Recently, a new type of K ؉ transporter, Ktr, has been identified in the bacterium Vibrio alginol... more Recently, a new type of K ؉ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T.
Proceedings of the National Academy of Sciences of the United States of America, Feb 1, 1989
Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an os... more Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proU-lacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-4acZ fusion gene at low copy number showed that the plasmidencoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium
All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure i... more All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to highosmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria.
When faced with increased osmolarity in the environment, many bacterial cells accumulate the comp... more When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxyectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata. These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-L-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to L-aspartate. We observed that the EutD–EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.
Bacilli are exposed, either suddenly or on a sustained basis, to changes in the osmotic condition... more Bacilli are exposed, either suddenly or on a sustained basis, to changes in the osmotic conditions of the varied ecological niches that they can colonize. This can be vividly visualized by considering the main habitat of Bacillus subtilis, the upper layers of the soil (Earl et al. 2008). Rainfall and drying of the soil cause fluctuations in water availability and in the osmotic potential of this ecological niche. Such changes pose a considerable challenge to the microbial cell since they elicit water fluxes across the cytoplasmic membrane. These water fluxes are driven by the differential in the osmotic potential between the cell's interior and that of the surrounding microenvironment (Bremer and Kr€ amer 2000). Water entry at low osmolarity can increase turgor to such an extent that the elastic and stress-bearing peptidoglycan sacculus can no longer cope with it and the cell will rupture. Water efflux at high osmolarity will trigger dehydration of the cytoplasm and the ensuing reduction or even collapse of turgor will cause growth arrest or even cell death. Turgor, an intracellular hydrostatic pressure considered to be essential for cell expansion and growth, is difficult to determine experimentally but has been estimated at 1.9 MPa (19.37 atm) for B. subtilis (Whatmore and Reed 1990), a pressure that is close to ten times the pressure present in a standard car tire. To maintain turgor within physiologically acceptable boundaries, the B. subtilis cell needs to take active countermeasures to redirect the flow of water in or out of the cell when it faces decreases or increases in the external osmolarity. A considerable number of bacteria, including several Bacilli (but not B. subtilis), possess AqpZ-type aquaporins, water-selective channels embedded in the cytoplasmic membrane that can mediate accelerated water fluxes along osmotic gradients. However, the potential role of AqpZ-type water channels in the osmotic adjustment
Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spati... more Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut‐shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA‐binding mode suggests that RemA organizes its target DNA into nucleosome‐like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic‐stress adjustment.
L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-pr... more L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the ⌬ 1-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.
Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to ... more Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt- stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate (DMSP) restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically-identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of L-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress. This article is protected by copyright. All rights reserved.
Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import... more Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.
SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH... more SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH), osmostress‐adaptive synthesis of the compatible solute proline. The proA‐encoded gamma‐glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB‐ProA‐ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress‐responsive proline production routes. A key player for enhanced osmostress‐adaptive proline production is the osmotically regulated proHJ promoter. We used site‐directed mutagenesis to study the salient features of this stress‐responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA‐type promoters serve to keep transcription low under non‐inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA‐type promoters of either 16‐bp (the natural proHJ promoter), or 18‐bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress‐inducibility of transcription.
The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotei... more The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein (YqiH), a secreted N-acetylmuramoyl-L-alanine amidase (YqiI), and a cytoplasmic glycerophosphodiester phosphodiesterase (YqiK). Reverse transcriptase PCR (RT-PCR) analysis showed that the yqiHIK genes are transcribed as an operon. Consistent with the in silico prediction, we found that the purified YqiI protein exhibited hydrolytic activity toward peptidoglycan sacculi. Transcription studies with yqiH-treA reporter fusion strains revealed that the expression of yqiHIK is subjected to finely tuned osmotic control, but enhanced expression occurs only in severely osmotically stressed cells. Primer extension analysis pinpointed the osmotically responsive yqiHIK promoter, and site-directed mutagenesis was employed to assess functionally important sequences required for promoter activity and osmotic control. Promoter variants with constitutive activity were isolated. A deletion analysis of the yqiHIK regulatory region showed that a 53-bp AT-rich DNA segment positioned 180 bp upstream of the ؊35 sequence is critical for the activity and osmotic regulation of the yqiHIK promoter. Hence, the expression of yqiHIK is subjected to genetic control at a distance. Upon the onset of growth of cells of the B. subtilis wild-type strain in high-salinity medium (1.2 M NaCl), we observed gross morphological deformations of cells that were then reversed to a rod-shaped morphology again when the cells had adjusted to the high-salinity environment. The products of the yqiHIK gene cluster were not critical for reestablishing rod-shaped morphology, but the deletion of this operon yielded a B. subtilis mutant impaired in growth in a defined minimal medium and at high salinity.
