KR102454110B1 - Recombinant plasmids and mutant strains for screening inhibitors of ppGpp biosynthesis-related gene expression - Google Patents

Abstract

The present invention relates to a recombinant plasmid for screening ppGpp biosynthesis-related gene expression inhibitors, a method for preparing a mutant strain for screening ppGpp biosynthesis-related gene expression inhibitors using the recombinant plasmid, the mutant strain prepared by the above method, and ppGpp biosynthesis using the mutant strain It relates to a screening method for a related gene expression inhibitor, wherein the recombinant plasmid and the mutant strain include the promoter region of the ppGpp biosynthesis-related gene and the antibiotic resistance gene without a promoter, so that the expression of the antibiotic resistance gene is regulated by the promoter of the ppGpp biosynthesis-related gene. In the process of screening for inhibitors using the mutant strain, it is possible to easily search for inhibitors of ppGpp biosynthesis-related gene expression by measuring the growth of bacteria in a medium to which antibiotics are added.

Description

Recombinant plasmids and mutant strains for screening inhibitors of ppGpp biosynthesis-related gene expression}

The present invention relates to a recombinant plasmid and a mutant strain for screening for inhibitors of ppGpp biosynthesis-related gene expression involved in the signaling pathway of organisms, and more specifically, to a recombinant plasmid for screening for ppGpp biosynthesis-related gene expression inhibitors, the recombination It relates to a method for producing a mutant strain for screening ppGpp biosynthesis-related gene expression inhibitors using a plasmid, the mutant strain prepared by the above method, and a screening method for a ppGpp biosynthesis-related gene expression inhibitor using the mutant strain.

Acinetobacter baumannii is a gram-negative bacterium belonging to Gammaproteobacteria that is widely distributed in natural environments such as soil and water, and shows weak pathogenicity to healthy people with strong immunity, but the immune system is damaged or It is an opportunistic pathogen with high toxicity in weakened individuals. In particular, due to the characteristics of multiple drug resistance (MDR) with resistance to antibiotics such as carbapenems, aminoglycosides, and fluoroquinolones, hospitals such as intensive care unit infections It is attracting attention as a major pathogen of hospital-acquired infection, and Acinetobacter baumani infection causes various infections including pneumonia, endocarditis, bloodstream infection, urinary tract infection, and peritonitis. Antibiotics such as colistin, tigecycline, imipenem, and meropenem are mainly prescribed to treat Acinetobacter baumani infection, but even these antibiotics show resistance when used for a long time. The development of new inhibitors against Acinetobacter baumani is still required.

On the other hand, ppGpp is a secondary messenger that serves as a signaling molecule involved in various stress responses such as bacterial growth regulation, tolerance to antibiotics, and expression of toxic genes. Activation of ppGpp has a great impact on the clinical success and effectiveness of antibacterial treatment. In the absence of ppGpp in bacteria, the amino acid synthesized in the body is insufficient, making survival difficult, and abnormal cell division or immobility is caused. can be In a recent study by the present inventors, it was confirmed that ppGpp affects antibiotic sensitivity by regulating the expression of EP (efflux pump)-related genes in A. baumannii , a multidrug-resistant bacterium. suggested that the related A1S_0579 gene could be a target for novel antibiotics (J Antimicrob Chemother. 2020. HW Jung, et al.).

Accordingly, the present inventors completed the present invention by preparing a reporter strain useful for searching for a substance that inhibits the expression of A1S_0579 gene related to ppGpp biosynthesis as an antibiotic of A. baumannii .

HW Jung, et al. Role of ppGpp-regulated efflux genes in Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy. Volume 75, Issue 5, May 2020, Pages 1130-1134,

An object of the present invention is to provide a recombinant plasmid for screening ppGpp biosynthesis-related gene expression inhibitors, which includes the promoter region of the A1S_0579 gene related to ppGpp biosynthesis and an antibiotic resistance gene.

Another object of the present invention is to provide a method for preparing a mutant strain for screening for ppGpp biosynthesis-related gene inhibitors using the recombinant plasmid.

Another object of the present invention is to provide a mutant strain for screening for ppGpp biosynthesis-related gene inhibitors prepared by the above method.

Another object of the present invention is to provide a method for screening a ppGpp biosynthesis-related gene inhibitor using the mutant strain.

The A1S_0579 gene of the present invention was confirmed to be related to ppGpp biosynthesis through our previous study (J Antimicrob Chemother. 2020. HW Jung, et al.). ppGpp was found to play an important role in affecting the survival of bacteria as a substance involved in the control of the synthesis and morphogenesis of secondary metabolites in bacteria. Therefore, it is judged that the inhibitor of A1S_0579 gene expression related to ppGpp biosynthesis has a very useful value as a material for preventing or treating infection of the corresponding bacteria.

One aspect of the present invention is a recombinant plasmid for ppGpp biosynthesis-related gene expression inhibitors including the promoter region of the A1S_0579 gene and antibiotic resistance gene in order to identify and search for inhibitors targeting the A1S_0579 gene related to ppGpp biosynthesis. provides

The promoter of the A1S_0579 gene may be the nucleotide sequence shown in SEQ ID NO: 1.

According to one embodiment of the present invention, the recombinant plasmid for screening for the ppGpp biosynthesis-related gene expression inhibitor comprises a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1; and antibiotic resistance genes may be sequentially operably linked.

As used herein, “promoter” refers to a polynucleotide sequence that allows and regulates the transcription of a gene operably linked thereto. A promoter contains a recognition sequence for binding RNA polymerase and an initiation site for transcription (transcription initiation site). In order to express the target gene in a particular cell type or host cell, it is necessary to select a suitable functional promoter. They are deposited in databanks such as, for example, GenBank, and may be commercially available or available as separate elements or elements cloned into polynucleotide sequences from individual sources.

The promoter is a promoter region of a ppGpp biosynthesis-related gene in a microorganism, and the microorganism is a pathogenic bacteria, such as Actinomyces , Bacillus , Borrelia , Chlamydia . , Clostridium genus, Enterococcus genus, Escherichia genus, Helicobacter genus, Klebsiella genus, Legionell a genus, Mycobacterium Mycobacterium genus, Pseudomonas genus, Salmonella genus, Shigell a genus, Staphylococcus genus, Streptococcus genus, Vibrio genus, or Acineto It may be a microorganism of the genus Acinetobacter , preferably Acinetobacter baumannii .

The "promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1" used in the present invention is located in the front part of the DNA nucleotide sequence carrying the genetic information of the A1S_0579 gene including the nucleotide sequence of SEQ ID NO: 1, which is the target gene, and transcription of the gene It is a regulatory region, and the expression of a gene operably linked to the A1S_0579 gene promoter is regulated by the A1S_0579 gene promoter region.

As used herein, "antibiotic resistance gene" means that an antibiotic resistance gene known in the art is introduced into a recombinant plasmid without a promoter regulating its expression. Specifically, the antibiotic resistance gene introduced into the recombinant plasmid may be an open reading frame (ORF) region. Since the expression of the antibiotic resistance gene linked to the promoter consisting of the nucleotide sequence of SEQ ID NO: 1 is regulated by the promoter consisting of the nucleotide sequence of SEQ ID NO: 1, the expression level of the antibiotic resistance gene can be easily measured in the recombinant plasmid of the present invention. It acts as a reporter gene.

