyr Val

448

449 Tyr Ile Asp Gln Asp Gly Leu Thr Leu His Ile Gln Thr Thr Thr Asn

464

465 Gly Asp Phe Ile Asn Arg His Thr Phe Gly Tyr Asn Asp Leu Val Tyr

480

481 Asp Ser Lys Ser Gly Tyr Gly Phe Thr Trp Ser Gly Asn Glu Gly Phe

496

497 Tyr Leu Asp Tyr His Asp Gly Asn Tyr Tyr Thr Phe His Asn Ala Ile

512

513 Ile Asn Tyr Tyr Pro Ser Gly Tyr Gly Gly Gly Ser Val Pro Asn Gly

528

529 Thr Trp Ala Leu Glu Gln Arg Ile Asn Glu Gly Trp Ala Ile Ala Pro

544

545 Leu Leu Asp Thr Leu His Thr Val Thr Val Lys Gly Ser Tyr Ile Ala

560

561 Trp Glu Gly Glu Thr Pro Thr Gly Tyr Asn Leu Tyr Ile Pro Asp Gly

576

577 Thr Val Leu Leu Asp Trp Phe Asp Lys Ile Asn Phe Ala Ile Gly Leu

592

593 Asn Lys Leu Glu Ser Val Phe Thr Ser Pro Asp Trp Pro Thr Leu Thr

608

609 Thr Ile Lys Asn Phe Ser Lys Ile Ala Asp Asn Arg Lys Phe Tyr Gln

624

625 Glu Ile Asn Ala Glu Thr Ala Asp Gly Arg Asn Leu Phe Lys Arg Tyr

640

641 Ser Thr Gln Thr Phe Gly Leu Thr Ser Gly Ala Thr Tyr Ser Thr Thr

656

657 Tyr Thr Leu Ser Glu Ala Asp Phe Ser Thr Asp Pro Asp Lys Asn Tyr

672

673 Leu Gln Val Cys Leu Asn Val Val Trp Asp His Tyr Asp Arg Pro Ser

688

689 Gly Lys Lys Gly Ala Tyr Ser Trp Val Ser Lys Trp Phe Asn Val Tyr

704

705 Val Ala Leu Gln Asp Ser Lys Ala Pro Asp Ala Ile Pro Arg Leu Val

720

721 Ser Arg Tyr Asp Ser Lys Arg Gly Leu Val Gln Tyr Leu Asp Phe Trp

736

737 Thr Ser Ser Leu Pro Ala Lys Thr Arg Leu Asn Thr Thr Phe Val Arg

752

753 Thr Leu Ile Glu Lys Ala Asn Leu Gly Leu Asp Ser Leu Leu Asp Tyr

768

769 Thr Leu Gln Ala Asp Pro Ser Leu Glu Ala Asp Leu Val Thr Asp Gly

784

785 Lys Ser Glu Pro Met Asp Phe Asn Gly Ser Asn Gly Leu Tyr Phe Trp

800

801 Glu Leu Phe Phe His Leu Pro Phe Leu Val Ala Thr Arg Phe Ala Asn

816

817 Glu Gln Gln Phe Ser Pro Ala Gln Lys Ser Leu His Tyr Ile Phe Asp

832

833 Pro Ala Met Lys Asn Lys Pro His Asn Ala Pro Ala Tyr Trp Asn Val

848

849 Arg Pro Leu Val Glu Gly Asn Ser Asp Leu Ser Arg His Leu Asp Asp

864

865 Ser Ile Asp Pro Asp Thr Gln Ala Tyr Ala His Pro Val Ile Tyr Gln

880

881 Lys Ala Val Phe Ile Ala Tyr Val Ser Asn Leu Ile Ala Gln Gly Asp

896

897 Met Trp Tyr Arg Gln Leu Thr Arg Asp Gly Leu Thr Gln Ala Arg Val

912

913 Tyr Tyr Asn Leu Ala Ala Glu Leu Leu Gly Pro Arg Pro Asp Val Ser

928

929 Leu Ser Ser Ile Trp Thr Pro Gln Thr Leu Asp Thr Leu Ala Ala Gly

944

945 Gln Lys Ala Val Leu Arg Asp Phe Glu His Gln Leu Ala Asn Ser Asp

960

961 Thr Ala Leu Pro Ala Leu Pro Gly Arg Asn Val Ser Tyr Leu Lys Leu

976

977 Ala Asp Asn Gly Tyr Phe Asn Glu Pro Leu Asn Val Leu Met Leu Ser

992

993 His Trp Asp Thr Leu Asp Ala Arg Leu Tyr Asn Leu Arg His Asn Leu

1008

1009 Thr Val Asp Gly Lys Pro Leu Ser Leu Pro Leu Tyr Ala Ala Pro Val

1024

1025 Asp Pro Val Ala Leu Leu Ala Gln Arg Ala Gln Ser Gly Thr Leu Thr

1040

1041 Asn Gly Val Ser Gly Ala Met Leu Thr Val Pro Pro Tyr Arg Phe Ser

1056

1057 Ala Met Leu Pro Arg Ala Tyr Ser Ala Val Gly Thr Leu Thr Ser Phe

1072

1073 Gly Gln Asn Leu Leu Ser Leu Leu Glu Arg Ser Glu Arg Ala Cys Gln

1088

1089 Glu Glu Leu Ala Gln Gln Gln Leu Leu Asp Met Ser Ser Tyr Ala Ile

1104

1105 Thr Leu Gln Gln Gln Ala Leu Asp Gly Leu Ala Ala Asp Arg Leu Ala

1120

1121 Leu Leu Ala Ser Gln Ala Thr Ala Gln Gln Arg His Asp His Tyr Tyr

1136

1137 Thr Leu Tyr Gln Asn Asn Ile Ser Ser Ala Glu Gln Leu Val Met Asp

1152

1153 Thr Gln Thr Ser Ala Gln Ser Leu Ile Ser Ser Ser Thr Gly Val Gln

1168

1169 Thr Ala Ser Gly Ala Leu Lys Val Ile Pro Asn Ile Phe Gly Leu Ala

1184

1185 Asp Gly Gly Ser Arg Tyr Glu Gly Val Thr Glu Ala Ile Ala Ile Gly

1200

1201 Leu Met Ala Ala Gly Gln Ala Thr Ser Val Val Ala Glu Arg Leu Ala

1216

1217 Thr Thr Glu Asn Tyr Arg Arg Arg Arg Glu Glu Trp Gln Ile Gln Tyr

1232

1233 Gln Gln Ala Gln Ser Glu Val Asp Ala Leu Gln Lys Gln Leu Asp Ala

1248

1249 Leu Ala Val Arg Glu Lys Ala Ala Gln Thr Ser Leu Gln Gln Ala Lys

1264

1265 Ala Gln Gln Val Gln Ile Arg Thr Met Leu Thr Tyr Leu Thr Thr Arg

1280

1281 Phe Thr Gln Ala Thr Leu Tyr Gln Trp Leu Ser Gly Gln Leu Ser Ala

1296

1297 Leu Tyr Tyr Gln Ala Tyr Asp Ala Val Val Ala Leu Cys Leu Ser Ala

1312

1313 Gln Ala Cys Trp Gln Tyr Glu Leu Gly Asp Tyr Ala Thr Thr Phe Ile

1328

1329 Gln Thr Gly Thr Trp Asn Asp His Tyr Arg Gly Leu Gln Val Gly Glu

1344

1345 Thr Leu Gln Leu Asn Leu His Gln Met Glu Ala Ala Tyr Leu Val Arg

1360

1361 His Glu Arg Arg Leu Asn Val Ile Arg Thr Val Ser Leu Lys Ser Leu

1376

1377 Leu Gly Asp Asp Gly Phe Gly Lys Leu Lys Thr Glu Gly Lys Val Asp

1392

1393 Phe Pro Leu Ser Glu Lys Leu Phe Asp Asn Asp Tyr Pro Gly His Tyr

1408

1409 Leu Arg Gln Ile Lys Thr Val Ser Val Thr Leu Pro Thr Leu Val Gly

1424

1425 Pro Tyr Gln Asn Val Lys Ala Thr Leu Thr Gln Thr Ser Ser Ser Ile

1440

1441 Leu Leu Ala Ala Asp Ile Asn Gly Val Lys Arg Leu Asn Asp Pro Thr

1456

1457 Gly Lys Glu Gly Asp Ala Thr His Ile Val Thr Asn Leu Arg Ala Ser

1472

1473 Gln Gln Val Ala Leu Ser Ser Gly Ile Asn Asp Ala Gly Ser Phe Glu

1488

1489 Leu Arg Leu Glu Asp Glu Arg Tyr Leu Ser Phe Glu Gly Thr Gly Ala

1504

1505 Val Ser Lys Trp Thr Leu Asn Phe Pro Arg Ser Val Asp Glu His Ile

1520

1521 Asp Asp Lys Thr Leu Lys Ala Asp Glu Met Gln Ala Ala Leu Leu Ala

1536

1537 Asn Met Asp Asp Val Leu Val Gln Val His Tyr Thr Ala Cys Asp Gly

1552

1553 Gly Ala Ser Phe Ala Asn Gln Val Lys Lys Thr Leu Ser 1565

(2) INFORMATION FOR SEQ ID NO: 60

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3132 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60 (tccC)