A decrease in the water content of the soil imposes a considerable stress on the soil-living bact... more A decrease in the water content of the soil imposes a considerable stress on the soil-living bacterium Bacillus subtifis: water exits from the cetts, resulting in decreased turgor and cessation of growth. Under these adverse circumstances, B. subtilis actively modulates the osmolarity of its cytoplasm to maintain turgor within acceptable boundaries. A rapid uptake of potassium ions via turgor-responsive transport systems is the primary stress response to a sudden increase in the external osmolarity. This is followed by the massive accumulation of the so-called compatible solutes, i.e., organic osmolytes that are highly congruous with cellular functions and hence can be accumulated by bacterial cells up to molar concentrations. Initially, the compatible solute proline is accumulated via de novo synthesis, but B. subtilis can also acquire proline from the environment by an osmoregulated transport system, OpuE. The preferred compatible solute of B. subtitis is the potent osmoprotectant gIycine betaine. This trimethylammonium compound can be taken up by the ceil through three high-affinity transport systems: the multicomponent ABC transporters OpuA and OpuC, and the single-component transporter OpuD. The OpuC systems also mediates the accumulation of a variety of naturally occurring betaines, each of which can confer a considerable degree of osmotic tolerance. In addition to the uptake of glycine betaine from the environment, B. subtilis can also synthesize this osmoprotectant but it requires exogenously provided choline as its precursor. Two evolutionarily closely related ABC transport systems, OpuB and OpuC, mediate the uptake of choline which is then converted by the GbsA and GbsB enzymes in a two-step oxidation process into glycine betaine. Our data show that the intracellular accumulation of osmoprotectants is of central importance for the cellular defence of B. subtitis against high osmolarity stress.
In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating o... more In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The B σ-dependent general stress response typically triggered by salt shocks was not induced, whereas the W σ regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes ("ripening") of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism.
Expression of the proU operon of Escherichia coli is directly proportional to the osmolarity of t... more Expression of the proU operon of Escherichia coli is directly proportional to the osmolarity of the growth medium. The basal level of proU transcription is very low, but a large increase is triggered by a sudden rise in the external osmolarity. This increased expression is maintained for as long as the osmotic stimulus persists. We have capitalized upon these regulatory features of the proU operon and have constructed a series of expression vectors (pOSEX) permitting osmotically controlled expression of heterologous genes governed by regulatory signals of proU. The pOSEX vectors carry the proU promoter, an upstream region required for high-level expression, and part of the first structural gene (proI/), which acts as a silencer and is necessary to maintain low-level expression in low osmolarity media. An extended multiple cloning site (MCS) positioned at the 3' end of proV' permits the cloning of heterologous genes into the pOSEX plasmids, and efficient transcription terminators derived from the rrnB operon prevent deleterious readthrough transcription into the vector portion. The properties of the pOSEX expression vectors were tested by positioning a promoterless lacZ (encoding B-galactosidase) gene from E. coli and the g&A (encoding carboxytransferase) gene from the Gram+ bacterium Acidaminococcus fermentans under the control of the proU regulatory region. Efficient, osmoregulated and finely tuned expression of both la& and g&A was achieved, and the amount of B-galactosidase and carboxytransferase synthesized were simply controlled by adjusting the osmolarity of the growth medium with various concentrations of NaCl.
Applied and Environmental Microbiology, Feb 1, 1999
We report here that the naturally occurring choline ester choline-O-sulfate serves as an effectiv... more We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K i of approximately 4 M. Uptake studies with [1,2-dimethyl-14 C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K m value of 4 ؎ 1 M and a maximum rate of transport (V max) of 54 ؎ 3 nmol/min ⅐ mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13 C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and ␥-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83-90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.
Recently, a new type of K ؉ transporter, Ktr, has been identified in the bacterium Vibrio alginol... more Recently, a new type of K ؉ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T.