The "antibiotic resistance gene" used in the present invention is a gene having resistance to antibiotics, and since bacteria or microorganisms having this gene survive even in an environment treated with the antibiotic, a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 in the present invention It is used as a selection marker (or reporter marker) in the process of obtaining a recombinant plasmid containing an antibiotic resistance gene whose expression is regulated by

The antibiotic resistance gene may be a resistance gene against antibiotics such as ampicillin, tetracyclin, kanamycin, chloramphenicol, streptomycin, neomycin, etc., For example, it may be an nptI gene, a bla gene (ampicillin resistance gene), or a cat gene (chloramphenicol resistance gene). Specifically, the antibiotic resistance gene may be an nptI gene (SEQ ID NO: 2).

The nptI gene is a gene encoding a neomycin phosphotransferase protein, and in microorganisms kanamycin, trimethoprim, neomycin, gentamicin, paromomycin ) can confer resistance to antibiotics such as

As used in the present invention, "operably linked" means that the expression control sequence of a gene and the nucleotide sequence encoding the target protein are operably linked to perform a function in general, thereby affecting the expression of the coding nucleotide sequence. can go crazy An operable linkage with a recombinant plasmid can be prepared using a genetic recombination technique known in the art, and site-specific DNA cleavage and ligation can be made using a cleavage and ligation enzyme in the art.

Since the recombinant plasmid for searching for ppGpp biosynthesis-related gene expression inhibitors includes a replication origin (ori) and an element regulating it to enable self-replication in microorganisms, the promoter consisting of the nucleotide sequence of SEQ ID NO: 1 to be introduced by the present inventors and a DNA fragment comprising an antibiotic resistance gene can be expressed by itself. In this case, the DNA fragment may exist in the form of one or more copies, that is, multiple copies in the microorganism.

A recombinant plasmid for screening ppGpp biosynthesis-related gene expression inhibitors according to an embodiment of the present invention comprises: a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1 of A. baumannii ; And antibiotic resistance genes are sequentially linked, and may include a nucleotide sequence represented by SEQ ID NO: 3. In this case, a sequence having high homology with the nucleotide sequence of SEQ ID NO: 3, for example, a sequence having 70% or more, 80% or more, or 90% or more homology thereof is also interpreted as being included in the scope of the present invention. can be When a transformant or mutant strain is prepared using such a recombinant plasmid, the antibiotic resistance gene is expressed by a single copy or multiple copies of a DNA fragment introduced into a chromosome.

Meanwhile, in the present specification, "DNA fragment", "gene fragment" and "gene" may be used interchangeably with the same meaning, and "recombinant plasmid" and "recombinant vector" may be used interchangeably with the same meaning.

According to another embodiment of the present invention, the recombinant plasmid for screening for the ppGpp biosynthesis-related gene expression inhibitor includes a nucleic acid region homologous to the 5' end of the target gene; a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1; antibiotic resistance gene; and a nucleic acid region homologous to the 3' end of the target gene may be sequentially operably linked.

"Target gene" as used in the present invention means a location in the genome of a microorganism into which the recombinant plasmid is to be introduced, specifically, the recombinant plasmid of the present invention is introduced at the 5'- and 3'-terminus of the target gene. Thus, a mutant strain containing the recombinant plasmid can be prepared. Specifically, the target gene may be a glmS gene.

As used herein, "a nucleic acid region homologous to the 5' end of the target gene" and "a nucleic acid region homologous to the 3' end of the target gene" refer to a nucleic acid sequence having homology with each end of the target gene, Specifically, it may be a sequence for the purpose of introducing the recombinant plasmid of the present invention into a predetermined position in the genome of a microorganism through homologous recombination by homology. The nucleic acid region homologous to the 5' end of the target gene may consist of the nucleotide sequence shown in SEQ ID NO: 4, and the nucleic acid region homologous to the 3' end of the target gene may be composed of the nucleotide sequence shown in SEQ ID NO: 5 can In this case, the homology may be 70% or more, 80% or more, or 90% or more, and the nucleic acid region may be 500 to 1,500 bp, 700 to 1,300 bp, or 900 to 1,100 bp.

The promoter and antibiotic resistance gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 are the same as described above.

The recombinant plasmid may further include a gene for selecting microorganisms, and the gene may be a cat gene. Since the cat gene imparts resistance to the antibiotic chloramphenicol to microorganisms, in the present invention, it can be used as a selection marker for a mutant strain transformed by being included in the recombinant plasmid.

In addition, the recombinant plasmid may further include a sacB gene after a nucleic acid region homologous to the 3' end of the target gene. The sacB gene refers to a gene (DNA fragment) encoding sucrase, which is known as an enzyme that degrades sucrose.

Specifically, when the microorganism containing the suicide vector is cultured in a medium containing sucrose, the sucrose expressed by the sacB gene decomposes sucrose to produce a product that is toxic to the microorganism, which Microorganisms that have not induced single cross recombination in the genomic DNA of the host microorganism, ie, exhibit antibiotic resistance, are excluded. On the other hand, when single cross recombination is induced in the genomic DNA of the host microorganism, the region including the entire DNA fragment of the vector is removed from the genomic DNA of the microorganism to return to the wild-type multidrug-resistant microorganism, or the desired ompA promoter among the DNA fragments of the vector Regions except for and Rluc8 genes can be removed.

Recombinant plasmid for searching for ppGpp biosynthesis-related gene expression inhibitors is a suicide vector or suicide plasmid vector, which does not replicate itself in microorganisms, and is a gene included therein, that is, the nucleotide sequence of SEQ ID NO: 1 to be introduced by the present inventors. Although the DNA fragment comprising the promoter and the antibiotic resistance gene is not expressed by itself, when the DNA fragment is inserted into the chromosome of a microorganism through homologous recombination, only the promoter and the antibiotic resistance gene comprising the nucleotide sequence of SEQ ID NO: 1 are inserted characterized by being

A recombinant plasmid for screening ppGpp biosynthesis-related gene expression inhibitors according to another embodiment of the present invention comprises: a nucleic acid region homologous to the 5' end of a target gene; A promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1 of A. baumannii ; antibiotic resistance gene; and nucleic acid regions homologous to the 3' end of the target gene are sequentially operably linked, and may include the nucleotide sequence represented by SEQ ID NO: 6. In this case, a sequence having high homology with the nucleotide sequence of SEQ ID NO: 6, for example, a sequence having 70% or more, 80% or more, or 90% or more homology thereof is also interpreted as being included in the scope of the present invention. can be When a transformant or a mutant strain is prepared using such a recombinant plasmid, the antibiotic resistance gene is expressed only by the single-copy DNA fragment introduced into the chromosome even if the multi-copy vector is included.

Another aspect of the present invention provides a method for preparing a mutant strain for screening ppGpp biosynthesis inhibitors, comprising the step of obtaining a transformant by introducing the recombinant plasmid into a microorganism.

According to an embodiment of the present invention, when the recombinant plasmid of the present invention is introduced into a microorganism in which a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1 and an antibiotic resistance gene are sequentially operably linked, SEQ ID NO: A DNA fragment including a promoter consisting of the nucleotide sequence of 1 and an antibiotic resistance gene may be inserted into the genomic DNA of a microorganism to prepare a transformant.

According to another embodiment of the present invention, a nucleic acid region homologous to the 5' end of the target gene, a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1, an antibiotic resistance gene, and a nucleic acid region homologous to the 3' end of the target gene When the recombinant plasmid (suicide vector) of the present invention sequentially operably linked is introduced into a microorganism, the glmS upstream gene corresponding to the nucleic acid region homologous to the 5' terminus of the target gene included in the recombinant plasmid is first single-crossed recombination This can be induced to prepare a transformant in which the entire DNA fragment contained in the recombinant plasmid is inserted into the genomic DNA of the microorganism. When the transformant is cultured for a certain period of time, secondary single crossover recombination may occur according to a certain probability. When the transformant is inoculated in a medium containing an excess of sucrose and cultured, the microorganism in which the second single cross recombination is not induced breaks down the sucrose by sucrose expressed from the sacB gene contained in the recombinant plasmid, and , can be excluded due to toxic products produced therefrom. When the second single crossover recombination is induced, the region containing the entire DNA fragment of the suicide vector is removed from the genomic DNA of the microorganism and returned to the wild-type microorganism, or the region containing only the nptI gene among the DNA fragments of the recombinant plasmid is removed can be

Whether or not the production of the transformant was successful may be confirmed by resistance to antibiotics (eg, kanamycin and/or trimethoprim) caused by the nptI gene.

