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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., (© 2018 John Wiley & Sons Ltd.)

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., (© 2014 John Wiley & Sons Ltd.)

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., (© 2012 Blackwell Publishing Ltd.)

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.

Adaptation of bacteria to the prevailing environmental and nutritional conditions is often mediated by two-component signal transduction systems (TCS). The Bacillus subtilis YycFG TCS has attracted special attention as it is essential for viability and its regulon is poorly defined. Here we show that YycFG is a regulator of cell wall metabolism. We have identified five new members of the YycFG regulon: YycF activates expression of yvcE, lytE and ydjM and represses expression of yoeB and yjeA. YvcE(CwlO) and LytE encode endopeptidase-type autolysins that participate in peptidoglycan synthesis and turnover respectively. We show that a yvcE lytE double mutant strain is not viable and that cells lacking LytE and depleted for YvcE exhibit defects in lateral cell wall synthesis and cell elongation. YjeA encodes a peptidoglycan deacetylase that modifies peptidoglycan thereby altering its susceptibility to lysozyme digestion and YdjM is also predicted to have a role in cell wall metabolism. A genetic analysis shows that YycFG essentiality is polygenic in nature, being a manifestation of disrupted cell wall metabolism caused by aberrant expression of a number of YycFG regulon genes.

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 YycG/YycF two-component system, originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low G + C Gram-positive bacteria. This system is required for cell viability, although the basis for this and the nature of the YycF regulon remained elusive. Using a combined hybrid regulator/transcriptome approach involving the inducible expression of a PhoP'-'YycF chimerical protein in B. subtilis, we have shown that expression of yocH, which encodes a potential autolysin, is specifically activated by YycF. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the regulatory regions of yocH as well as ftsAZ, previously reported to be controlled by YycF. Nucleotide sequence analysis and site-directed mutagenesis allowed us to define a potential consensus recognition sequence for the YycF response regulator, composed of two direct repeats: 5'-TGT A/T A A/T/C-N5-TGT A/T A A/T/C-3'. A DNA-motif analysis indicates that there are potentially up to 10 genes within the B. subtilis YycG/YycF regulon, mainly involved in cell wall metabolism and membrane protein synthesis. Among these, YycF was shown to bind directly to the region upstream from the ykvT gene, encoding a potential cell wall hydrolase, and the intergenic region of the tagAB/tagDEF divergon, encoding essential components of teichoic acid biosynthesis. Definition of a potential YycF recognition sequence allowed us to identify likely members of the YycF regulon in other low G + C Gram-positive bacteria, including several pathogens such as Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae.

The accumulation of malfolded proteins in the cell envelope of the Gram-positive eubacterium Bacillus subtilis was previously shown to provoke a so-called secretion stress response. In the present studies, proteomic approaches were employed to identify changes in the extracellular proteome of B. subtilis in response to secretion stress. The data shows that, irrespective of the way in which secretion stress is imposed on the cells, the levels of only two extracellular proteins, HtrA and YqxI, display major variations in a parallel manner. Whereas the extracellular level of the HtrA protease is determined through transcriptional regulation, the level of YqxI in the growth medium is determined post-transcriptionally in an HtrA-dependent manner. In the absence of secretion stress, the extracellular levels of HtrA and YqxI are low because of extracytoplasmic proteolysis. Finally, the protease active site of HtrA is dispensable for post-transcriptional YqxI regulation. It is known that Escherichia coli HtrA has combined protease and chaperone-like activities. As this protein shares a high degree of similarity with B. subtilis HtrA, it can be hypothesized that both activities are conserved in B. subtilis HtrA. Thus, a chaperone-like activity of B. subtilis HtrA could be involved in the appearance of YqxI on the extracellular proteome.