Proceedings of the National Academy of Sciences of the United States of America, Feb 1, 1989
Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an os... more Osmoregulated expression of proU has been reconstituted in a cell-free system. proU encodes an osmotically inducible, high-affinity transport system for the osmoprotectant glycine betaine in Escherichia coli. Previously, a proU-lacZ fusion gene had been cloned, resulting in plasmid pOS3. In vivo osmoregulation of this extrachromosomal proU-4acZ fusion gene at low copy number showed that the plasmidencoded fusion contained all the necessary sequences in cis for correctly receiving osmoregulatory signals during induction by osmotic stress and repression by glycine betaine. Using a cell-free (S-30) extract, plasmid pOS3 was then used to program protein synthesis in vitro. The ionic compound potassium
All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure i... more All microorganisms possess a positive turgor, and maintenance of this outward-directed pressure is essential since it is generally considered as the driving force for cell expansion. Exposure of microorganisms to highosmolality environments triggers rapid fluxes of cell water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of the cytoplasm. To counteract the outflow of water, microorganisms increase their intracellular solute pool by amassing large amounts of organic osmolytes, the so-called compatible solutes. These osmoprotectants are highly congruous with the physiology of the cell and comprise a limited number of substances including the disaccharide trehalose, the amino acid proline, and the trimethylammonium compound glycine betaine. The intracellular amassing of compatible solutes as an adaptive strategy to high-osmolality environments is evolutionarily well-conserved in Bacteria, Archaea, and Eukarya. Furthermore, the nature of the osmolytes that are accumulated during water stress is maintained across the kingdoms, reflecting fundamental constraints on the kind of solutes that are compatible with macromolecular and cellular functions. Generally, compatible solutes can be amassed by microorganisms through uptake and synthesis. Here we summarise the molecular mechanisms of compatible solute accumulation in Escherichia coli and Bacillus subtilis, model organisms for the gram-negative and gram-positive branches of bacteria.
When faced with increased osmolarity in the environment, many bacterial cells accumulate the comp... more When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxyectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata. These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-L-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to L-aspartate. We observed that the EutD–EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.
Bacilli are exposed, either suddenly or on a sustained basis, to changes in the osmotic condition... more Bacilli are exposed, either suddenly or on a sustained basis, to changes in the osmotic conditions of the varied ecological niches that they can colonize. This can be vividly visualized by considering the main habitat of Bacillus subtilis, the upper layers of the soil (Earl et al. 2008). Rainfall and drying of the soil cause fluctuations in water availability and in the osmotic potential of this ecological niche. Such changes pose a considerable challenge to the microbial cell since they elicit water fluxes across the cytoplasmic membrane. These water fluxes are driven by the differential in the osmotic potential between the cell's interior and that of the surrounding microenvironment (Bremer and Kr€ amer 2000). Water entry at low osmolarity can increase turgor to such an extent that the elastic and stress-bearing peptidoglycan sacculus can no longer cope with it and the cell will rupture. Water efflux at high osmolarity will trigger dehydration of the cytoplasm and the ensuing reduction or even collapse of turgor will cause growth arrest or even cell death. Turgor, an intracellular hydrostatic pressure considered to be essential for cell expansion and growth, is difficult to determine experimentally but has been estimated at 1.9 MPa (19.37 atm) for B. subtilis (Whatmore and Reed 1990), a pressure that is close to ten times the pressure present in a standard car tire. To maintain turgor within physiologically acceptable boundaries, the B. subtilis cell needs to take active countermeasures to redirect the flow of water in or out of the cell when it faces decreases or increases in the external osmolarity. A considerable number of bacteria, including several Bacilli (but not B. subtilis), possess AqpZ-type aquaporins, water-selective channels embedded in the cytoplasmic membrane that can mediate accelerated water fluxes along osmotic gradients. However, the potential role of AqpZ-type water channels in the osmotic adjustment
Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spati... more Biofilms can be viewed as tissue‐like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut‐shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA‐binding mode suggests that RemA organizes its target DNA into nucleosome‐like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic‐stress adjustment.
L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-pr... more L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the ⌬ 1-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.
Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to ... more Infections by the pathogenic gut bacterium Clostridioides difficile cause severe diarrheas up to a toxic megacolon and are currently among the major causes of lethal bacterial infections. Successful bacterial propagation in the gut is strongly associated with the adaptation to changing nutrition-caused environmental conditions; e.g. environmental salt stresses. Concentrations of 350 mM NaCl, the prevailing salinity in the colon, led to significantly reduced growth of C. difficile. Metabolomics of salt- stressed bacteria revealed a major reduction of the central energy generation pathways, including the Stickland-fermentation reactions. No obvious synthesis of compatible solutes was observed up to 24 h of growth. The ensuing limited tolerance to high salinity and absence of compatible solute synthesis might result from an evolutionary adaptation to the exclusive life of C. difficile in the mammalian gut. Addition of the compatible solutes carnitine, glycine-betaine, γ-butyrobetaine, crotonobetaine, homobetaine, proline-betaine and dimethylsulfoniopropionate (DMSP) restored growth (choline and proline failed) under conditions of high salinity. A bioinformatically-identified OpuF-type ABC-transporter imported most of the used compatible solutes. A long-term adaptation after 48 h included a shift of the Stickland fermentation-based energy metabolism from the utilization to the accumulation of L-proline and resulted in restored growth. Surprisingly, salt stress resulted in the formation of coccoid C. difficile cells instead of the typical rod-shaped cells, a process reverted by the addition of several compatible solutes. Hence, compatible solute import via OpuF is the major immediate adaptation strategy of C. difficile to high salinity-incurred cellular stress. This article is protected by copyright. All rights reserved.
Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import... more Under hyperosmotic conditions, bacteria accumulate compatible solutes through synthesis or import. Bacillus subtilis imports a large set of osmostress protectants via five osmotically controlled transport systems (OpuA to OpuE). Biosynthesis of the particularly effective osmoprotectant glycine betaine requires the exogenous supply of choline. While OpuB is rather specific for choline, OpuC imports a broad spectrum of compatible solutes, including choline and glycine betaine. One previously mapped antisense RNA of B. subtilis, S1290, exhibits strong and transient expression in response to a suddenly imposed salt stress. It covers the coding region of the opuB operon and is expressed from a strictly SigB-dependent promoter. By inactivation of this promoter and analysis of opuB and opuC transcript levels, we discovered a time-delayed osmotic induction of opuB that crucially depends on the S1290 antisense RNA and on the degree of the imposed osmotic stress. Time-delayed osmotic induction of opuB is apparently caused by transcriptional interference of RNA-polymerase complexes driving synthesis of the converging opuB and S1290 mRNAs. When our data are viewed in an ecophysiological framework, it appears that during the early adjustment phase of B. subtilis to acute osmotic stress, the cell prefers to initially rely on the transport activity of the promiscuous OpuC system and only subsequently fully induces opuB. Our data also reveal an integration of osmostress-specific adjustment systems with the SigB-controlled general stress response at a deeper level than previously appreciated.
SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH... more SummaryThe ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ‐ProA‐ProH), osmostress‐adaptive synthesis of the compatible solute proline. The proA‐encoded gamma‐glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB‐ProA‐ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress‐responsive proline production routes. A key player for enhanced osmostress‐adaptive proline production is the osmotically regulated proHJ promoter. We used site‐directed mutagenesis to study the salient features of this stress‐responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA‐type promoters serve to keep transcription low under non‐inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA‐type promoters of either 16‐bp (the natural proHJ promoter), or 18‐bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress‐inducibility of transcription.
The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotei... more The yqiHIK gene cluster from Bacillus subtilis is predicted to encode an extracellular lipoprotein (YqiH), a secreted N-acetylmuramoyl-L-alanine amidase (YqiI), and a cytoplasmic glycerophosphodiester phosphodiesterase (YqiK). Reverse transcriptase PCR (RT-PCR) analysis showed that the yqiHIK genes are transcribed as an operon. Consistent with the in silico prediction, we found that the purified YqiI protein exhibited hydrolytic activity toward peptidoglycan sacculi. Transcription studies with yqiH-treA reporter fusion strains revealed that the expression of yqiHIK is subjected to finely tuned osmotic control, but enhanced expression occurs only in severely osmotically stressed cells. Primer extension analysis pinpointed the osmotically responsive yqiHIK promoter, and site-directed mutagenesis was employed to assess functionally important sequences required for promoter activity and osmotic control. Promoter variants with constitutive activity were isolated. A deletion analysis of the yqiHIK regulatory region showed that a 53-bp AT-rich DNA segment positioned 180 bp upstream of the ؊35 sequence is critical for the activity and osmotic regulation of the yqiHIK promoter. Hence, the expression of yqiHIK is subjected to genetic control at a distance. Upon the onset of growth of cells of the B. subtilis wild-type strain in high-salinity medium (1.2 M NaCl), we observed gross morphological deformations of cells that were then reversed to a rod-shaped morphology again when the cells had adjusted to the high-salinity environment. The products of the yqiHIK gene cluster were not critical for reestablishing rod-shaped morphology, but the deletion of this operon yielded a B. subtilis mutant impaired in growth in a defined minimal medium and at high salinity.