A method of introducing the recombinant plasmid into a microorganism to obtain a transformant may use a method of introducing a nucleic acid into a cell known in the art, and a suitable standard technique as known in the art depending on the host microorganism or host cell This can be done by selecting . For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method ( cationic liposome), lithium superoxide-DMSO method, and the like.

As used in the present invention, "transformation (transformation)" means introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or by chromosomal integration completion. As used in the present invention, "transformant" refers to a nucleic acid molecule sequence comprising a promoter consisting of the nucleotide sequence of SEQ ID NO: 1 in the vector and an antibiotic resistance gene after the plasmid or vector is transformed into a host cell. It undergoes homologous recombination with the sequence of an endogenous gene region on the genome and may be inserted into a chromosome or retained in the form of a plasmid.

In addition, another aspect of the present invention provides a mutant strain for screening ppGpp biosynthesis-related gene inhibitors prepared by the above method.

According to one embodiment of the present invention, the mutant strain transformed with the recombinant plasmid is a reporter strain, and the kanamycin antibiotic resistance gene (nptI) is regulated by the promoter consisting of the nucleotide sequence of SEQ ID NO: 1, so the presence of kanamycin While growth was possible in the environment, strains without recombinant plasmid did not grow. This means that the antibiotic resistance gene nptI present in the recombinant plasmid is regulated by the expression control system of the A1S_0579 gene related to the ppGpp biosynthesis of A. baumannii . Therefore, the reporter strain constructed in the present invention can be usefully used for high-speed and large-scale screening of A1S_0579 gene expression inhibitors.

In addition, another aspect of the present invention comprises the steps of: a) culturing the mutant strain in a solid medium containing antibiotics; And b) provides a screening method for a ppGpp biosynthesis-related gene inhibitor comprising the step of contacting the test substance with the mutant strain.

The antibiotic of step a) is an antibiotic that exhibits resistance to the antibiotic resistance gene introduced into the recombinant plasmid, and includes kanamycin, trimethoprim, neomycin, gentamicin (G418), paromomycin. (paromomycin) and the like, preferably kanamycin.

This ppGpp biosynthesis gene-related inhibitor screening method is applicable because, when the test substance is an inhibitor that inhibits the expression of ppGpp biosynthesis-related genes, the antibiotic resistance gene whose expression is regulated by the promoter of the ppGpp biosynthesis-related gene cannot be expressed. Mutant strains cannot grow on a solid medium containing antibiotics. Therefore, when the mutant strain cannot be grown by the test substance, the contacted candidate substance can be judged as an expression inhibitor of the ppGpp biosynthesis-related gene. Therefore, the screening method according to the present invention has the advantage of being able to search for inhibitors of the expression of ppGpp biosynthesis-related genes by simply measuring the growth of the corresponding strain.

The recombinant plasmid and mutant strain for screening ppGpp biosynthesis-related gene expression inhibitors according to the present invention include the promoter region of the ppGpp biosynthesis-related gene and the antibiotic resistance gene. Inhibitors of ppGpp biosynthesis-related gene expression can be easily searched for by measuring the growth of bacteria in an antibiotic-added medium in the process of searching for inhibitors using mutant strains.

1 is a schematic diagram of the development of a recombinant plasmid pWH1266_A1S0579p-nptI according to an embodiment of the present invention.
2 shows the results of confirming the transgenes through DNA sequencing of the A1S_0579 promoter region and nptI of the recombinant plasmid pWH1266_A1S0579p-nptI according to an embodiment of the present invention.
3 is a schematic diagram of the development of a recombinant plasmid pDM4_A1S0579p-nptI according to an embodiment of the present invention.
4 shows the results of confirming the transgenes through DNA sequencing of the A1S_0579 promoter region and nptI of the recombinant plasmid pDM4_A1S0579p-nptI according to an embodiment of the present invention.
5 is a result of confirming whether growth in a medium containing kanamycin antibiotics for a mutant strain transformed with (A) a wild-type strain and a recombinant plasmid (B) pWH1266_A1S0579p-nptI according to an embodiment of the present invention.
6 is a result of confirming whether growth in a medium containing kanamycin antibiotics for (A) wild-type strain and recombinant plasmid (B) pDM4_A1S0579p-nptI gene-inserted mutant strain according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Preparation of A1S_0579-nptI

1-1. pWH1266_A1S0579p-nptⅠ recombinant plasmid preparation

For cloning into the pWH1266 plasmid, the A1S_0579 promoter region and nptI (kanamycin antibiotic resistance gene) were linked through overlap extension PCR. In this case, the primers in Table 1 below were used for PCR. At each end of the recombinant gene, the base sequence CTGCAG recognized by PstI was added, and the recombinant gene was treated with PstI, and then cloned by ligation to the pWH1266 plasmid treated with the same restriction enzyme. The recombinant plasmid map is shown in FIG. 1 . The newly constructed plasmid was confirmed through DNA sequencing. The sequencing results are shown in FIG. 2 .

primer
(SEQ ID NO:) Primer sequence (5'→3') purpose GlmS_Up_F_speⅠ
(SEQ ID NO: 7) GGACTAGTTGGTTTGAGCAATTGACTTGG GlmS upstream region amplification GlmS_Up_R
(SEQ ID NO: 8) CTTCACTTGCTGCCTTAATAATGATCTTTTTTGAATTACTCTACA A1S_0579p_F
(SEQ ID NO: 9) GTAATTCAAAAAAGATCATTATTAAGGCAGCAAGTGAAGAAATGGTGCAG A1S_0579 Promoter region amplification A1S_0579p_R
(SEQ ID NO: 10) TGGCTCATACCATTCTCCTGGATATGTAACTC NptⅠ_F
(SEQ ID NO: 11) CAGGAGAATGGTATGAGCCATATTCAACGGGAAA nptⅠ amplification NptⅠ_R
(SEQ ID NO: 12) GCAGGTGATGTCTGCCTCGTGAAGAAGG GlmS_Down_F
(SEQ ID NO: 13) AGGCAGACATCACCTGCTTTAATAATTGATTGATTAAGC GlmS subregion amplification GlmS_Down_R_ApaⅠ
(SEQ ID NO: 14) GTTGGGCCCAGTCGGTTTTAGCAGACCG A1S_0579p_F_pstⅠ
(SEQ ID NO: 15) AACTGCAGGCAAGTGAAGAAATGGTGCAG A1S_0579 promoter-nptI amplification NptⅠ_R_pstⅠ
(SEQ ID NO: 16) AACTGCAGGTCTGCCTCGTGAAGAAGG

1-2. Preparation of pDM4_A1S0579p-nptI recombinant plasmid

In order to clone into the pDM4 plasmid, the GlmS up (up) region, the A1S_0579 promoter region, nptI, and the GlmS down (down) region were ligated in order through overlap extension PCR. At each end of the recombinant gene, ACTAGT and GGGCCC recognizing SpeI and ApaI were added, and the recombinant gene was treated with SpeI and ApaI, and then ligated to the pDM4 plasmid treated with the same restriction enzyme and cloned. The recombinant plasmid map is shown in FIG. 3 . The newly constructed plasmid was confirmed through DNA sequencing. The sequencing results are shown in FIG. 4 .

