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 V^G composed of poly(glycerol phosphate), a wall teichoic acid (WTA) biosynthetic intermediate that inhibits PhoR autokinase activity. However, the reduction in Lipid V^G 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 V^G composed of poly(glycerol phosphate) but not by Lipid V^R 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.     

Changes in wall teichoic acid during the rod-sphere transition of Bacillus subtilis 168.

Wall teichoic acid (WTA) is essential for the growth of Bacillus subtilis 168. To clarify the function of this polymer, the WTAs of strains 168, 104 rodB1, and 113 tagF1 (rodC1) grown at 32 and 42 degrees C were characterized. At the restrictive temperature, the rodB1 and tagF1 (rodC1) mutants undergo a rod-to-sphere transition that is correlated with changes in the WTA content of the cell wall. The amount of WTA decreased 33% in strain 104 rodB1 and 84% in strain 113 tagF1 (rodC1) when they were grown at the restrictive temperature. The extent of alpha-D-glucosylation (0.84) was not affected by growth at the higher temperature in these strains. The degree of D-alanylation decreased from 0.22 to 0.10 in the rodB1 mutant but remained constant (0.12) in the tagF1 (rodC1) mutant at both temperatures. Under these conditions, the degree of D-alanylation in the parent strain decreased from 0.27 to 0.21. The chain lengths of WTA in strains 168 and 104 rodB1 grown at both temperatures were approximately 53 residues, with a range of 45 to 60. In contrast, although the chain length of WTA from the tagF1 (rodC1) mutant at 32 degrees C was similar to that of strains 168 and 104 rodB1, it was approximately eight residues at the restrictive temperature. The results suggested that the rodB1 mutant is partially deficient in completed poly(glycerophosphate) chains. The precise biochemical defect in this mutant remains to be determined. The results for strain 113 tagF1(rodC1) are consistent with the temperature-sensitive defect in the CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (H. M. Pooley, F.-X. Abellan, and D. Karamata, J. Bacteriol. 174:646-649, 1992).     

Interactions between the YycFG and PhoPR two-component systems in Bacillus subtilis: the PhoR kinase phosphorylates the non-cognate YycF response regulator upon phosphate limitation

Two-component signal transduction systems (TCS) are an important mechanism by which bacteria sense and respond to their environment. Although each two-component system appears to detect and respond to a specific signal(s), it is now evident that they do not always act independently of each other. In this paper we present data indicating regulatory links between the PhoPR two-component system that participates in the cellular response to phosphate limitation, and the essential YycFG two-component system in Bacillus subtilis. We show that the PhoR sensor kinase can activate the YycF response regulator during a phosphate limitation-induced stationary phase, and that this reaction occurs in the presence of the cognate YycG sensor kinase. Phosphorylation of YycF by PhoR also occurs in vitro, albeit at a reduced level. However, the reciprocal cross-phosphorylation does not occur. A second level of interaction between PhoPR and YycFG is indicated by the fact that cells depleted for YycFG have a severely deficient PhoPR-dependent phosphate limitation response and that YycF can bind directly to the promoter of the phoPR operon. YycFG-depleted cells neither activate expression of phoA and phoPR nor repress expression of the essential tagAB and tagDEF operons upon phosphate limitation. This effect is specific to the PhoPR-dependent phosphate limitation response because PhoPR-independent phosphate limitation responses can be initiated in YycFG-depleted cells.     

The TagB protein in Bacillus subtilis 168 is an intracellular peripheral membrane protein that can incorporate glycerol phosphate onto a membrane-bound acceptor in vitro

Genes involved in the synthesis of poly(glycerol phosphate) wall teichoic acid have been identified in the tag locus of the model Gram-positive organism Bacillus subtilis 168. The functions of most of these gene products are predictable from sequence similarity to characterized proteins and have provided limited insight into the intracellular synthesis and translocation of wall teichoic acid. Nevertheless, critical steps of poly(glycerol phosphate) teichoic acid polymerization continue to be a puzzle. TagB and TagF, encoded in the tag locus, do not show sequence similarity to characterized proteins. We recently showed that recombinant TagF could catalyze glycerol phosphate polymerization in vitro. Based largely on homology to TagF, the TagB protein has been proposed to catalyze either an intracellular glycerophosphotransfer reaction or the extracellular teichoic acid/peptidoglycan ligation reaction. Here we have taken steps to characterize TagB, particularly through in vivo localization studies and in vitro biochemical assay, in order to make a case for either role in teichoic acid biogenesis. We have shown that TagB associates peripherally with the intracellular face of the cell membrane in vivo. We have also produced recombinant TagB and used it to demonstrate the enzymatic incorporation of labeled glycerol phosphate onto a membrane-bound acceptor. The data collected from this and the accompanying study are strongly supportive of a role for TagB in B. subtilis 168 teichoic acid biogenesis as the CDP-glycerol:N-acetyl-beta-d-mannosaminyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenyl glycerophosphotransferase. Here we use the trivial name "Tag primase."     

The Parkinson's disease‐associated GPR37 receptor‐mediated cytotoxicity is controlled by its intracellular cysteine‐rich domain

GPR37, also known as parkin‐associated endothelin‐like receptor (Pael‐R), is an orphan G protein‐coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C‐terminal tail, in particular on a cystein‐enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine‐rich domain is linked to the previously described receptor‐mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C‐terminal cysteine‐rich domain protected against GPR37‐mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C‐terminal tail cysteine‐rich domain, which played a critical role in receptor cell surface expression, function and GPR37‐mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinson's disease) of this rather unknown member of the GPCR family.     

CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase, which is involved in the synthesis of the major wall teichoic acid in Bacillus subtilis 168, is encoded by tagF (rodC).

Assays of CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (CGPTase) (EC 2.7.8.12) in membranes isolated from Bacillus subtilis 168 wild type and 11 strains bearing conditional lethal thermosensitive mutations in tagB, tagD, or tagF revealed that CGPTase deficiency was associated only with mutant tagF alleles. In vitro, thermosensitivity of CGPTase strongly suggests that the structural gene for this enzyme is tagF. We discuss apparent discrepancies between biochemical evidence favoring a membrane location for TagF and a previous report that suggested a cytoplasmic location based on sequence analysis.     

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 ～27% the rate of exponentially growing cells, and at ～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～P in vitro and that PhoP occupies its promoter in phosphate‐limited cells. However, iseA(yoeB) expression cannot be repressed by PhoP～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.     

Evidence that Bacillus subtilis sporulation induced by the stringent response is caused by the decrease in GTP or GDP.

Partial amino acid deprivation of Bacillus subtilis, which evokes the stringent response, initiates sporulation not because the highly phosphorylated guanine nucleotides guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) increase but because GTP decreases. This was shown with a mutant (Myc) partially resistant to mycophenolate, an inhibitor of IMP dehydrogenase. Upon amino acid deprivation, the Myc mutant (62032) showed the usual increase in ppGpp and pppGpp but a reduced decrease in GTP, and only few cells sporulated. Extensive sporulation was restored by the addition of mycophenolate or decoyinine, and inhibitor of GMP synthetase, which caused a further decrease in GTP.     

Autoregulation of the Bacillus subtilis response regulator gene degU is coupled with the proteolysis of DegU‐P by ClpCP

The response regulator DegU and its cognate kinase DegS constitute a two‐component system in Bacillus subtilis that regulates many cellular processes, including exoprotease production and competence development. Using DNA footprint assay, gel shift assay and mutational analyses of P3degU‐lacZ fusions, we showed that phosphorylated DegU (DegU‐P) binds to two direct repeats (DR1 and DR2) of the consensus DegU‐binding sequence in the P3degU promoter. The alteration of chromosomal DR2 severely decreased degU expression, demonstrating its importance in positive autoregulation of degU. Observation of DegU protein levels suggested that DegU is degraded. Western blot analysis of DegU in disruption mutants of genes encoding various ATP‐dependent proteases strongly suggested that ClpCP degrades DegU. Moreover, when de novo protein synthesis was blocked, DegU was rapidly degraded in the wild‐type but not in the clpC and clpP strains, and DegU with a mutated phosphorylation site was much stable. These results suggested preferential degradation of DegU‐P by ClpCP, but not of unphosphorylated DegU. We confirmed that DegU‐P was degraded preferentially using an in vitro ClpCP degradation system. Furthermore, a mutational analysis showed that the N‐terminal region of DegU is important for proteolysis.     

SigM,an extracytoplasmic function sigma factor of Bacillus subtilis,is activated in response to cell wall antibiotics,ethanol, heat,acid, and superoxide stress

The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (P(A)) is sigma A dependent, and the other (P(M)) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the P(M) fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 micro g per ml), and by heat shock of 50 degrees C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 micro M paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H(2)O(2), the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 micro M monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 micro g of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival-a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.     

Synthetic lethality of the lytE cwlO genotype in Bacillus subtilis is caused by lack of D,L-endopeptidase activity at the lateral cell wall

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two D,L-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the D,L-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these D,L-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of D,L-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.     

Demonstration of the formation of 1,3-diphosphoglyceric acid as an intermediate in the high-energy phosphate metabolism during reinitiation of development in Artemia

Extensive labelling of the glycolytic intermediate 2,3-diphosphoglycerate by ³²PO^3?₄ during the early periods of development in Artemia is reported. At 30 min of activation this is the major labelled compound. The mobilization of inorganic phosphate through glycolysis leading to the formation of 1,3-diphosphoglycerate results in the formation of a high-energy phosphate donor. The label from this compound could be chased to high-energy phosphates (adenine derivatives). The location and subsequent high degree of labelling of 2,3-diphosphoglycerate in the yolk platelets further demonstrate the important role played by this organelle in the metabolic events accompanying the breakdown of dormancy in Artemia.     

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.     

Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate): gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity

The resistance spectrum to bacteriophage phi 3T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate), a so-called minor teichoic acid of strain 168, forms part of the receptor for this phage, and a serologically related group of phages. A representative sample of 25 mutants specifically resistant to phi 3T, obtained from a mutagenized culture by direct selection, were all found to have a greatly reduced galactosamine content. Relevant mutations in these strains were shown by PBS1 transduction and transformation to belong to two linkage groups; a minority, associated with an atypical colony morphology, were localized between sacA and purA, whereas the majority mapped between gtaB and tagB1 (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate). The former mutations mapped in a new locus, gneA, characterized by a deficiency in UDP-N-acetylglucosamine 4-epimerase, while the latter ones, as well as the previously identified pha-3 (Estrela et al., 1986, Journal of General Microbiology 132, 411-415), map is a locus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosamine-containing teichoic acid. A possible biological role of this polymer is discussed.