ABSTRACT
Phosphorous is essential for cell viability. To ensure an adequate supply under phosphate limiting conditions, bacteria induce a cohort of enzymes to scavenge for phosphate, and a high affinity transporter for its uptake into the cell. This response is controlled by a two-component signal transduction system named PhoBR in Escherichia coli and PhoPR in Bacillus subtilis. PhoR is a sensor kinase whose activity is responsive to phosphate availability. Under phosphate limiting conditions, PhoR exists in kinase mode that phosphorylates its cognate response regulator (PhoB, PhoP). When activated, PhoBâ¼P/PhoPâ¼P execute changes in gene expression that adapt cells to the phosphate limited state. Under phosphate replete conditions, PhoR exists in phosphatase mode that maintains PhoB/PhoP in an inactive, non-phosphorylated state. The mechanism by which phosphate availability is sensed and how it controls the balance between PhoR kinase and phosphatase activities has been studied in E. coli and B. subtilis. Two different mechanisms have emerged. In the most common mechanism, PhoR activity is responsive to phosphate transport through a PstSCAB/PhoU signaling complex that relays the conformational status of the transporter to PhoR. In the second mechanism currently confined to B. subtilis, PhoR activity is responsive to wall teichoic acid metabolism whereby biosynthetic intermediates can promote or inhibit PhoR autokinase activity. Variations of both mechanisms are found that allow each bacterial species to adapt to phosphate availability in their particular environmental niche.
ABSTRACT
The PhoPR-mediated response to phosphate limitation (PHO response) in Bacillus subtilis subsp subtilis is amplified and maintained by reducing the level of Lipid VG composed of poly(glycerol phosphate), a wall teichoic acid (WTA) biosynthetic intermediate that inhibits PhoR autokinase activity. However, the reduction in Lipid VG level is effected by activated PhoPâ¼P, raising the question of how the PHO response is first initiated. Furthermore, that WTA is composed of poly(ribitol phosphate) in Bacillus subtilis subsp spizizenii prompted an investigation of how the PHO response is regulated in that bacterium. We report that the PHO responses of B. subtilis subsp subtilis and subsp spizizenii are distinct. The PhoR kinases of the two B. subtilis subspecies are functionally equivalent and are activated either by the TagA/TarA or TagB/TarB enzyme product. However, they are inhibited by Lipid VG composed of poly(glycerol phosphate) but not by Lipid VR composed of poly(ribitol phosphate). Therefore, the distinctive PHO responses of these B. subtilis subspecies stem from the differential sensitivity of PhoR kinases to the polyol composition of Lipid V and from the genomic organization of WTA biosynthetic genes and the regulation of their expression.
ABSTRACT
Many bacterial species are highly social, adaptively shaping their local environment through the production of secreted molecules. This can, in turn, alter interaction strengths among species and modify community composition. However, the relative importance of such behaviours in determining the structure of complex communities is unknown. Here we show that single-locus changes affecting biofilm formation phenotypes in Bacillus subtilis modify community structure to the same extent as loss of an apex predator and even to a greater extent than loss of B. subtilis itself. These results, from experimentally manipulated multitrophic microcosm assemblages, demonstrate that bacterial social traits are key modulators of the structure of their communities. Moreover, they show that intraspecific genetic variability can be as important as strong trophic interactions in determining community dynamics. Microevolution may therefore be as important as species extinctions in shaping the response of microbial communities to environmental change.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Biofilms , Food Chain , Microbial Consortia , Microbial Interactions , Aeromonas , Alkaline Phosphatase , Ciliophora , Hymenostomatida , Klebsiella , Mutation , Paramecium , Serratia marcescensABSTRACT