Example 2. Preparation of reporter strains for screening ppGpp synthesis inhibitors

2-1. Multi-copy reporter strain production (pWH1266_A1S0579p-nptⅠ introduced)

The recombinant plasmid pWH1266_A1S0579p-nptI prepared in Example 1-1 was transformed into A. baumannii ATCC 17978 Wild-type cells through electroporation. Transformed A. baumannii was selected in LB (Luria Bertani) solid medium supplemented with kanamycin (50 μg/ml) antibiotic. It was confirmed whether the kanamycin antibiotic resistance gene (nptI) possessed by the reporter strain constructed through the selection process is regulated by the A1S_0579 promoter.

As a result, referring to FIG. 5 , A. baumannii without recombinant plasmid pWH1266_A1S0579p-nptI did not grow in the medium to which kanamycin was added, but A. baumannii into which pWH1266_A1S0579p-nptI was introduced was grown in the medium to which kanamycin was added. was confirmed to be These results suggest that the kanamycin resistance gene (nptI) present in pWH1266_A1S0579p-nptI is regulated by the A1S_0579 gene expression regulation system of A. baumannii .

2-2. Single-copy reporter strain production (introduced pDM4_A1S0579p-nptⅠ)

The recombinant plasmid pDM4_A1S0579p-nptI suicide vector prepared in Example 1-2 is an E. coli SM10 λ pir (thi thr leu tonA lacY) donor strain with λ pir that enables replication of the R6K suicide vector. supE recA::RP4-2-Tc::Mu Km λ pir) was transformed. Transformed E. coli SM10 λ pir was selected in LB (Luria Bertani) solid medium to which the antibiotic chloramphenicol (20 μg/ml) was added. The recombinant plasmid was transformed through conjugation with the selected E. coli SM10 λ pir pDM4_A1S0579p-nptI as the donor strain and the A. baumannii ATCC 17978 Wild-type as the acceptor strain. Transformed A. baumannii were selected on LB (Luria Bertani) solid medium supplemented with kanamycin (50 μg/ml) and trimethoprim (10 μg/ml) antibiotics. The selected strains were selected in LB (Luria Bertani) solid medium to which kanamycin (50 μg/ml) antibiotics and 10% sucrose were added. It was confirmed whether the kanamycin antibiotic resistance gene (nptI) possessed by the mutant reporter strain constructed through the selection process is regulated by the A1S_0579 promoter.

As a result, referring to FIG. 6 , A. baumannii without recombinant plasmid pDM4_A1S0579p-nptI did not grow in the medium to which kanamycin was added, but A. baumannii into which pDM4_A1S0579p-nptI was introduced was grown in the medium to which kanamycin was added. was confirmed to be These results suggest that the kanamycin resistance gene (nptI) present in pDM4_A1S0579p-nptI is regulated by the A1S_0579 gene expression regulation system of A. baumannii .