1 ATG AGT CCG TCT GAG ACT ACT CTT TAT ACT CAA ACC CCA ACA GTC AGC 48

1 Met Ser Pro Ser Glu Thr Thr Leu Tyr Thr Gln Thr Pro Thr Val Ser 16

49 GTG TTA GAT AAT CGC GGT CTG TCC ATT CGT GAT ATT GGT TTT CAC CGT 96

17 Val Leu Asp Asn Arg Gly Leu Ser Ile Arg Asp Ile Gly Phe His Arg 32

97 ATT GTA ATC GGG GGG GAT ACT GAC ACC CGC GTC ACC CGT CAC CAG TAT

144

33 Ile Val Ile Gly Gly Asp Thr Asp Thr Arg Val Thr Arg His Gln Tyr 48

145 GAT GCC CGT GGA CAC CTG AAC TAC AGT ATT GAC CCA CGC TTG TAT GAT

192

49 Asp Ala Arg Gly His Leu Asn Tyr Ser Ile Asp Pro Arg Leu Tyr Asp 64

193 GCA AAG CAG GCT GAT AAC TCA GTA AAG CCT AAT TTT GTC TGG CAG CAT

240

65 Ala Lys Gln Ala Asp Asn Ser Val Lys Pro Asn Phe Val Trp Gln His 80

241 GAT CTG GCC GGT CAT GCC CTG CGG ACA GAG AGT GTC GAT GCT GGT CGT

288

81 Asp Leu Ala Gly His Ala Leu Arg Thr Glu Ser Val Asp Ala Gly Arg 96

289 ACT GTT GCA TTG AAT GAT ATT GAA GGT CGT TCG GTA ATG ACA ATG AAT

336

97 Thr Val Ala Leu Asn Asp Ile Glu Gly Arg Ser Val Met Thr Met Asn

112

337 GCG ACC GGT GTT CGT CAG ACC CGT CGC TAT GAA GGC AAC ACC TTG CCC

384

113 Ala Thr Gly Val Arg Gln Thr Arg Arg Tyr Glu Gly Asn Thr Leu Pro

128

385 GGT CGC TTG TTA TCT GTG AGC GAG CAA GTT TTC AAC CAA GAG AGT GCT

432

129 Gly Arg Leu Leu Ser Val Ser Glu Gln Val Phe Asn Gln Glu Ser Ala

144

433 AAA GTG ACA GAG CGC TTT ATC TGG GCT GGG AAT ACA ACC TCG GAG AAA

480

145 Lys Val Thr Glu Arg Phe Ile Trp Ala Gly Asn Thr Thr Ser Glu Lys

160

481 GAG TAT AAC CTC TCC GGT CTG TGT ATA CGC CAC TAC GAC ACA GCG GGA

528

161 Glu Tyr Asn Leu Ser Gly Leu Cys Ile Arg His Tyr Asp Thr Ala Gly

176

529 GTG ACC CGG TTG ATG AGT CAG TCA CTG GCG GGC GCC ATG CTA TCC CAA

576

177 Val Thr Arg Leu Met Ser Gln Ser Leu Ala Gly Ala Met Leu Ser Gln

192

577 TCT CAC CAA TTG CTG GCG GAA GGG CAG GAG GCT AAC TGG AGC GGT GAC

624

193 Ser His Gln Leu Leu Ala Glu Gly Gln Glu Ala Asn Trp Ser Gly Asp

208

625 GAC GAA ACT GTC TGG CAG GGA ATG CTG GCA AGT GAG GTC TAT ACG ACA

672

209 Asp Glu Thr Val Trp Gln Gly Met Leu Ala Ser Glu Val Tyr Thr Thr

224

673 CAA AGT ACC ACT AAT GCC ATC GGG GCT TTA CTG ACC CAA ACC GAT GCG

720

225 Gln Ser Thr Thr Asn Ala Ile Gly Ala Leu Leu Thr Gln Thr Asp Ala

240

721 AAA GGC AAT ATT CAG CGT CTG GCT TAT GAC ATT GCC GGT CAG TTA AAA

768

241 Lys Gly Asn Ile Gln Arg Leu Ala Tyr Asp Ile Ala Gly Gln Leu Lys

256

769 GGG AGT TGG TTG ACG GTG AAA GGC CAG AGT GAA CAG GTG ATT GTT AAG

816

257 Gly Ser Trp Leu Thr Val Lys Gly Gln Ser Glu Gln Val Ile Val Lys

272

817 TCC CTG AGC TGG TCA GCC GCA GGT CAT AAA TTG CGT GAA GAG CAC GGT

864

273 Ser Leu Ser Trp Ser Ala Ala Gly His Lys Leu Arg Glu Glu His Gly

288

865 AAC GGC GTG GTT ACG GAG TAC AGT TAT GAG CCG GAA ACT CAA CGT CTG

912

289 Asn Gly Val Val Thr Glu Tyr Ser Tyr Glu Pro Glu Thr Gln Arg Leu

304

913 ATA GGT ATC ACC ACC CGG CGT GCC GAA GGG AGT CAA TCA GGA GCC AGA

960

305 Ile Gly Ile Thr Thr Arg Arg Ala Glu Gly Ser Gln Ser Gly Ala Arg

320

961 GTA TTG CAG GAT CTA CGC TAT AAG TAT GAT CCG GTG GGG AAT GTT ATC

1008

321 Val Leu Gln Asp Leu Arg Tyr Lys Tyr Asp Pro Val Gly Asn Val Ile 336

1009 AGT ATC CAT AAT GAT GCC GAA GCT ACC CGC TTT TGG CGT AAT CAG AAA