A decrease in the water content of the soil imposes a considerable stress on the soil-living bact... more A decrease in the water content of the soil imposes a considerable stress on the soil-living bacterium Bacillus subtifis: water exits from the cetts, resulting in decreased turgor and cessation of growth. Under these adverse circumstances, B. subtilis actively modulates the osmolarity of its cytoplasm to maintain turgor within acceptable boundaries. A rapid uptake of potassium ions via turgor-responsive transport systems is the primary stress response to a sudden increase in the external osmolarity. This is followed by the massive accumulation of the so-called compatible solutes, i.e., organic osmolytes that are highly congruous with cellular functions and hence can be accumulated by bacterial cells up to molar concentrations. Initially, the compatible solute proline is accumulated via de novo synthesis, but B. subtilis can also acquire proline from the environment by an osmoregulated transport system, OpuE. The preferred compatible solute of B. subtitis is the potent osmoprotectant gIycine betaine. This trimethylammonium compound can be taken up by the ceil through three high-affinity transport systems: the multicomponent ABC transporters OpuA and OpuC, and the single-component transporter OpuD. The OpuC systems also mediates the accumulation of a variety of naturally occurring betaines, each of which can confer a considerable degree of osmotic tolerance. In addition to the uptake of glycine betaine from the environment, B. subtilis can also synthesize this osmoprotectant but it requires exogenously provided choline as its precursor. Two evolutionarily closely related ABC transport systems, OpuB and OpuC, mediate the uptake of choline which is then converted by the GbsA and GbsB enzymes in a two-step oxidation process into glycine betaine. Our data show that the intracellular accumulation of osmoprotectants is of central importance for the cellular defence of B. subtitis against high osmolarity stress.
In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating o... more In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The B σ-dependent general stress response typically triggered by salt shocks was not induced, whereas the W σ regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes ("ripening") of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism.
Expression of the proU operon of Escherichia coli is directly proportional to the osmolarity of t... more Expression of the proU operon of Escherichia coli is directly proportional to the osmolarity of the growth medium. The basal level of proU transcription is very low, but a large increase is triggered by a sudden rise in the external osmolarity. This increased expression is maintained for as long as the osmotic stimulus persists. We have capitalized upon these regulatory features of the proU operon and have constructed a series of expression vectors (pOSEX) permitting osmotically controlled expression of heterologous genes governed by regulatory signals of proU. The pOSEX vectors carry the proU promoter, an upstream region required for high-level expression, and part of the first structural gene (proI/), which acts as a silencer and is necessary to maintain low-level expression in low osmolarity media. An extended multiple cloning site (MCS) positioned at the 3' end of proV' permits the cloning of heterologous genes into the pOSEX plasmids, and efficient transcription terminators derived from the rrnB operon prevent deleterious readthrough transcription into the vector portion. The properties of the pOSEX expression vectors were tested by positioning a promoterless lacZ (encoding B-galactosidase) gene from E. coli and the g&A (encoding carboxytransferase) gene from the Gram+ bacterium Acidaminococcus fermentans under the control of the proU regulatory region. Efficient, osmoregulated and finely tuned expression of both la& and g&A was achieved, and the amount of B-galactosidase and carboxytransferase synthesized were simply controlled by adjusting the osmolarity of the growth medium with various concentrations of NaCl.
Applied and Environmental Microbiology, Feb 1, 1999
We report here that the naturally occurring choline ester choline-O-sulfate serves as an effectiv... more We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K i of approximately 4 M. Uptake studies with [1,2-dimethyl-14 C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K m value of 4 ؎ 1 M and a maximum rate of transport (V max) of 54 ؎ 3 nmol/min ⅐ mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13 C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and ␥-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83-90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.
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Papers by Erhard Bremer