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Recombinant plasmids and mutant strains for screening inhibitors of ppGpp biosynthesis-related gene expression <130> PN200045 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 399 <212> DNA <213> Artificial Sequence <220> <223> A1S_0579 Promoter <400> 1 gcaagtgaag aaatggtgca gcgggcaaca gataatgcaa agcgtaataa tctggtgcaa 60 gcaagcttct tttcacaaga tttaacaaaa gatttttcgc atcattcttg ggcaaatcaa 120 ggatttgatg cattattgat tgatcctcct cgtgctggtg catatgaaat tatgcagtat 180 gtgccgaact ttggagcaaa aagaatcgtt tatgtatcat gtaatccagc aacattagca 240 agggatgcag gtgttttggt tcaacatggt taccagttaa aaaaagcagc ggttatggac 300 atgtttacgc acacagaaca tgttgaatcg attgctttat ttgagaaaat tcaagagata 360 aacgattaaa aacatgagtt acatatccag gagaatggt 399 <210> 2 <211> 1095 <212> DNA <213> Artificial Sequence <220> <223> npt I <400> 2 atgagccata ttcaacggga aacgtcttgc tcgaggccgc gattaaattc caacatggat 60 gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcag g tgcgacaatc 120 tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc 180 gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct 240 cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg 300 atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt 360 gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct 420 tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg 480 gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa 540 gaaatgcata agcttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca 600 cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc 660 ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct 720 ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa 780 ttgcagtttc atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa 840 cactggcaga gcattacgct gacttgacgg gacggcggct ttgttgaata aatcgaactt 900 ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa 960 agc aaaagtt caaaatcacc aactggtcca cctacaacaa agctctcatc aaccgtggct 1020 ccctcacttt ctggctggat gatggggcga ttcaggcctg gtatgagtca gcaacacctt 1080 cttcacgagg cagac 1095 <210> 3 <211> 10411 <212> DNA <213> Artificial Sequence <220> <223> pWH1266_A1S0579p-npt I <400> 3 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa 60 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg 120 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt 180 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata 240 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg 300 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac 360 cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac 420 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca 480 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg 540 actgttgggc gccatctcct tgcatgcacc attgccttgtgcgctgctg cctg ctgcatgcacc attagccttgtgcg gcctgc ggct at aagggagagc gtcgaccgat 660 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt 720 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct 780 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct 840 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa 900 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt 960 cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc 1020 cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca 1080 tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcattggacc 1140 gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat 1200 tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg 1260 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga 1320 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac 1380 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg 1440 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggc t aggctggcgg 1500 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc 1560 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg 1620 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca 1680 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga 1740 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa 1800 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt 1860 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc 1920 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt 1980 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac 2040 gaccacgctg atgagcttta ccgcagctgc tgctgttgct gctgttcaag tgtcaattgc 2100 tctactctgt tgctcgtctc aatgatagac atatcaagcc tttcagcttt tttattgcgc 2160 tttttgattt catcattacg cttgacgatc tccagttcct cccccctgcg cttggcgtgg 2220 gtggcttcaa gtccctcctt gaatgtcggc tcgatgtcta gtccacgatc cttatgactg 2280 cggtgatcaa ctctcacctc aagccctgct cgctctaggt ggacgttcgt caga tccgcc 2340 actttttccc tgattttttt cagcgttgag ttctgatcta gctccctgac tttcttccct 2400 agcccttgcg gtgtaaggct tcttgttgtc attagtatgt gggcgtgatg gtttctttca 2460 tcacttcccc catgcacatg aggggcatgg atcgctacgt ccaccgccac tccataggct 2520 ttaactaagg actggcacaa ctcggtaaca agtgcctgac gctgtgtctt atccagttca 2580 tgcggcaatg ctatctctac ttcctttgct aatcttgcct cctgtttgat gtcaccgttc 2640 ttttttagtt cggattgctc tacccgattc catagggttt gacgatctaa catgtcagga 2700 cttgccccag ttggggcaaa aatttgggtg tattcaatgc ctgttttttt ggtgtagtcc 2760 tgctcttttc cgtacgtatc acagtacaat ttttcgcctg ctcggtatgc tgcacatgcc 2820 acgattgagc gaccatctga cctcgaaatg ttctgcattt cacaatggta aattgccatt 2880 ttcatcacct gtttattaac agccgcccca agttttgggc ggttggggtg tcggggtttc 2940 cctgaccgta cttgcaaaat ttggcgtagc aaaaattttg cgtaagtgcg cccttcggga 3000 actctgcgag gctaaaagca aaagcaagag caataaggct ttgactttgt tttttgattt 3060 tgctcgctgc gctcggatct tgatgtaagt cgagtttttg aagtagaaca cttttcacat 3120 tgatgatggt tcatacttcg gaaaataggt tgagacacag gcattaaaaa tggtcaaaaa 3180 aacagctatg gaattgggtc aaatgctcga tgaagaaaag gaaaaattgg aacgtcagga 3240 aaaggcaaga aaggatctag ctgacgctgt ggtgaaaggt cgagagcaaa aacaggcaag 3300 gtcagacgat gcaaaacgca agatcctgat cggggcttac ttactgaaga aacatgataa 3360 tgacatcaag aaacttgtgg aacaaaaccc cgattttatt gggtacatta gagaaaatga 3420 taagcactta ttttcatagg cattagtagt tataaaaaac cctcataaat cgagggtttc 3480 ttttttatct gcattacttt ttgaatcaat gtactttttt agcttgctat ttttcactgc 3540 atacagccgc ttatactctt tctcaatcaa tgcggtcaaa atggcttttt ggctctcccc 3600 tgctgcgccc accatgtccg caagcatatc cgacacaccc ttatctataa acacctccaa 3660 acgttgacta tcaaggcttt tgcgtttttc acgatatgct ttttgtcgtt cggcattggt 3720 ttttggcggc tcttttagca tgtcctgtgt tttctgatcg ttcgggtctt tcatgtcttt 3780 aactcccata tagtatatca tagttacagg ataacattgt ttttagttac aggataactt 3840 tttataaaat ttatttgtaa aaatttatgc tatcctgcaa ctatttttaa attacaggat 3900 aactatttaa cgttaccctg taactatagt tatcctgtaa cgctaaatta attaccctgt 3960 aacatatatt ttgttatcct gtaacaacta aatattgaaa taatactacc aaagttaccc 4020 tgtaacgaaa ataactaggt taaaaaatga tcggatttta acattttgcg ttgttccaaa 4080 agttatcaac agcctagaac gtcataggaa gcgattacag acactttata gctatcagca 4140 tgggaacata agggcaggat gaaatatggg tcttaaaacg caaatggtga ggttttagag 4200 gtattttttg aagatgatta aggcggtttg tttttaaaat ttttggcggc tctcaggctg 4260 cttacatttt aaccagttca gtgaaaagtt ctttttcagc aaatttctgt ttagcaccat 4320 agctaaaact tgcgtggaac atattaagaa ttgaccgaaa tgacacaatc tcaattatat 4380 tttttttgaa aagttttctt tatcaaatat tttaaatcat tgatttatat ataagtatac 4440 attcatttta ataattaatc tttatttaac aatgatttat ctatattcaa ttgtttaatt 4500 attcttacta atattatctc tatatcaata ttttttattt aaaaacatat gtttagtagt 4560 gcttttgatt aaagtaccag agggagggag cagagctgaa tgggaaatac tcaccctaga 4620 gcgattctta aaaatcaccc taaagtattc ccattcgatg taccgtcggt cggtcgcttt 4680 cgcatcaggg atgacatcac tgtatcaagc tgccactgtt atgattacga ttgatagcac 4740 cgcctgaaca cgctcataac cgccaattaa atgactactc attgcgtccg ctactctgtt 4800 cagttcccct agtaatagcg tttttccgat gtgtgctagc gtcactgtac ctcatcaccc 4860 acacat ggac agattgagtt acagacattg gctaaatttt tggggtctat gcttgacaaa 4920 gcgagttaaa accttagaat ttaagacagg tacattaagc ccctgtggtg aaatcattag 4980 cggtcgtcaa accttaatgc tttcgattgc cgatatgtca gtatcgaaga tctgtacccc 5040 ataaattacg gtaaagcccc aagcaattgc aaggggcttt tatctttttt aacaaaaaaa 5100 atttataaat caggatttta taacactaaa taatccaaag tacatgagta agtcatgacc 5160 actcctgcga tgtgtgtagc actctgagta tccgtattat atcagtgatg tcatatacaa 5220 ccacataatg cggatgtatc actaactccc tagtatcttt ctgtctgcct gtgcgcccca 5280 tcagtcgatt atcaacaact aaatctgtct tttcttcaat taaatcatca atttcaatag 5340 ctgctatagg gttgcgttct tcaagataat catagatatt tctacgatct tggcgagctg 5400 tttcagtcca ctcaatcttc attcttcgct gccagtttcg ccaatgttgc agcacgtcta 5460 gcggcaaact ctgcctttac gtcctcatgt ggaatcaact tgcctgcatt ggcttcattt 5520 aagccgatct gtacctgctg cttgaaccat ttgtcatagt ctgcggcttc ctgttgcttc 5580 ttaacaaagt cacgcatgaa gtcacggatt agctgcgccc ccgaccgatc aacagtctta 5640 gcaaggcttg aaaattcttg ctttaggtca tggtctaccc gaaaggtaaa agtcgcttct 5700 gtcatgtttt t cactccaat gtgttacatt tgaatgctat tgtaacacat taaaaagtta 5760 ggtcattgtc ttttttcggc ttaggctgtt cttggtcata acgtggttta aatgatggct 5820 gtcgctcatt ctcaagtaaa attttccgtt tattaacagc ctgttgcatc tgctcatagt 5880 aaggcatcaa ctgcggtaaa atatcagcaa gtgcctgtct gtctttatcc aggatacggc 5940 tcaattgccc gatttttttc aagtcacgct cataggctat tctgtcgctg atctgctcgt 6000 atttttgctt tgaacacctt atgtactctt gcatatcgtt acttgcccat gcctgcgcct 6060 gaagacgtgt aaacatggaa atgtctgaag caacatggtc gctgatatgg tcatactcag 6120 catctttgac atagccgtat tttgataaat atctttcata gtcctgccgt ttctttttgt 6180 catcttcctg tttttgttta gtttctcttt ctatctgctg ttgttttcgt tgctgttcct 6240 gttgctcctc ttttatctct ctgtgataaa tggaaaggct agattgatca ttatcccaac 6300 gtgtgagcat gtcctcaaag ccgtaataat cagcactaaa ccaacctgtt ttcactttca 6360 ctggtggcgg cagtggctct ccgatctccc ttagcagtgc tgtagctgct cgtccctctt 6420 tgatcacttt atcaagctgc aagcggtcga tattgtcttg caaagccttg taaaatctat 6480 gctctgctcg cttctcatcc gtttttactt caatcaaata cacatctata agcgtttggc 6540 ggtgttgttg gtggtcg tat agtgcctgtt gtgcggtggc atagactgcg ggggcttggt 6600 ctacggcatc ctccagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 6660 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 6720 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 6780 