1056

337 Ser Ile His Asn Asp Ala Glu Ala Thr Arg Phe Trp Arg Asn Gln Lys 352

1057 GTG GAG CCG GAG AAT CGC TAT GTT TAT GAT TCT CTG TAT CAG CTT ATG

1104

353 Val Glu Pro Glu Asn Arg Tyr Val Tyr Asp Ser Leu Tyr Gln Leu Met 368

1105 AGT GCG ACA GGG CGT GAA ATG GCT AAT ATC GGT CAG CAA AGC AAC CAA

1152

369 Ser Ala Thr Gly Arg Glu Met Ala Asn Ile Gly Gln Gln Ser Asn Gln

384

1153 CTT CCC TCA CCC GTT ATA CCT GTT CCT ACT GAC GAC AGC ACT TAT ACC

1200

385 Leu Pro Ser Pro Val Ile Pro Val Pro Thr Asp Asp Ser Thr Tyr Thr 400

1201 AAT TAC CTT CGT ACC TAT ACT TAT GAC CGT GGC GGT AAT TTG GTT CAA

1248

401 Asn Tyr Leu Arg Thr Tyr Thr Tyr Asp Arg Gly Gly Asn Leu Val Gln 416

1249 ATC CGA CAC AGT TCA CCC GCG ACT CAA AAT AGT TAC ACC ACA GAT ATC

1296

417 Ile Arg His Ser Ser Pro Ala Thr Gln Asn Ser Tyr Thr Thr Asp Ile 432

1297 ACC GTT TCA AGC CGC AGT AAC CGG GCG GTA TTG AGT ACA TTA ACG ACA

1344

433 Thr Val Ser Ser Arg Ser Asn Arg Ala Val Leu Ser Thr Leu Thr Thr 448

1345 GAT CCA ACC CGA GTG GAT GCG CTA TTT GAT TCC GGC GGT CAT CAG AAG

1392

449 Asp Pro Thr Arg Val Asp Ala Leu Phe Asp Ser Gly Gly His Gln Lys 464

1393 ATG TTA ATA CCG GGG CAA AAT CTG GAT TGG AAT ATT CGG GGT GAA TTG

1440

465 Met Leu Ile Pro Gly Gln Asn Leu Asp Trp Asn Ile Arg Gly Glu Leu 480

1441 CAA CGA GTC ACA CCG GTG AGC CGT GAA AAT AGC AGT GAC AGT GAA TGG

1488

481 Gln Arg Val Thr Pro Val Ser Arg Glu Asn Ser Ser Asp Ser Glu Trp 496

1489 TAT CGC TAT AGC AGT GAT GGC ATG CGG CTG CTA AAA GTG AGT GAA CAG

1536

497 Tyr Arg Tyr Ser Ser Asp Gly Met Arg Leu Leu Lys Val Ser Glu Gln 512

1537 CAG ACG GGC AAC AGT ACT CAA GTA CAA CGG GTG ACT TAT CTG CCG GGA

1584

513 Gln Thr Gly Asn Ser Thr Gln Val Gln Arg Val Thr Tyr Leu Pro Gly 528

1585 TTA GAG CTA CGG ACA ACT GGG GTT GCA GAT AAA ACA ACC GAA GAT TTG

1632

529 Leu Glu Leu Arg Thr Thr Gly Val Ala Asp Lys Thr Thr Glu Asp Leu 544

1633 CAG GTG ATT ACG GTA GGT GAA GCG GGT CGC GCA CAG GTA AGG GTA TTG

1680

545 Gln Val Ile Thr Val Gly Glu Ala Gly Arg Ala Gln Val Arg Val Leu 560

1681 CAC TGG GAA AGT GGT AAG CCG ACA GAT ATT GAC AAC AAT CAG GTG CGC

1728

561 His Trp Glu Ser Gly Lys Pro Thr Asp Ile Asp Asn Asn Gln Val Arg 576

1729 TAC AGC TAC GAT AAT CTG CTT GGC TCC AGC CAG CTT GAA CTG GAT AGC

1776

577 Tyr Ser Tyr Asp Asn Leu Leu Gly Ser Ser Gln Leu Glu Leu Asp Ser 592

1777 GAA GGG CAG ATT CTC AGT CAG GAA GAG TAT TAT CCG TAT GGC GGT ACG

1824

593 Glu Gly Gln Ile Leu Ser Gln Glu Glu Tyr Tyr Pro Tyr Gly Gly Thr 608

1825 GCG ATA TGG GCG GCG AGA AAT CAG ACA GAA GCC AGC TAC AAA TTT ATT

1872

609 Ala Ile Trp Ala Ala Arg Asn Gln Thr Glu Ala Ser Tyr Lys Phe Ile 624

1873 CGT TAC TCC GGT AAA GAG CGG GAT GCC ACT GGA TTG TAT TAT TAC GGC

1920

625 Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr Gly Leu Tyr Tyr Tyr Gly 640

1921 TAC CGT TAT TAT CAA CCT TGG GTG GGT CGA TGG TTG AGT GCT GAT CCG

1968

641 Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg Trp Leu Ser Ala Asp Pro 656

1969 GCG GGA ACC GTG GAT GGG CTG AAT TTG TAC CGA ATG GTG AGG AAT AAC

2016

657 Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr Arg Met Val Arg Asn Asn 672