UNLABELLED: The PhoPR two-component signal transduction system controls one of three responses activated by Bacillus subtilis to adapt to phosphate-limiting conditions (PHO response). The response involves the production of enzymes and transporters that scavenge for phosphate in the environment and assimilate it into the cell. However, in B. subtilis and some other Firmicutes bacteria, cell wall metabolism is also part of the PHO response due to the high phosphate content of the teichoic acids attached either to peptidoglycan (wall teichoic acid) or to the cytoplasmic membrane (lipoteichoic acid). Prompted by our observation that the phosphorylated WalR (WalRâ¼P) response regulator binds to more chromosomal loci than are revealed by transcriptome analysis, we established the PhoPâ¼P bindome in phosphate-limited cells. Here, we show that PhoPâ¼P binds to the chromosome at 25 loci: 12 are within the promoters of previously identified PhoPR regulon genes, while 13 are newly identified. We extend the role of PhoPR in cell wall metabolism showing that PhoPâ¼P binds to the promoters of four cell wall-associated operons (ggaAB, yqgS, wapA, and dacA), although none show PhoPR-dependent expression under the conditions of this study. We also show that positive autoregulation of phoPR expression and full induction of the PHO response upon phosphate limitation require PhoPâ¼P binding to the 3' end of the phoPR operon. IMPORTANCE: The PhoPR two-component system controls one of three responses mounted by B. subtilis to adapt to phosphate limitation (PHO response). Here, establishment of the phosphorylated PhoP (PhoPâ¼P) bindome enhances our understanding of the PHO response in two important ways. First, PhoPR plays a more extensive role in adaptation to phosphate-limiting conditions than was deduced from transcriptome analyses. Among 13 newly identified binding sites, 4 are cell wall associated (ggaAB, yqgS, wapA, and dacA), revealing that PhoPR has an extended involvement in cell wall metabolism. Second, amplification of the PHO response must occur by a novel mechanism since positive autoregulation of phoPR expression requires PhoPâ¼P binding to the 3' end of the operon.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Chromosomes, Bacterial/metabolism , DNA, Bacterial/metabolism , Genome, Bacterial , Phosphates/metabolism , Amino Acid Sequence , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Chromosomes, Bacterial/genetics , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial/physiology , Molecular Sequence Data , Operon , Phosphorylation , Protein Binding , RNA, Bacterial/genetics , RNA, Bacterial/metabolismABSTRACT
The PhoPR two-component signal transduction system controls one of the major responses to phosphate limitation in Bacillus subtilis. When activated it directs expression of phosphate scavenging enzymes, lowers synthesis of the phosphate-rich wall teichoic acid (WTA) and initiates synthesis of teichuronic acid, a non-phosphate containing replacement anionic polymer. Despite extensive knowledge of this response, the signal to which PhoR responds has not been identified. Here we report that one of the main functions of the PhoPR two-component system in B. subtilis is to monitor WTA metabolism. PhoR autokinase activity is controlled by the level of an intermediate in WTA synthesis that is sensed through the intracellular PAS domain. The pool of this intermediate generated by WTA synthesis in cells growing under phosphate-replete conditions is sufficient to inhibit PhoR autokinase activity. However WTA synthesis is lowered upon phosphate limitation by the combined effects of PhoP â¼ P-mediated activation of tuaA-H transcription and repression of tagAB. These transcriptional changes combine to lower the level of the inhibitory WTA metabolite thereby increasing PhoR autokinase activity. This amplifies the PHO response with full induction being achieved â¼ 90 min after the onset of phosphate limitation.
Subject(s)
Bacillus subtilis/growth & development , Bacterial Proteins/genetics , Phosphates/metabolism , Teichoic Acids/metabolism , Alkanes/pharmacology , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Phosphorylation , Promoter Regions, Genetic , Signal TransductionABSTRACT
The Bacillus subtilis cell wall is a dynamic structure, composed of peptidoglycan and teichoic acid, that is continually remodeled during growth. Remodeling is effected by the combined activities of penicillin binding proteins and autolysins that participate in the synthesis and turnover of peptidoglycan, respectively. It has been established that one or the other of the CwlO and LytE D,L-endopeptidase-type autolysins is essential for cell viability, a requirement that is fulfilled by coordinate control of their expression by WalRK and SigI RsgI. Here we report on the regulation of cwlO expression. The cwlO transcript is very unstable, with its degradation initiated by RNase Y cleavage within the 187-nucleotide leader sequence. An antisense cwlO transcript of heterogeneous length is expressed from a SigB promoter that has the potential to control cellular levels of cwlO RNA and protein under stress conditions. We discuss how a multiplicity of regulatory mechanisms makes CwlO expression and activity responsive to the prevailing growth conditions.
Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Endopeptidases/biosynthesis , N-Acetylmuramoyl-L-alanine Amidase/biosynthesis , RNA Stability , RNA, Messenger/metabolism , Base Sequence , Endopeptidases/genetics , Molecular Sequence Data , N-Acetylmuramoyl-L-alanine Amidase/genetics , Nucleic Acid Conformation , RNA, Antisense/biosynthesis , RNA, Bacterial/metabolism , Ribonucleases/metabolismABSTRACT
The WalRK (YycFG) two-component system co-ordinates cell wall metabolism with growth by regulating expression of autolysins and proteins that modulate autolysin activity. Here we extend its role in cell wall metabolism by showing that WalR binds to 22 chromosomal loci in vivo. Among the newly identified genes of the WalRK bindome are those that encode the wall-associated protein WapA, the penicillin binding proteins PbpH and Pbp5, the minor teichoic acid synthetic enzymes GgaAB and the regulators σ(I) RsgI. The putative WalR binding sequence at many newly identified binding loci deviates from the previously defined consensus. Moreover, expression of many newly identified operons is controlled by multiple regulators. An unusual feature is that WalR binds to an extended DNA region spanning multiple open reading frames at some loci. WalRK directly activates expression of the sigIrsgI operon from a newly identified σ(A) promoter and represses expression from the previously identified σ(I) promoter. We propose that this regulatory link between WalRK and σ(I) RsgI expression ensures that the endopeptidase requirement (CwlO or LytE) for cell viability is fulfilled during growth and under stress conditions. Thus the WalRK and σ(I) RsgI regulatory systems cooperate to control cell wall metabolism in growing and stressed cells.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/metabolism , Cell Wall/metabolism , N-Acetylmuramoyl-L-alanine Amidase/biosynthesis , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Base Sequence , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial , Promoter Regions, Genetic , Protein Binding , Sequence Analysis, DNA , Sigma Factor/metabolism , Transcription, Genetic , beta-Lactam Resistance/geneticsABSTRACT
In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferring the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61â Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg(2+) ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3' of the sugar ring and O2B of the ß-phosphate, implying an internal hydrogen bond. The stability of the WalK-ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.
Subject(s)
Adenosine Triphosphate/metabolism , Bacillus subtilis/enzymology , Protein Kinases/chemistry , Protein Kinases/metabolism , Bacillus subtilis/chemistry , Bacillus subtilis/metabolism , Binding Sites , Crystallography, X-Ray , Histidine Kinase , Hydrogen Bonding , Magnesium/metabolism , Models, Molecular , Protein Structure, TertiaryABSTRACT
Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.
Subject(s)
Adaptation, Physiological , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Gene Regulatory Networks , Glucose/metabolism , Malates/metabolism , Metabolic Networks and Pathways/genetics , Algorithms , Bacterial Proteins/metabolism , Computer Simulation , Data Interpretation, Statistical , Gene Expression Regulation, Bacterial , Genome, Bacterial , Metabolome , Metabolomics , Models, Biological , Operon , Promoter Regions, Genetic , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.
Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/physiology , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Transcription, Genetic , Transcriptome , Adaptation, Physiological , Algorithms , Binding Sites , Gene Expression Profiling , Gene Regulatory Networks , Oligonucleotide Array Sequence Analysis , RNA, Antisense/genetics , RNA, Antisense/metabolism , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Regulon , Sigma Factor/metabolism , Terminator Regions, GeneticABSTRACT
The CssRS two-component system responds to heat and secretion stresses in Bacillus subtilis by controlling expression of HtrA and HtrB chaperone-type proteases and positively autoregulating its own expression. Here we report on the features of the CssS extracellular loop domain that are involved in signal perception and on CssS subcellular localization. Individual regions of the CssS extracellular loop domain contribute differently to signal perception and activation. The conserved hydrophilic 26-amino-acid segment juxtaposed to transmembrane helix 1 is involved in the switch between the deactivated and activated states, while the conserved 19-amino-acid hydrophobic segment juxtaposed to transmembrane 2 is required for signal perception and/or transduction. Perturbing the size of the extracellular loop domain increases CssS kinase activity and makes it unresponsive to secretion stress. CssS is localized primarily at the septum but is also found in a punctate pattern with lower intensity throughout the cell cylinder. Moreover, the CssRS-controlled HtrA and HtrB proteases are randomly distributed in foci throughout the cell surface, with more HtrB than HtrA foci in unstressed cells.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Protein Kinases/chemistry , Protein Kinases/metabolism , Signal Transduction , Amino Acid Sequence , Bacillus subtilis/chemistry , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Histidine Kinase , Molecular Sequence Data , Protein Kinases/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Bacterial L-forms are cell wall-less forms of bacteria that usually grow with a conventional cell wall. Despite being important for research, L-forms are difficult to generate reproducibly and research in this area is challenging. Domínguez-Cuevas et al. (2011) report a method to rapidly, quantitatively and reproducibly generate populations of L-forms in Bacillus subtilis. Importantly, the methodology may be applicable to other bacteria heralding a new era of L-form research. Moreover, the genetic requirements of this method provide insights into how Lipid II synthesis and autolysin expression/activity are normally balanced and the central role of the WalRK two-component system in this process.