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt 6840 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 6900 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 6960 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 7020 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 7080 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 7140 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 7200 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 7260 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7320 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7380 taactatcgt cttgagtcca ac ccggtaag acacgactta tcgccactgg cagcagccac 7440 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7500 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 7560 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7620 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7680 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 7740 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 7800 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 7860 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 7920 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 7980 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 8040 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 8100 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 8160 aggcaagtga agaaatggtg cagcgggcaa cagataatgc aaagcgtaat aatctggtgc 8220 aagcaagctt cttttcacaa gatttaac aa aagatttttc gcatcattct tgggcaaatc 8280 aaggatttga tgcattattg attgatcctc ctcgtgctgg tgcatatgaa attatgcagt 8340 atgtgccgaa ctttggagca aaaagaatcg tttatgtatc atgtaatcca gcaacattag 8400 caagggatgc aggtgttttg gttcaacatg gttaccagtt aaaaaaagca gcggttatgg 8460 acatgtttac gcacacagaa catgttgaat cgattgcttt atttgagaaa attcaagaga 8520 taaacgatta aaaacatgag ttacatatcc aggagaatgg tatgagccat attcaacggg 8580 aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata 8640 aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc 8700 ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag 8760 atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt 8820 ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg aaaacagcat 8880 tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt 8940 tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc gatcgcgtat 9000 ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg agtgattttg 9060 atgacgagcg taatggctgg cctgttgaac aag tctggaa agaaatgcat aagcttttgc 9120 cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg 9180 acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc 9240 aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc 9300 tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt catttgatgc 9360 tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag agcattacgc 9420 tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca 9480 gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac 9540 caactggtcc acctacaaca aagctctcat caaccgtggc tccctcactt tctggctgga 9600 tgatggggcg attcaggcct ggtatgagtc agcaacacct tcttcacgag gcagacctgc 9660 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 9720 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 9780 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 9840 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 9900 aaccaagtca ttctgagaat agtgtatgcg gcgaccgag t tgctcttgcc cggcgtcaac 9960 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 10020 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 10080 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 10140 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 10200 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 10260 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 10320 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 10380 gcgtatcacg aggccctttc gtcttcaaga a 10411 <210> 4 <211> 934 <212> DNA <213> Artificial Sequence <220> <223> GlmS upstream <400> 4 tggtttgagc aattgacttg gtgtgccttg ccaagttgag attgcgagtg aattccgtta 60 tcgctcacca gtgattgtgg aaaatacact ttacatctgt atttcacaat caggcgaaac 120 agcggatacc ttagctgcac tacgtgaaac tcaaaaacgt gctaaagcaa ataatatcga 180 tattcagact ttaacgatct gtaacgtcgc aacttcttca atggtgcgtg aaactgatca 240 ccacttattacag accagctttcag tggttg accagcttgcag cacgac 300 tcagcttgct gcattaatgt tgttaatctt gaaaattggt caagtgaaac agcgtattag 360 caatgtgatg attgaagagc ttgctcgtga attgtggcat agcccgaaag tgattcttga 420 tacacttaag aatgatccag aaattttacg tttatcagaa ttgttcgttg aaaaacaaca 480 ctgtttattc ttaggccgtg gcacacacta tccaatcgct ttggaaggtg cattaaagct 540 taaagaaatt tcatatattc acgcagaagg ttatgcagct ggtgagttga aacacggccc 600 attggcactt gttgatactg aaatgccaat cgtgattctt gcaccaaatg acgaaatgct 660 tgataagctt aagtcaaata tggaagaagt tcaggctcgt ggcggtgaat tattcgtttt 720 cgctgatgaa aatagtggtg tagttgaaaa agaccgtcaa catgtcgttc atattccagc 780 agtaaacgaa tggcttgcac caatcattta tagcgtacca gttcagttat tgtcttatca 840 cgttgctgta ttacgtggta cagacgttga ccagccacgt aacttggcga agtcagtaac 900 tgtagagtaa ttcaaaaaag atcattatta aggc 934 <210> 5 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> GlmS downstream <400> 5 atcacctgct ttaataattg attgattaag cccgatatgc attaattgcc tattgggctt 60 ttttattgcc ttcatatcgc tacacagaag ctttcctacg aaagaaaata gagaactctg 120 cttaggtccc cgtgtca caa cggacttttt acttttgcgc tgatttaaag tttgagagat 180 cattgcgaca cagagataaa gaagctaagc gaacttagtc cattttattt ttgtcgcagc 240 gttgaatgct gctgaacaag ttaggggcaa agtcctgtgg gagaacagga ctttgcaaac 300 tggtgtgttt taaacgttgt acggtttgag gtgaatacac ctgtgcaagc tgttggcaaa 360 cacaattaca actggcttac atgtgttcac ctgacaaaca aattctttta aattcctgct 420 gagcattggc atcatcattt gtaatactgt aagcaccgac cacaaggttt aaaatcaata 480 taaatttatg aagtttcata ttattctcat gattatttat aaatttaaat aaaataaaaa 540 tatggttcaa ttaaatatta aaataatttt ttaaataata attaatcctc ccacaaacat 600 aggaaaataa aataattaaa attttaaaat agaaataatt tataaattta aaaaaacagg 660 tcgcaaaaaa cccaaatttt aaataaataa aatagttaac taaaagagaa aatgagtttt 720 aataatggca tggccaactc cttaaaaatg gagttctacc accagattac cgcgaactaa 780 tttagggtgg tagaacgaaa gagggttagc agactgacct agcgaccagc acgcgcgaac 840 gcgtccccct tccgccctac catagagaaa aagggggcac aaaggttcca cacgggtaat 900 actgaggaag tataacccga cacgctaggg tacggtctgc taaaaccgac t 951 <210> 6 <211> 10483 <212> DNA <213> Artificial Sequence <220> <223> pDM4_A1S0579p-npt I <400> 6 gatccttttt gtccggtgtt gggttgaagg tgaagccggt cggggccgca gcgggggccg 60 gcttttcagc cttgcccccc tgcttcggcc gccgtggctc cggcgt cttg ggtgccgt gcc gccgc aggt c gggt gcc gcct 120 cgggt gggt gccg ggcgtg cgagcggaac gtctcgtagg agaacttgac cttccccgtt tcccgcatgt 240 gctcccaaat ggtgacgagc gcatagccgg acgctaacgc cgcctcgaca tccgccctca 300 ccgccaggaa cgcaaccgca gcctcatcac gccggcgctt cttggccgcg cgggattcaa 360 cccactcggc cagctcgtcg gtgtagctct ttggcatcgt ctctcgcctg tcccctcagt 420 tcagtaattt cctgcatttg cctgtttcca gtcggtagat attccacaaa acagcaggga 480 agcagcgctt ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat 540 atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt 600 ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt 660 gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc 720 gaggctggcc ggctaccgcc ggcgtaacag atgagggcaa gcggatggct gatgaaacca 780 agccaaccag gaagggcagc ccacctatca aggtgtactg ccttccagac gaacgaagag 840 cgattgagga aaaggcggcg gcggccggca tgagcctgtc ggcctacctg ctggccgtcg 900 gccagggcta caaaatcacg ggcgtcgtgg actatgagca cgtccgcgag ctggcccgca 960 tcaatggcga cctgggccgc ctgggcggcc tgctgaaact ctggctcacc gacgacccgc 1020 gcacggcgcg gttcggtgat g ccacgatcc tcgccctgct ggcgaagatc gaagagaagc 1080 aggacgagct tggcaaggtc atgatgggcg tggtccgccc gagggcagag ccatgacttt 1140 tttagccgct aaaacggccg gggggtgcgc gtgattgcca agcacgtccc catgcgctcc 1200 atcaagaaga gcgacttcgc ggagctggtg aagtacatca ccgacgagca aggcaagacc 1260 gagcgcctgg gtcacgtgcg cgtcacgaac tgcgaggcaa acaccctgcc cgctgtcatg 1320 gccgaggtga tggcgaccca gcacggcaac acccgttccg aggccgacaa gacctatcac 1380 ctgctggtta gcttccgcgc gggagagaag cccgacgcgg agacgttgcg cgcgattgag 1440 gaccgcatct gcgctgggct tggcttcgcc gagcatcagc gcgtcagtgc cgtgcatcac 1500 gacaccgaca acctgcacat ccatatcgcc atcaacaaga ttcacccgac ccgaaacacc 1560 atccatgagc cgtatcgggc ctaccgcgcc ctcgctgacc tctgcgcgac gctcgaacgg 1620 gactacgggc ttgagcgtga caatcacgaa acgcggcagc gcgtttccga gaaccgcgcg 1680 aacgacatgg agcggcacgc gggcgtggaa agcctggtcg gctggatccg gccacgatgc 1740 gtccggcgta gaggatctga agatcagcag ttcaacctgt tgatagtacg tactaagctc 1800 tcatgtttca cgtactaagc tctcatgttt aacgtactaa gctctcatgt ttaacgaact 1860 aaaccctcat ggctaacgta ctaagct ctc atggctaacg tactaagctc tcatgtttca 1920 cgtactaagc tctcatgttt gaacaataaa attaatataa atcagcaact taaatagcct 1980 ctaaggtttt aagttttata agaaaaaaaa gaatatataa ggcttttaaa gcttttaagg 2040 tttaacggtt gtggacaaca agccagggat gtaacgcact gagaagccct tagagcctct 2100 caaagcaatt ttgagtgaca caggaacact taacggctga catgggaatt ccacatgtgg 2160 aattccacat gtggaattgt gagcggataa caatttgtgg aatcccggga gagctcaggt 2220 tacccgcatg caagatctat ctagaagggc ccagtcggtt ttagcagacc gtaccctagc 2280 gtgtcgggtt atacttcctc agtattaccc gtgtggaacc tttgtgcccc ctttttctct 2340 atggtagggc ggaaggggga cgcgttcgcg cgtgctggtc gctaggtcag tctgctaacc 2400 ctctttcgtt ctaccaccct aaattagttc gcggtaatct ggtggtagaa ctccattttt 2460 aaggagttgg ccatgccatt attaaaactc attttctctt ttagttaact attttattta 2520 tttaaaattt gggttttttg cgacctgttt ttttaaattt ataaattatt tctattttaa 2580 aattttaatt attttatttt cctatgtttg tgggaggatt aattattatt taaaaaatta 2640 ttttaatatt taattgaacc atatttttat tttatttaaa tttataaata atcatgagaa 2700 taatatgaaa cttcataaat ttatattgat tt taaacctt gtggtcggtg cttacagtat 2760 tacaaatgat gatgccaatg ctcagcagga atttaaaaga atttgtttgt caggtgaaca 2820 catgtaagcc agttgtaatt gtgtttgcca acagcttgca caggtgtatt cacctcaaac 2880 cgtacaacgt ttaaaacaca ccagtttgca aagtcctgtt ctcccacagg actttgcccc 2940 taacttgttc agcagcattc aacgctgcga caaaaataaa atggactaag ttcgcttagc 3000 ttctttatct ctgtgtcgca atgatctctc aaactttaaa tcagcgcaaa agtaaaaagt 