2017 CCC ATC ACA TTG ACT GAC CAT GAC GGA TTA GCA CCG TCT CCA AAT AGA

2064

673 Pro Ile Thr Leu Thr Asp His Asp Gly Leu Ala Pro Ser Pro Asn Arg 688

2065 AAT CGA AAT ACA TTT TGG TTT GCT TCA TTT TTG TTT CGT AAA CCT GAT

2112

689 Asn Arg Asn Thr Phe Trp Phe Ala Ser Phe Leu Phe Arg Lys Pro Asp 704

2113 GAG GGA ATG TCC GCG TCA ATG AGA CGG GGA CAA AAA ATT GGC AGA GCC

2160

705 Glu Gly Met Ser Ala Ser Met Arg Arg Gly Gln Lys Ile Gly Arg Ala 720

2161 ATT GCC GGC GGG ATT GCG ATT GGC GGT CTT GCG GCT ACC ATT GCC GCT

2208

721 Ile Ala Gly Gly Ile Ala Ile Gly Gly Leu Ala Ala Thr Ile Ala Ala 736

2209 ACG GCT GGC GCG GCT ATC CCC GTC ATT CTG GGG GTT GCG GCC GTA GGC

2256

737 Thr Ala Gly Ala Ala Ile Pro Val Ile Leu Gly Val Ala Ala Val Gly 752

2257 GCG GGG ATT GGC GCG TTG ATG GGA TAT AAC GTC GGT AGC CTG CTG GAA

2304

753 Ala Gly Ile Gly Ala Leu Met Gly Tyr Asn Val Gly Ser Leu Leu Glu 768

2305 AAA GGC GGG GCA TTA CTT GCT CGA CTC GTA CAG GGG AAA TCG ACG TTA

2352

769 Lys Gly Gly Ala Leu Leu Ala Arg Leu Val Gln Gly Lys Ser Thr Leu 784

2353 GTA CAG TCG GCG GCT GGC GCG GCT GCC GGA GCG AGT TCA GCC GCG GCT

2400

785 Val Gln Ser Ala Ala Gly Ala Ala Ala Gly Ala Ser Ser Ala Ala Ala 800

2401 TAT GGC GCA CGG GCA CAA GGT GTC GGT GTT GCA TCA GCC GCC GGG GCG

2448

801 Tyr Gly Ala Arg Ala Gln Gly Val Gly Val Ala Ser Ala Ala Gly Ala 816

2449 GTA ACA GGG GCT GTG GGA TCA TGG ATA AAT AAT GCT GAT CGG GGG ATT

2496

817 Val Thr Gly Ala Val Gly Ser Trp Ile Asn Asn Ala Asp Arg Gly Ile 832

2497 GGC GGC GCT ATT GGG GCC GGG AGT GCG GTA GGC ACC ATT GAT ACT ATG

2544

833 Gly Gly Ala Ile Gly Ala Gly Ser Ala Val Gly Thr Ile Asp Thr Met 848

2545 TTA GGG ACT GCC TCT ACC CTT ACC CAT GAA GTC GGG GCA GCG GCG GGT

2592

849 Leu Gly Thr Ala Ser Thr Leu Thr His Glu Val Gly Ala Ala Ala Gly 864

2593 GGG GCG GCG GGT GGG ATG ATC ACC GGT ACG CAA GGG AGT ACT CGG GCA

2640

865 Gly Ala Ala Gly Gly Met Ile Thr Gly Thr Gln Gly Ser Thr Arg Ala 880

2641 GGT ATC CAT GCC GGT ATT GGC ACC TAT TAT GGC TCC TGG ATT GGT TTT

2688

881 Gly Ile His Ala Gly Ile Gly Thr Tyr Tyr Gly Ser Trp Ile Gly Phe 896

2689 GGT TTA GAT GTC GCT AGT AAC CCC GCC GGA CAT TTA GCG AAT TAC GCA

2736

897 Gly Leu Asp Val Ala Ser Asn Pro Ala Gly His Leu Ala Asn Tyr Ala 912

2737 GTG GGT TAT GCC GCT GGT TTG GGT GCT GAA ATG GCT GTC AAC AGA ATA

2784

913 Val Gly Tyr Ala Ala Gly Leu Gly Ala Glu Met Ala Val Asn Arg Ile 928

2785 ATG GGT GGT GGA TTT TTG AGT AGG CTC TTA GGC CGG GTT GTC AGC CCA

2832

929 Met Gly Gly Gly Phe Leu Ser Arg Leu Leu Gly Arg Val Val Ser Pro 944

2833 TAT GCC GCC GGT TTA GCC AGA CAA TTA GTA CAT TTC AGT GTC GCC AGA

2880

945 Tyr Ala Ala Gly Leu Ala Arg Gln Leu Val His Phe Ser Val Ala Arg 960

2881 CCT GTC TTT GAG CCG ATA TTT AGT GTT CTC GGC GGG CTT GTC GGT GGT

2928

961 Pro Val Phe Glu Pro Ile Phe Ser Val Leu Gly Gly Leu Val Gly Gly 976

2929 ATT GGA ACT GGC CTG CAC AGA GTG ATG GGA AGA GAG AGT TGG ATT TCC

2976

977 Ile Gly Thr Gly Leu His Arg Val Met Gly Arg Glu Ser Trp Ile Ser 992

2977 AGA GCG TTA AGT GCT GCC GGT AGT GGT ATA GAT CAT GTC GCT GGC ATG

3024

993 Arg Ala Leu Ser Ala Ala Gly Ser Gly Ile Asp His Val Ala Gly Met

1008

3025 ATT GGT AAT CAG ATC AGA GGC AGG GTC TTG ACC ACA ACC GGG ATC GCT

3072

1009 Ile Gly Asn Gln Ile Arg Gly Arg Val Leu Thr Thr Thr Gly Ile Ala

1024

3073 AAT GCG ATA GAC TAT GGC ACC AGT GCT GTG GGA GCC GCA CGA CGA GTT

3120

1025 Asn Ala Ile Asp Tyr Gly Thr Ser Ala Val Gly Ala Ala Arg Arg Val

1040

3121 TTT TCT TTG TAA 3132

1041 Phe Ser Leu End 1043

(2) INFORMATION FOR SEQ ID NO: 61

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1043 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61 (TccC peptide)