Subject(s)
Bacillus subtilis/metabolism , Cell Wall/metabolism , L Forms/metabolism , Protoplasts/metabolismABSTRACT
The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (ΔphoPR) cells.
Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Wall/metabolism , Gene Expression , Phosphates/metabolism , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Gene Expression Profiling , Gene Expression Regulation, Bacterial , Genetic Heterogeneity , Microarray Analysis , N-Acetylmuramoyl-L-alanine Amidase/genetics , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Promoter Regions, Genetic , Protein Kinases/metabolism , TranscriptomeABSTRACT
The vancomycin resistance operons from Enterococci, Staphylococci and Actinomycetes encode a VanRS two-component signal transduction system (TCS) and a suite of enzymes to modify the peptidoglycan biosynthetic precursor lipid II and to eliminate the D-Ala-D-Ala from the cell. Commingling of these regulatory and enzymatic activities with host functions has the potential to significantly impact host gene expression and cell wall metabolism. Here we report the effects of individually expressing the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins in Bacillus subtilis. VanY(B) WH(B) BX(B) expression confers resistance to 2 µg ml(-1) of vancomycin with concomitant reduced Van-FL staining and leads to a cell division defect. In contrast to E. faecalis and S. aureus, VanS(B) is active in B. subtilis without vancomycin addition. Individual expression of the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins repress and increase, respectively, expression of PhoPR regulon genes in the phosphate-limited state. When vancomycin-resistant cells are exposed to elevated vancomycin levels, mutant strains with increased resistance to vancomycin and a growth dependency on vanY(B) WH(B) BX(B) expression frequently arise. Mutation of the endogenous Ddl ligase is the necessary and sufficient cause of both phenotypes. We discuss how these effects may influence establishment of van operons in new host bacteria.
Subject(s)
Bacillus subtilis/drug effects , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Cell Wall/chemistry , Gene Expression , Vancomycin Resistance , Vancomycin/pharmacology , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/chemistry , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Cell Wall/metabolism , Enterococcus faecalis/genetics , Gene Expression Regulation, Bacterial , Operon , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staphylococcus/geneticsABSTRACT
We report the development of a suite of six integrative vectors for construction of single copy transcriptional fusions with the gfpmut3, cfp and iyfp reporter genes in Bacillus subtilis. The promoter fusions are constructed using the highly efficient ligation-independent cloning (LIC) technique making them suitable for high-throughput applications. The plasmids insert into the chromosome by a double cross-over event at the amyE or bglS loci and integration at each site can be verified by a plate-based screening assay. The vectors allow expression of two different promoters to be determined in the same strain using the cfp and iyfp reporter genes since CFP and iYFP are spectrally distinct and have comparable half-lives of approximately 2h in exponentially growing B. subtilis cells. We demonstrate the versatility of these vectors by measuring expression of the tuaA and phoA operons singularly and in combination, during growth in phosphate limiting conditions.
Subject(s)
Bacillus subtilis/genetics , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Mutagenesis, Insertional/methods , Plasmids/genetics , Bacterial Proteins/genetics , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genes, Reporter , Luminescent Proteins/genetics , Polymerase Chain Reaction , Transcription, GeneticABSTRACT
BACKGROUND: Lysyl-tRNA synthetase (LysRS) is unique within the aminoacyl-tRNA synthetase family in that both class I (LysRS1) and class II (LysRS2) enzymes exist. LysRS1 enzymes are found in Archaebacteria and some eubacteria while all other organisms have LysRS2 enzymes. All sequenced strains of Bacillus cereus (except AH820) and Bacillus thuringiensis however encode both a class I and a class II LysRS. The lysK gene (encoding LysRS1) of B. cereus strain 14579 has an associated T box element, the first reported instance of potential T box control of LysRS expression. RESULTS: A global study of 891 completely sequenced bacterial genomes identified T box elements associated with control of LysRS expression in only four bacterial species: B. cereus, B. thuringiensis, Symbiobacterium thermophilum and Clostridium beijerinckii. Here we investigate the T box element found in the regulatory region of the lysK gene in B. cereus strain 14579. We show that this T box element is functional, responding in a canonical manner to an increased level of uncharged tRNALys but, unusually, also responding to an increased level of uncharged tRNAAsn. We also show that B. subtilis strains with T box regulated expression of the endogenous lysS or the heterologous lysK genes are viable. CONCLUSIONS: The T box element controlling lysK (encoding LysRS1) expression in B. cereus strain 14579 is functional, but unusually responds to depletion of charged tRNALys and tRNAAsn. This may have the advantage of making LysRS1 expression responsive to a wider range of nutritional stresses. The viability of B. subtilis strains with a single LysRS1 or LysRS2, whose expression is controlled by this T box element, makes the rarity of the occurrence of such control of LysRS expression puzzling.