3060 ccgttgtgac acggggacct aagcagagtt ctctattttc tttcgtagga aagcttctgt 3120 gtagcgatat gaaggcaata aaaaagccca ataggcaatt aatgcatatc gggcttaatc 3180 aatcaattat taaagcaggt gatgtctgcc tcgtgaagaa ggtgttgctg actcatacca 3240 ggcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga tgagagcttt 3300 gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac ggtctgcgtt 3360 gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag 3420 ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt aaccaattct 3480 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca 3540 ataccatatt tttgaaaaag ccgtttctgt aatgaagg ag aaaactcacc gaggcagttc 3600 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa 3660 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg 3720 actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc 3780 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat 3840 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc 3900 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga 3960 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca 4020 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag 4080 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga 4140 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg 4200 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc 4260 ggcctcgagc aagacgtttc ccgttgaata tggctcatac cattctcctg gatatgtaac 4320 tcatgttttt aatcgtttat ctcttgaatt ttctcaaata aagcaatcga ttcaacatgt 4380 tctgtgtgcg taaacatgtc cataaccgct gcttttttta act ggtaacc atgttgaacc 4440 aaaacacctg catcccttgc taatgttgct ggattacatg atacataaac gattcttttt 4500 gctccaaagt tcggcacata ctgcataatt tcatatgcac cagcacgagg aggatcaatc 4560 aataatgcat caaatccttg atttgcccaa gaatgatgcg aaaaatcttt tgttaaatct 4620 tgtgaaaaga agcttgcttg caccagatta ttacgctttg cattatctgt tgcccgctgc 4680 accatttctt cacttgctgc cttaataatg atcttttttg aattactcta cagttactga 4740 cttcgccaag ttacgtggct ggtcaacgtc tgtaccacgt aatacagcaa cgtgataaga 4800 caataactga actggtacgc tataaatgat tggtgcaagc cattcgttta ctgctggaat 4860 atgaacgaca tgttgacggt ctttttcaac tacaccacta ttttcatcag cgaaaacgaa 4920 taattcaccg ccacgagcct gaacttcttc catatttgac ttaagcttat caagcatttc 4980 gtcatttggt gcaagaatca cgattggcat ttcagtatca acaagtgcca atgggccgtg 5040 tttcaactca ccagctgcat aaccttctgc gtgaatatat gaaatttctt taagctttaa 5100 tgcaccttcc aaagcgattg gatagtgtgt gccacggcct aagaataaac agtgttgttt 5160 ttcaacgaac aattctgata aacgtaaaat ttctggatca ttcttaagtg tatcaagaat 5220 cactttcggg ctatgccaca attcacgagc aagctcttca atcatcaca t tgctaatacg 5280 ctgtttcact tgaccaattt tcaagattaa caacattaat gcagcaagct gagtcgtgaa 5340 cgcttttgtt gatgcaaccc caatctctgg gcctgcaagc gtcaataagt ggtgatcagt 5400 ttcacgcacc attgaagaag ttgcgacgtt acagatcgtt aaagtctgaa tatcgatatt 5460 atttgcttta gcacgttttt gagtttcacg tagtgcagct aaggtatccg ctgtttcgcc 5520 tgattgtgaa atacagatgt aaagtgtatt ttccacaatc actggtgagc gataacggaa 5580 ttcactcgca atctcaactt ggcaaggcac accaagtcaa ttgctcaaac caactagtga 5640 cgcgtactcg agggtcgacg gtatcgataa gcttgatata cactccgcta gcgctgatgt 5700 ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag ctgaacagga gggacagctg 5760 atagaaacag aagccactgg agcacctcaa aaacaccatc atacactaaa tcagtaagtt 5820 ggcagcatca cccgacgcac tttgcgccga ataaatacct gtgacggaag atcacttcgc 5880 agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg 5940 aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc 6000 actaccgggc gtattttttg agttatcgag attttcagga gctaargaag ctaaaatgga 6060 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aaga acattt 6120 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac 6180 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat 6240 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct 6300 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt 6360 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca 6420 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat 6480 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa 6540 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa 6600 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg 6660 cagaatgctt aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt 6720 ttaaggcagt tattggtgcc cttaaacgcc tggttgctac gcctgaataa gtgataataa 6780 gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg tcgtcggttc agggcagggt 6840 cgttaaatag ccgcttatgt ctattgctgg tttaccggtt tattgactac cggaagcagt 6900 gtgaccgtgt gcttctcaaa tgcctgaggc cagwttgctc agctctcccg tggaggtaat 6960 aattgacgat atgatcattt attctgcctc ccagagcctg ataaaaacgg ttagcgcttc 7020 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatcatc 7080 gaattcctgc agccaagcta gacctaggcc ttaagatcct ttttaaccca tcacatatac 7140 ctgccgttca ctattattta gtgaaatgag atattatgat attttctgaa ttgtgattaa 7200 aaaggcaact ttatgcccat gcaacagaaa ctataaaaaa tacagagaat gaaaagaaac 7260 agatagattt tttagttctt taggcccgta gtctgcaaat ccttttatga ttttctatca 7320 aacaaaagag gaaaatagac cagttgcaat ccaaacgaga gtctaataga atgaggtcga 7380 aaagtaaatc gcgcgggttt gttactgata aagcaggcaa gacctaaaat gtgtaaaggg 7440 caaagtgtat actttggcgt caccccttac atattttagg tcttttttta ttgtgcgtaa 7500 ctaacttgcc atcttcaaac aggagggctg gaagaagcag accgctaaca cagtacataa 7560 aaaaggagac atgaacgatg aacatcaaaa agtttgcaaa acaagcaaca gtattaacct 7620 ttactaccgc actgctggca ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa 7680 agccatataa ggaaacatac ggcatttccc atattacacg ccatgatatg ctgcaaatcc 7740 ctgaacagca aaaaaatgaa aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa 7800 atatctcttc tgcaaaaggc ctggacgttt gggacagctg gccattacaa aacgctgacg 7860 gcactgtcgc aaactatcac ggctaccaca tcgtctttgc attagccgga gatcctaaaa 7920 atgcggatga cacatcgatt tacatgttct atcaaaaagt cggcgaaact tctattgaca 7980 gctggaaaaa cgctggccgc gtctttaaag acagcgacaa attcgatgca aatgattcta 8040 tcctaaaaga ccaaacacaa gaatggtcag gttcagccac atttacatct gacggaaaaa 8100 tccgtttatt ctacactgat ttctccggta aacattacgg caaacaaaca ctgacaactg 8160 cacaagttaa cgtatcagca tcagacagct ctttgaacat caacggtgta gaggattata 8220 aatcaatctt tgacggtgac ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag 8280 gcaactacag ctcaggcgac aaccatacgc tgagagatcc tcactacgta gaagataaag 8340 gccacaaata cttagtattt gaagcaaaca ctggaactga agatggctac caaggcgaag 8400 aatctttatt taacaaagca tactatggca aaagcacatc attcttccgt caagaaagtc 8460 aaaaacttct gcaaagcgat aaaaaacgca cggctgagtt agcaaacggc gctctcggta 8520 tgattgagct aaacgatgat tacacactga aaaaagtgat gaaaccgctg attgcatcta 8580 acacagtaac agatgaaatt gaacgcgcga acgtctttaa aatgaacggc aaatggtacc 8640 tgttca ctga ctcccgcgga tcaaaaatga cgattgacgg cattacgtct aacgatattt 8700 acatgcttgg ttatgtttct aattctttaa ctggcccata caagccgctg aacaaaactg 8760 gccttgtgtt aaaaatggat cttgatccta acgatgtaac ctttacttac tcacacttcg 8820 ctgtacctca agcgaaagga aacaatgtcg tgattacaag ctatatgaca aacagaggat 8880 tctacgcaga caaacaatca acgtttgcgc caagcttcct gctgaacatc aaaggcaaga 8940 aaacatctgt tgtcaaagac agcatccttg aacaaggaca attaacagtt aacaaataaa 9000 aacgcaaaag aaaatgccga tatcctatng gcattttctt ttatttctta tcaacataaa 9060 ggtgaatccc atatgaacta tataaaagca ggcaaatggc taaccgtatt cctaaccttt 9120 tggtaatgac tccaacttat tgatagtgtt ttatgttcag ataatgcccg atgactttgt 9180 catgcagctc caccgatttt gagaacgaca gcgacttccg tcccagccgt gccaggtgct 9240 gcctcagatt caggttatgc cgctcaattc gctgcgtata tcgcttgctg attacgtgca 9300 gctttccctt caggcgggat tcatacagcg gccagccatc cgtcatccat atcaccacgt 9360 caaagggtga cagcaggctc ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata 9420 cgtgcgcaac aaccgtcttc cggagactgt catacgcgta aaacagccag cgctggcgcg 9480 atttagcccc g acatagccc cactgttcgt ccatttccgc gcagacgatg acgtcactgc 9540 ccggctgtat gcgcgaggtt accgactgcg gcctgagttt tttaagtgac gtaaaatcgt 9600 gttgaggcca acgcccataa tgcgggctgt tgcccggcat ccaacgccat tcatggccat 9660 atcaatgatt ttctggtgcg taccgggttg agaagcggtg taagtgaact gcagcaatgg 9720 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 9780 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 9840 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 9900 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 9960 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 10020 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta tggtgcactc 10080 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg 10140 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 10200 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 10260 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag caaggagatg gcgcccaaca 10320 gtcccccggc c acggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga 10380 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac 10440 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gag 10483 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer - GlmS_Up_F_spe I <400> 7 ggactagttg gtttgagcaa ttgacttgg 29 <210> 8 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Primer - GlmS_Up_R <400> 8 cttcacttgc tgccttaata atgatctttt ttgaattact ctaca 45 <210> 9 <211> 50212 > DNA <213> Artificial Sequence <220> <223> Primer - A1S_0579p_F <400> 9 gtaattcaaa aaagatcatt attaaggcag caagtgaaga aatggtgcag 50 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer - A1S_0579p_R <400> 10 tggctcatac cattctcctg gatatgtaac tc 32 <210> 11 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer - Npt I_F <400> 11 caggagaatg gtatgagcca tattcaacgg gaaa 34 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer - Npt I_R <400> 12 gcaggtgatg tctgcctcgt ga agaagg 28 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer - GlmS_Down_F <400> 13 aggcagacat cacctgcttt aataattgat tgattaagc 39 <210> 14 <211> 28 <212> DNA < 213> Artificial Sequence <220> <223> Primer - GlmS_Down_R_Apa I <400> 14 gttgggccca gtcggtttta gcagaccg 28 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer - A1S_0579p_F_pst I < 400> 15 aactgcaggc aagtgaagaa atggtgcag 29 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer - Npt I_R_pst I<400> 16 aactgcaggt ctgcctcgtg aagaagg 27