1 Met Ser Pro Ser Glu Thr Thr Leu Tyr Thr Gln Thr Pro Thr Val Ser 16

17 Val Leu Asp Asn Arg Gly Leu Ser Ile Arg Asp Ile Gly Phe His Arg 32

33 Ile Val Ile Gly Gly Asp Thr Asp Thr Arg Val Thr Arg His Gln Tyr 48

49 Asp Ala Arg Gly His Leu Asn Tyr Ser Ile Asp Pro Arg Leu Tyr Asp 64

65 Ala Lys Gln Ala Asp Asn Ser Val Lys Pro Asn Phe Val Trp Gln His 80

81 Asp Leu Ala Gly His Ala Leu Arg Thr Glu Ser Val Asp Ala Gly Arg 96

97 Thr Val Ala Leu Asn Asp Ile Glu Gly Arg Ser Val Met Thr Met Asn 112

113 Ala Thr Gly Val Arg Gln Thr Arg Arg Tyr Glu Gly Asn Thr Leu Pro 128

129 Gly Arg Leu Leu Ser Val Ser Glu Gln Val Phe Asn Gln Glu Ser Ala 144

145 Lys Val Thr Glu Arg Phe Ile Trp Ala Gly Asn Thr Thr Ser Glu Lys 160

161 Glu Tyr Asn Leu Ser Gly Leu Cys Ile Arg His Tyr Asp Thr Ala Gly 176

177 Val Thr Arg Leu Met Ser Gln Ser Leu Ala Gly Ala Met Leu Ser Gln 192

193 Ser His Gln Leu Leu Ala Glu Gly Gln Glu Ala Asn Trp Ser Gly Asp 208

209 Asp Glu Thr Val Trp Gln Gly Met Leu Ala Ser Glu Val Tyr Thr Thr 224

225 Gln Ser Thr Thr Asn Ala Ile Gly Ala Leu Leu Thr Gln Thr Asp Ala 240

241 Lys Gly Asn Ile Gln Arg Leu Ala Tyr Asp Ile Ala Gly Gln Leu Lys 256

257 Gly Ser Trp Leu Thr Val Lys Gly Gln Ser Glu Gln Val Ile Val Lys 272

273 Ser Leu Ser Trp Ser Ala Ala Gly His Lys Leu Arg Glu Glu His Gly 288

289 Asn Gly Val Val Thr Glu Tyr Ser Tyr Glu Pro Glu Thr Gln Arg Leu 304

305 Ile Gly Ile Thr Thr Arg Arg Ala Glu Gly Ser Gln Ser Gly Ala Arg 320

321 Val Leu Gln Asp Leu Arg Tyr Lys Tyr Asp Pro Val Gly Asn Val Ile 336

337 Ser Ile His Asn Asp Ala Glu Ala Thr Arg Phe Trp Arg Asn Gln Lys 352

353 Val Glu Pro Glu Asn Arg Tyr Val Tyr Asp Ser Leu Tyr Gln Leu Met 368

369 Ser Ala Thr Gly Arg Glu Met Ala Asn Ile Gly Gln Gln Ser Asn Gln 384

385 Leu Pro Ser Pro Val Ile Pro Val Pro Thr Asp Asp Ser Thr Tyr Thr 400

401 Asn Tyr Leu Arg Thr Tyr Thr Tyr Asp Arg Gly Gly Asn Leu Val Gln 416

417 Ile Arg His Ser Ser Pro Ala Thr Gln Asn Ser Tyr Thr Thr Asp Ile 432

433 Thr Val Ser Ser Arg Ser Asn Arg Ala Val Leu Ser Thr Leu Thr Thr 448

449 Asp Pro Thr Arg Val Asp Ala Leu Phe Asp Ser Gly Gly His Gln Lys 464

465 Met Leu Ile Pro Gly Gln Asn Leu Asp Trp Asn Ile Arg Gly Glu Leu 480

481 Gln Arg Val Thr Pro Val Ser Arg Glu Asn Ser Ser Asp Ser Glu Trp 496

497 Tyr Arg Tyr Ser Ser Asp Gly Met Arg Leu Leu Lys Val Ser Glu Gln 512

513 Gln Thr Gly Asn Ser Thr Gln Val Gln Arg Val Thr Tyr Leu Pro Gly 528

529 Leu Glu Leu Arg Thr Thr Gly Val Ala Asp Lys Thr Thr Glu Asp Leu 544

545 Gln Val Ile Thr Val Gly Glu Ala Gly Arg Ala Gln Val Arg Val Leu 560

561 His Trp Glu Ser Gly Lys Pro Thr Asp Ile Asp Asn Asn Gln Val Arg 576

577 Tyr Ser Tyr Asp Asn Leu Leu Gly Ser Ser Gln Leu Glu Leu Asp Ser 592

593 Glu Gly Gln Ile Leu Ser Gln Glu Glu Tyr Tyr Pro Tyr Gly Gly Thr 608

609 Ala Ile Trp Ala Ala Arg Asn Gln Thr Glu Ala Ser Tyr Lys Phe Ile 624

625 Arg Tyr Ser Gly Lys Glu Arg Asp Ala Thr Gly Leu Tyr Tyr Tyr Gly 640

641 Tyr Arg Tyr Tyr Gln Pro Trp Val Gly Arg Trp Leu Ser Ala Asp Pro 656

657 Ala Gly Thr Val Asp Gly Leu Asn Leu Tyr Arg Met Val Arg Asn Asn 672

673 Pro Ile Thr Leu Thr Asp His Asp Gly Leu Ala Pro Ser Pro Asn Arg 688

689 Asn Arg Asn Thr Phe Trp Phe Ala Ser Phe Leu Phe Arg Lys Pro Asp 704

705 Glu Gly Met Ser Ala Ser Met Arg Arg Gly Gln Lys Ile Gly Arg Ala 720

721 Ile Ala Gly Gly Ile Ala Ile Gly Gly Leu Ala Ala Thr Ile Ala Ala 736

737 Thr Ala Gly Ala Ala Ile Pro Val Ile Leu Gly Val Ala Ala Val Gly 752

753 Ala Gly Ile Gly Ala Leu Met Gly Tyr Asn Val Gly Ser Leu Leu Glu 768

769 Lys Gly Gly Ala Leu Leu Ala Arg Leu Val Gln Gly Lys Ser Thr Leu 784

785 Val Gln Ser Ala Ala Gly Ala Ala Ala Gly Ala Ser Ser Ala Ala Ala 800

801 Tyr Gly Ala Arg Ala Gln Gly Val Gly Val Ala Ser Ala Ala Gly Ala 816

817 Val Thr Gly Ala Val Gly Ser Trp Ile Asn Asn Ala Asp Arg Gly Ile 832

833 Gly Gly Ala Ile Gly Ala Gly Ser Ala Val Gly Thr Ile Asp Thr Met 848

849 Leu Gly Thr Ala Ser Thr Leu Thr His Glu Val Gly Ala Ala Ala Gly 864

865 Gly Ala Ala Gly Gly Met Ile Thr Gly Thr Gln Gly Ser Thr Arg Ala 880

881 Gly Ile His Ala Gly Ile Gly Thr Tyr Tyr Gly Ser Trp Ile Gly Phe 896

897 Gly Leu Asp Val Ala Ser Asn Pro Ala Gly His Leu Ala Asn Tyr Ala 912

913 Val Gly Tyr Ala Ala Gly Leu Gly Ala Glu Met Ala Val Asn Arg Ile 928

929 Met Gly Gly Gly Phe Leu Ser Arg Leu Leu Gly Arg Val Val Ser Pro 944

945 Tyr Ala Ala Gly Leu Ala Arg Gln Leu Val His Phe Ser Val Ala Arg 960

961 Pro Val Phe Glu Pro Ile Phe Ser Val Leu Gly Gly Leu Val Gly Gly 976

977 Ile Gly Thr Gly Leu His Arg Val Met Gly Arg Glu Ser Trp Ile Ser 992

993 Arg Ala Leu Ser Ala Ala Gly Ser Gly Ile Asp His Val Ala Gly Met 1008

1009 Ile Gly Asn Gln Ile Arg Gly Arg Val Leu Thr Thr Thr Gly Ile Ala 1024

1025 Asn Ala Ile Asp Tyr Gly Thr Ser Ala Val Gly Ala Ala Arg Arg Val 1040

