The distinct PhoPR mediated responses to phosphate limitation in Bacillus subtilis subspecies subtilis and spizizenii stem from differences in wall teichoic acid composition and metabolism.

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 PhoP∼P, 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.

Genome-wide analysis of phosphorylated PhoP binding to chromosomal DNA reveals several novel features of the PhoPR-mediated phosphate limitation response in Bacillus subtilis.

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 (WalR∼P) response regulator binds to more chromosomal loci than are revealed by transcriptome analysis, we established the PhoP∼P bindome in phosphate-limited cells. Here, we show that PhoP∼P 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 PhoP∼P 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 PhoP∼P 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 (PhoP∼P) 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 PhoP∼P binding to the 3' end of the operon.

PhoR autokinase activity is controlled by an intermediate in wall teichoic acid metabolism that is sensed by the intracellular PAS domain during the PhoPR-mediated phosphate limitation response of Bacillus subtilis.

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 ∼ 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 ∼ 90 min after the onset of phosphate limitation.

A highly unstable transcript makes CwlO D,L-endopeptidase expression responsive to growth conditions in Bacillus subtilis.

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.

The WalRK (YycFG) and σ(I) RsgI regulators cooperate to control CwlO and LytE expression in exponentially growing and stressed Bacillus subtilis cells.

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.

Bacterial L-forms on tap: an improved methodology to generate Bacillus subtilis L-forms heralds a new era of research.

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.

Signal perception by the secretion stress-responsive CssRS two-component system in Bacillus subtilis.

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.

ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain.

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 1.61 Šresolution. 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.

Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology.

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.

Peptidoglycan metabolism is controlled by the WalRK (YycFG) and PhoPR two-component systems in phosphate-limited Bacillus subtilis cells.

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.

Cell envelope gene expression in phosphate-limited Bacillus subtilis cells.

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.

Amino acid identity at one position within the alpha1 helix of both the histidine kinase and the response regulator of the WalRK and PhoPR two-component systems plays a crucial role in the specificity of phosphotransfer.

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.

pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis.

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.

The T box regulatory element controlling expression of the class I lysyl-tRNA synthetase of Bacillus cereus strain 14579 is functional and can be partially induced by reduced charging of asparaginyl-tRNAAsn.

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.

Suite of novel vectors for ectopic insertion of GFP, CFP and IYFP transcriptional fusions in single copy at the amyE and bglS loci in Bacillus subtilis.

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.

From one amino acid to another: tRNA-dependent amino acid biosynthesis.

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway.

The WalK/WalR (aka YycG/YycF) two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and specific to low G+C Gram-positive bacteria, including a number of important pathogens. An unusual feature is that this system is essential for viability in most of these bacteria. Recent studies have revealed conserved functions for this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism, that we have accordingly renamed WalK/WalR. Here we review the cellular role of the WalK/WalR TCS in different bacterial species, focusing on the function of genes in its regulon, as well as variations in walRK operon structure and the composition of its regulon. We also discuss the nature of its essentiality and the potential type of signal being sensed. The WalK histidine kinase of B. subtilis has been shown to localize to the divisome and we suggest that the WalKR system acts as an information conduit between extracytoplasmic cellular structures and intracellular processes required for their synthesis, playing a vital role in effectively co-ordinating peptidoglycan plasticity with the cell division process.

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome.

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.

Activation of the PhoPR-Mediated Response to Phosphate Limitation Is Regulated by Wall Teichoic Acid Metabolism in Bacillus subtilis.

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, PhoB∼P/PhoP∼P 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.