Subject(s)
Bacillus cereus/enzymology , Bacterial Proteins/genetics , Gene Expression Regulation, Enzymologic , Lysine-tRNA Ligase/genetics , RNA, Transfer, Asn/metabolism , Regulatory Elements, Transcriptional , Amino Acid Sequence , Bacillus cereus/chemistry , Bacillus cereus/genetics , Bacillus cereus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Lysine-tRNA Ligase/chemistry , Lysine-tRNA Ligase/metabolism , Molecular Sequence Data , RNA, Transfer, Amino Acyl/metabolism , Sequence AlignmentABSTRACT
In Bacillus subtilis, the WalRK (YycFG) two-component system controls peptidoglycan metabolism in exponentially growing cells while PhoPR controls the response to phosphate limitation. Here we examine the roles of WalRK and PhoPR in peptidoglycan metabolism in phosphate-limited cells. We show that B. subtilis cells remain viable in a phosphate-limited state for an extended period and resume growth rapidly upon phosphate addition, even in the absence of a PhoPR-mediated response. Peptidoglycan synthesis occurs in phosphate-limited wild-type cells at approximately 27% the rate of exponentially growing cells, and at approximately 18% the rate of exponentially growing cells in the absence of PhoPR. In phosphate-limited cells, the WalRK regulon genes yocH, cwlO(yvcE), lytE and ydjM are expressed in a manner that is dependent on the WalR recognition sequence and deleting these genes individually reduces the rate of peptidoglycan synthesis. We show that ydjM expression can be activated by PhoP approximately P in vitro and that PhoP occupies its promoter in phosphate-limited cells. However, iseA(yoeB) expression cannot be repressed by PhoP approximately P in vitro, but can be repressed by non-phosphorylated WalR in vitro. Therefore, we conclude that peptidoglycan metabolism is controlled by both WalRK and PhoPR in phosphate-limited B. subtilis cells.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Cell Wall/metabolism , Peptidoglycan/metabolism , Phosphates/metabolism , Protein Kinases/metabolism , Bacillus subtilis/cytology , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Bacterial Proteins/genetics , Base Sequence , Gene Expression Regulation, Bacterial , Microbial Viability , Peptidoglycan/genetics , Phosphorylation , Promoter Regions, Genetic , Protein Kinases/genetics , RegulonABSTRACT
Two-component systems usually function as cognate pairs, thereby ensuring an appropriate response to the detected signal. The ability to exclusively phosphorylate a partner protein, often in the presence of many competing homologous substrates, demonstrates a high level of specificity that must derive from the interacting surfaces of the two-component system. Here, we identify positions within the histidine kinases and response regulators of the WalRK and PhoPR two-component systems of Bacillus subtilis that make a major contribution to the specificity of phosphotransfer. Changing the identity of the amino acid at position 11 within the alpha1 helix of WalK and at position 17 within the alpha1 helix of PhoP altered discrimination and allowed phosphotransfer to occur with the non-cognate partner. Changing amino acids at additional positions of the WalK kinase increased phosphotransfer, while changes at additional positions in PhoP only had an effect in the presence of the change at position 17. The importance of amino acid identity at these two positions is supported by the fact that the amino acid combinations of Ile and Ser in WalRK, and Leu and Gly in PhoPR, are very highly conserved among orthologues, while modelling indicates that these amino acid pairs are juxtaposed in the WalRK and PhoPR complexes.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Protein Kinases/chemistry , Amino Acids/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Histidine Kinase , Models, Molecular , Mutagenesis , Phosphorylation , Protein Interaction Domains and Motifs , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Structure, SecondaryABSTRACT
Plasmid pBaSysBioII was constructed for high-throughput analysis of gene expression in Bacillus subtilis. It is an integrative plasmid with a ligation-independent cloning (LIC) site, allowing the generation of transcriptional gfpmut3 fusions with desired promoters. Integration is by a Campbell-type event and is non-mutagenic, placing the fusion at the homologous chromosomal locus. Using phoA, murAA, gapB, ptsG and cggR promoters that are responsive to phosphate availability, growth rate and carbon source, we show that detailed profiles of promoter activity can be established, with responses to changing conditions being measurable within 1 min of the stimulus. This makes pBaSysBioII a highly versatile tool for real-time gene expression analysis in growing cells of B. subtilis.