1041 Phe Ser Leu 1043

(2) INFORMATION FOR SEQ ID NO: 62: TcaAiv

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: TcaAiv

Asn Ile Gly Gly Asp

1 5

(2) INFORMATION FOR SEQ ID NO: 63: TcaAii-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: TcaAii-syn

Cys Leu Arg Gly Asn Ser Pro Thr Asn Pro Asp Lys Asp Gly Ile

1 5 10 15

Phe Ala Gln Val Ala

20

(2) INFORMATION FOR SEQ ID NO: 64: TcaAiii-syn

(i) SEQUENCE CHARACTERISTICS;

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: Internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: TcaAiii-syn

Cys Tyr Thr Pro Asp Gln Thr Pro Ser Phe Tyr Glu Thr Ala Phe

1 5 10 15

Arg Ser Ala Asp Gly

20

(2) INFORMATION FOR SEQ ID NO: 65: TcaBi-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: Internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: TcaBi-syn

His Gly Gln Ser Tyr Asn Asp Asn Asn Tyr Cys Asn Phe Thr Leu

1 5 10 15

Ser Ile Asn Thr

19

(2) INFORMATION FOR SEQ ID NO: 66: TcaBii-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: TcaBii-syn

Cys Val Asp Pro Lys Thr Leu Gln Arg Gln Gln Ala Gly Gly Asp

1 5 10 15

Gly Thr Gly Ser Ser

20

(2) INFORMATION FOR SEQ ID NO: 67: TcaC-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: TcaC-syn

Cys Tyr Lys Ala Pro Gln Arg Gln Glu Asp Gly Asp Ser Asn Ala

1 5 10 15

Val Thr Tyr Asp Lys

20

(2) INFORMATION FOR SEQ ID NO: 68: TcbAii-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: TcbAii-syn

Cys Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr Phe

1 5 10 15

Ser Ser Lys Asp Asp

20

(2) INFORMATION FOR SEQ ID NO: 69: TcbAiii-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: TcbAiii-syn

Cys Phe Asp Ser Tyr Ser Gln Leu Tyr Glu Glu Asn Ile Asn Ala

1 5 10 15

Gly Glu Gln Arg Ala

20

(2) INFORMATION FOR SEQ ID NO: 70: TcdAii-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: TcdAii-syn

Cys Asn Pro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val

1 5 10 15

Tyr Gln Tyr Ser Gly Asn Thr

20

(2) INFORMATION FOR SEQ ID NO: 71: TcdAiii-syn

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: TcdAiii-syn

Val Ser Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn Asn Asn Leu

1 5 10 15

Ala Phe Gly Ala Gly

20

(2) INFORMATION FOR SEQ ID NO: 72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: 160 kDa-Hb

Met Gln Asp Ser Pro Glu Val Ala Ile Thr Thr Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: 170 kDa-WIR

Met Gln Arg Ser Ser Glu Val Ser

1 5

(2) INFORMATION FOR SEQ ID NO: 74:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: 180 kDa-H9

Met Gln Asp Ile Pro Glu Val Gln Leu Asn

1 5 10

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: 170 kDa-Hm (2)

INFORMATION FOR SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

Met Gln Asp Ser Pro Glu Val Ser Val Thr Gln Asn

1 5 10

(2) INFORMATION FOR SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: 74 kDa-H9

Ser Glu Ser Leu Phe Thr Gln Ser Leu Lys Glu Ala Arg Arg Asp

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: 71 kDa-Hb

Met Asn Leu Ile Glu Ala Lys Leu Gln Glu Asn Arg Asp Ala

1 5 10

(2) INFORMATION FOR SEQ ID NO: 78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: 170 kDa-H9

Met Leu Ser Thr Met Glu Lys Gln Leu Asn Glu Ser Gln Arg Asp

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: 109 kDa-Hm

Met Leu Asp Ile Met Glu Lys Gln Leu Asn Glu Ser Glu Arg Asp

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: 170 kDa-WX-1

Met Gln Asp Ser Arg Glu Val Ser

1 5

(2) INFORMATION FOR SEQ ID NO: 81:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: 69 kDa-H9

Leu Arg Ser Ala Xxx Ser Ala Leu Thr Thr Leu Leu

1 5 10

(2) INFORMATION FOR SEQ ID NO: 82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: 64 kDa-HP88

Leu Lys Leu Ala Asp Asn Gly Tyr Phe Asn Glu Pro Leu Asn Val

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: 70 kDa-NC-1

Leu Lys Leu Ala Asp Asn Ser Tyr Phe Asn Glu Pro Leu Asn

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: 60 kDa-WIR

Ser Lys Asp Glu Ser Lys Ala Asp Ser Gln Leu Val Tyr His Thr

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: 58 kDa-NC-1

Met Lys Lys Arg Gly Leu Thr Thr Asn Ala Gly Ala Pro Val

1 5 10

(2) INFORMATION FOR SEQ ID NO: 86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: 60 kDa-WX-12

Met Leu Asn Pro Ile Val Arg Lys Phe Glu Tyr Gly Glu His Thr

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: 60 kDa-Hm

Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys Leu Asp Leu Tyr Gly

1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: protein

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: 140 kDa-Hm

Asn Leu Ile Glu Ala Thr Leu Glu Gln Asn Leu Arg Asp Ala

1 5 10 15

Claims (99)