Claims (11)

a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1; And as a recombinant plasmid for screening ppGpp biosynthesis-related gene expression inhibitors in which antibiotic resistance genes are sequentially operably linked,
The promoter is the promoter region of the ppGpp biosynthesis-related gene of Acinetobacter baumannii ,
The ppGpp biosynthesis-related gene is A1S_0579 comprising the nucleotide sequence of SEQ ID NO: 1,
The recombinant plasmid will have the nucleotide sequence represented by SEQ ID NO: 3, the recombinant plasmid.
a nucleic acid region homologous to the 5' end of the target gene; a promoter consisting of the nucleotide sequence represented by SEQ ID NO: 1; antibiotic resistance gene; And as a recombinant plasmid for screening ppGpp biosynthesis-related gene expression inhibitors in which a nucleic acid region homologous to the 3' end of the target gene is sequentially operably linked,
The promoter is the promoter region of the ppGpp biosynthesis-related gene of Acinetobacter baumannii ,
The ppGpp biosynthesis-related gene is A1S_0579 comprising the nucleotide sequence of SEQ ID NO: 1,
The recombinant plasmid will have the nucleotide sequence shown in SEQ ID NO: 6, the recombinant plasmid.
delete The method according to claim 1 or 2,
The antibiotic resistance gene is the nptI gene, the recombinant plasmid.
delete 3. The method according to claim 2,
The target gene is a glmS gene, the recombinant plasmid.
delete A method for producing a mutant strain for screening for ppGpp biosynthesis-related gene expression inhibitors, comprising the step of introducing the recombinant plasmid of claim 1 or 2 into a microorganism to obtain a transformant.
A mutant strain for screening for ppGpp biosynthesis-related gene expression inhibitors prepared by the method of claim 8.
a) culturing the mutant strain of claim 9 in a solid medium containing antibiotics; and
b) contacting the mutant strain with a test substance
A screening method for ppGpp biosynthesis-related gene expression inhibitors comprising a.
11. The method of claim 10,
Wherein the antibiotic of step a) is kanamycin, the method.

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