A composition comprising an effective amount of a Photorhabdus protein toxin having a functional activity against an insect. The composition of claim 1, wherein the photolabduus toxin is produced by a purified culture of photolapus, a transgenic plant, a baculovirus, or a heterologous microbial host. The method of claim 2, wherein the toxin is a photo picture drawer Douce drawer Douce luminescent composition produced by a culture of purified (P. luminescens). The composition of claim 2, wherein the toxin is produced from a purified culture of photolabduum luminescence strain designated ATCC 55397. 3. The composition of claim 2, wherein the toxin is produced by purified culture of photolabduum luminescence strain designated W-14. The method of claim 1, wherein the toxin is WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX- 11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC # 43948, ATCC # 43949, ATCC # 43950, ATCC # 43951, ATCC # 43952, DEP1, DEP2, DEP3, p. Giallanda Rica, p. Hepialus, HB-Arg, HB Oswego, HB Leviston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A. Cowes, MP1, MP2, MP3, MP4 , MP5, GL98, GL101, GL138, GL55, GL217, or GL257 picture specified by the drawer Douce produced by a purified culture of a strain composition. The method of claim 2, wherein the toxin is WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX- 11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC # 43948, ATCC # 43949, ATCC # 43950, ATCC # 43951, ATCC # 43952, DEP1, DEP2, DEP3, p. Zilandrica, P. Hepialus, HB-Arg, HB Oswego, HB Leviston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A. A composition produced from a purified culture of photolabduum luminescence strains designated as cowes, MP1, MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL55, GL217 or GL257. The composition of claim 1, wherein the toxin is represented by the amino acid sequence of SEQ ID NO: 12. The method of claim 6, wherein the composition of the mixture composition of one or more toxins produced from cultured waters purification picture drawer Douce. The method of claim 1 or claim 6, wherein the insect is Lepidoptera (Lepidoptera), Coleoptera (Coleoptera), logging (Hymenoptera), Diptera (Diptera), room simok (Dictyoptera), mites neck (Acarina) or the same time the neck (Homoptera) Phosphorus composition. 7. The composition of claim 1 or 6, wherein the insect species belong to the coleoptera and are southern corn rootworm, western corn rootworm, colorado potato beetle, wheat worm, ball weevil or grass grub. 7. A composition according to claim 1 or 6, wherein the insect species belong to Lepidoptera and are beetle maggots, black beetles, cabbage beetle, codling moths, corn beetles, European corn borer, horned moth or gray tobacco moth. 7. The composition of claim 1 or 6, wherein the toxin is formulated as a spray pesticide. 7. The composition of claim 1 or 6, wherein the toxin is formulated as a bait matrix and delivered to the above ground or underground bait station. A method for controlling insects comprising orally delivering to the insect an effective amount of a protein toxin having a functional activity against the insect, produced by the purified bacterial culture of the genus Photorapdus . The method of claim 15, wherein the bacterium is a purified culture of photolabduum luminescence . 16. The method of claim 15, wherein the toxin is produced from a purified culture of photolabduum luminescence strain designated ATCC 55397. 17. The method of claim 16, wherein the toxin is produced from a purified culture of photolabduus luminescence strain designated W-14. The method of claim 15, wherein the toxin is WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX- 11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC # 43948, ATCC # 43949, ATCC # 43950, ATCC # 43951, ATCC # 43952, DEP1, DEP2, DEP3, p. Zilandrica, P. Hepialus, HB-Arg, HB Oswego, HB Leviston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A. Cowes, MP1, method of production from a culture purified in picture drawer Douce strain designated as MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL55, GL217 , or GL257. The method of claim 15, wherein the toxin is WX-1, WX-2, WX-3, WX-4, WX-5, WX-6, WX-7, WX-8, WX-9, WX-10, WX- 11, WX-12, WX-14, WX-15, H9, Hb, Hm, HP88, NC-1, W30, WIR, B2, ATCC # 43948, ATCC # 43949, ATCC # 43950, ATCC # 43951, ATCC # 43952, DEP1, DEP2, DEP3, p. Zilandrica, P. Hepialus, HB-Arg, HB Oswego, HB Leviston, K-122, HMGD, Indicus, GD, PWH-5, Megidis, HF-85, A. The method is produced from purified culture of Photorabdus luminescence strains designated as Kaus, MP1, MP2, MP3, MP4, MP5, GL98, GL101, GL138, GL155, GL217 or GL257. 20. The method of claim 19, wherein the mixture of one or more toxins is produced from a purified culture of photolabose and the toxins are delivered orally to insects. The method of claim 15, wherein the toxin is produced by a prokaryotic host transformed with a gene encoding the toxin. The method of claim 15, wherein the toxin is produced by a eukaryotic host transformed with a gene encoding the toxin. The method of claim 23, wherein the eukaryotic host is baculovirus. 20. The composition according to claim 15 or 19, wherein the insect is lepidoptera, coleoptera, felling, fly, rapeseed, mite or simulane. 20. The method of claim 15 or 19, wherein the insect species belong to the coleopteran and are southern corn rootworm, western corn rootworm, Colorado potato beetle, wheat worm, ball weevil or grass grub. 20. The method of claim 15 or 19, wherein the insect species belong to Lepidoptera and are beetle maggots, black beetles, cabbage beetle, codling moths, corn beetles, European corn borer, horned moth or gray tobacco moth. The method of claim 15 or 19, wherein the toxin is formulated as a spray pesticide. 20. The method of claim 15 or 19, wherein the toxin is formulated as a bait matrix and delivered to the above ground or underground bait station. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, used to separate genetic material from Portobactus or Portobactus luminescence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 , SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84. A protein subunit comprising constructing at least one RNA or DNA oligonucleotide molecule corresponding to at least a portion of a DNA coding region of an amino acid sequence selected from the group consisting of SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88; Isolation Method of Gene Coding. By purified bacterial culture of the genus Photorapdus comprising constructing a chimeric DNA construct having a 5 'to 3' promoter, a DNA sequence encoding a protein, and a transcription terminator and then delivering the chimeric DNA construct into a host A method of expressing a protein to be produced in a prokaryotic or eukaryotic host in an effective amount such that the protein can have a functional activity against insects. 32. The method of claim 31, wherein the protein has agonistic activity against insects selected from the group consisting of coleoptera, lepidoptera, fly, colo, lumber, locust and mite. The method of claim 31, wherein the protein encoded by the DNA sequence is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 , SEQ ID NO: 43, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88 having a N-terminal amino acid sequence selected from the group consisting of. The protein of claim 31, wherein the protein encoded by the DNA sequence is SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, A method comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 61. 5 'to 3' transcriptional promoters active in the host; A DNA sequence encoding a photolabduus protein having a functional activity against an insect; And a chimeric DNA construct suitable for expression in a prokaryotic or eukaryotic host, including a transcription terminator. The method of claim 35, wherein the protein encoded by the DNA sequence is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 , SEQ ID NO: 43, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: A chimeric DNA construct having an N-terminal amino acid sequence selected from the group consisting of 86, SEQ ID NO: 87, and SEQ ID NO: 88. The method of claim 35, wherein the protein encoded by the DNA sequence is SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, A chimeric DNA construct having an amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 61. 36. The method of claim 35, wherein the DNA sequence encoding the photolapid luminescence protein is SEQ ID NO: 11, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: A chimeric DNA construct selected from the group consisting of 54, SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 60. 36. The chimeric DNA construct of claim 35 wherein the host is a baculovirus or plant cell. An isolated and substantially purified formulation comprising a DNA molecule produced by a bacterium in the genus Photorapdus and capable of encoding an effective amount of a protein having a functional activity against insects. 41. The formulation according to claim 40, wherein the bacterium is photolabose luminescence . SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 , SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: N- selected from the group consisting of 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88. A purified formulation comprising a protein produced by Photorabdus or Photorabdus luminescence having a terminal amino acid sequence. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 , SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: A protein having an N-terminal amino acid sequence selected from the group consisting of 76, SEQ ID NO: 78, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88; Purified protein formulation comprising a. A protein selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 61 Purified protein formulation comprising a. A group consisting of SEQ ID NO: 11, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 60 isolated from the native host Purified DNA preparation comprising a DNA sequence selected from among. 18 kDa to about 230 kDa; About 160 kDa to about 230 kDa; 100 kDa to about 160 kDa; About 80 kDa to about 100 kDa; Or a purified protein preparation comprising photolabose luminescence protein having at least one subunit having an approximate molecular weight of about 50 kDa to about 80 kDa. Purified protein formulation comprising a photolapose luminescence protein having at least one subunit having an approximate molecular weight of about 280 kDa. Substantially pure microbial culture comprising ATCC 55397. 49. The culture of claim 48 wherein the culture is a variant of ATCC 55397 that produces protein toxins having functional activity against insects. A transgenic plant comprising in its genome a chimeric artificial genetic construct that confers on a plant the ability to express an effective amount of a photolapose protein having an action activity against an insect. 51. The transgenic plant of claim 50, wherein the plant is transformed using direct promotion of the genetic material coated on the microparticles into cells, Agrobacteria technique, Whisker technique or electroporation technique. 51. The method of claim 50, wherein the selection marker is selected from the group consisting of kanamycin, neomycin, glyphosate, hygromycin, methotrexate, phosphinothricin (bialophos), chlorosulfuron, bromocinyl, dalapon, and the like. The transgenic plant of choice. 51. The small subunit of claim 50, wherein the promoter is an octopin synthase, nopalin synthase, mannofin synthase, 35S, 19S, 35T, ribulose-1,6-bisphosphate (RUBP) carboxylase. ), Beta-conglycinin, pazeolin, alcohol dehydrogenase (ADH), heat shank, ubiquitin, zein, oleosin, napin or acyl carrier protein (ACP). 51. The transgenic plant of claim 50, wherein an embryonic tissue, callus tissue type I or II, embryonic tissue, meristem, or plant tissue during dedifferentiation is used to make the transgenic plant. 51. The transgenic plant of claim 50, wherein the chimeric gene is a DNA sequence encoding a photolabdus protein having a functional activity against an insect, wherein at least one codon of the gene is modified with a codon desired for the plant. An insect control method comprising orally delivering an effective amount of a protein toxin produced by a transgenic plant to be consumed by the insect. Picture drawer compositions of matter comprising a purified DNA sequence from a purified bacterial culture of the genus Douce. Substantially pure microbial culture comprising H9. Substantially pure microbial culture comprising Hb. Substantially pure microbial culture comprising Hm. Substantially pure microbial culture comprising HP88. Substantially pure microbial culture comprising NC-1. Substantially pure microbial culture comprising W30. Substantially pure microbial culture comprising WIR. Substantially pure microbial culture comprising B2. blood. Substantially pure microbial culture comprising giallandica . blood. Substantially pure microbial culture comprising Hepialus . Substantially pure microbial culture comprising HB-Arg. Substantially pure microbial culture comprising HB Oswego. Substantially pure microbial culture comprising HB Levistone. Substantially pure microbial culture comprising K-122. Substantially pure microbial culture comprising HMGD. Substantially pure microbial culture comprising indicus. Substantially pure microbial culture comprising GD. Substantially pure microbial culture comprising PWH-5. Substantially pure microbial culture comprising megidis. Substantially pure microbial culture comprising HF-85. a. Substantially pure microbial culture comprising a cow. Substantially pure microbial culture comprising MP1. Substantially pure microbial culture comprising MP2. Substantially pure microbial culture comprising MP3. Substantially pure microbial culture comprising MP4. Substantially pure microbial culture comprising MP5. Substantially pure microbial culture comprising GL98. Substantially pure microbial culture comprising GL155. Substantially pure microbial culture comprising GL101. Substantially pure microbial culture comprising GL138. Substantially pure microbial culture comprising GL217. Substantially pure microbial culture comprising GL257. a) isolating fragments of proteins that are at least six amino acids, b) immunizing a mammalian species with said protein fragment, c) a protein fragment which is part of a protein having a functional activity produced by a bacterium of the Enterobacteraeaceae, comprising recovering an antibody or a serum containing the protein fragment having a functional activity from the spleen of the mammalian species. Method for preparing an antibody. 91. The method of claim 90, wherein said protein fragment is selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71. The method of claim 90, wherein the bacteria are cheated picture drawer Douce. 91. The method of claim 90, wherein said bacterium is photolabose luminescence . a) isolating fragments of DNA sequence having at least 30 nucleotides, b) labeling said DNA fragment with radioactive or chemical agents, c) hybridizing said DNA fragment to a DNA library that is an enterobacteraeca cDNA or enterobactercae genome library, d) A method of selecting a DNA fragment encoding a portion of a protein having a functional activity produced by a bacterium of the bacterium Enterobacteraea comprising selecting a fragment hybridized to a DNA of a library encoding a protein having a functional activity. The method of claim 94, wherein the bacteria are cheated picture drawer Douce. 97. The method of claim 95, wherein said bacterium is photolabose luminescence . a) isolating at least two primers that are DNA fragments having at least 12 nucleotides, b) using the primer of step a) to amplify the DNA fragment derived from Enterobacterae by polymerase chain polymerization technique, to purify the DNA fragment, c) labeling the purified DNA fragment with a radioactive or chemical agent, d) hybridizing the purified DNA fragment to a DNA library that is an Enterobactercae cDNA or Enterobactercae genome library, e) selecting DNA fragments of the same or larger size than the purified DNA fragments from said library, wherein the selected DNA fragment or portion thereof encodes a protein having a functional activity. A method of selecting a DNA fragment encoding a portion of a protein having a functional activity produced by the bacteria of. The method of claim 97, wherein the bacteria are cheated picture drawer Douce. 99. The method of claim 98, wherein said bacterium is photolapux luminescence .