The phoPR operon encodes a response regulator, PhoP, and a histidine kinase, PhoR, which activate or repress genes of the Bacillus subtilis Pho regulon in response to an extracellular phosphate deficiency. Induction of phoPR upon phosphate starvation required activity of both PhoP and PhoR, suggesting autoregulation of the operon, a suggestion that is supported here by PhoP footprinting on the phoPR promoter. Primer extension analyses, using RNA from JH642 or isogenic sigE or sigB mutants isolated at different stages of growth and/or under different growth conditions, suggested that expression of the phoPR operon represents the sum of five promoters, each responding to a specific growth phase and environmental controls. The temporal expression of the phoPR promoters was investigated using in vitro transcription assays with RNA polymerase holoenzyme isolated at different stages of Pho induction, from JH642 or isogenic sigE or sigB mutants. In vitro transcription studies using reconstituted E A , E B , and E E holoenzymes identified P A4 and P A3 as E A promoters and P E2 as an E E promoter. Phosphorylated PhoP (PhoPϳP) enhanced transcription from each of these promoters. E B was sufficient for in vitro transcription of the P B1 promoter. P 5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoPϳP in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoPϳP at an E E promoter. Information concerning P B1 and P 5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.Inorganic phosphate (P i ) is the limiting nutrient for biological growth in the soil, the natural habitat of Bacillus subtilis. To thrive in this environment where P i levels are often 2 to 3 orders of magnitude lower than levels of other required ions (29), B. subtilis has evolved complex regulatory systems for utilization of this limiting nutrient. At least three global regulatory systems are responsible for changes in gene expression upon phosphate deprivation. One set of genes is controlled either positively or negatively by the PhoP-PhoR two-component regulators, genes referred to as the Pho regulon genes (for review, see reference 12). Other genes that are induced upon phosphate limitation are dependent on SigB (1), an alternative stress sigma factor. A third class of genes is expressed under phosphate-limiting growth conditions that are independent of either SigB or PhoP-PhoR (1). The regulatory coordination between these three sets of genes is unclear, although up-regulation of certain Pho regulon genes has been reported in a sigB mutant strain (12, 33).Pho regulon genes are the most extensively studied set of phosphate-regulated genes in B. subtilis. Identification of genes of known function that are directly regulated by PhoP-PhoR provides insight into one strategy B. subtilis may use to deal with conditions of limiting phosphate. A high-affinity P i tra...

The Bacillus subtilis PhoPR two-component system is directly responsible for activation or repression of Pho regulon genes in response to phosphate deprivation. The response regulator, PhoP, and the histidine kinase, PhoR, are encoded in a single operon with a complex promoter region that contains five known transcription start sites, which respond to at least two regulatory proteins. We report here the identification of another direct regulator of phoPR transcription, carbon catabolite protein A, CcpA. This regulator functions in the presence of glucose or other readily metabolized carbon sources. The maximum derepression of phoPR expression in a ccpA mutant compared to a wild-type stain was observed under excess phosphate conditions with glucose either throughout growth in a high-phosphate defined medium or in a low-phosphate defined medium during exponential growth, a growth condition when phoPR transcription is low in a wild-type strain due to the absence of autoinduction. Either HPr or Crh were sufficient to cause CcpA dependent repression of the phoPR promoter in vivo. A ptsH1 (Hpr) crh double mutant completely relieves phoPR repression during phosphate starvation but not during phosphate replete growth. In vivo and in vitro studies showed that CcpA repressed phoPR transcription by binding directly to the cre consensus sequence present in the promoter. Primer extension and in vitro transcription studies revealed that the CcpA regulation of phoPR transcription was due to repression of P A6 , a previously unidentified promoter positioned immediately upstream of the cre box. E A was sufficient for transcription of P A6 , which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered E A -responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between P A6 and the five downstream promoters characterized previously.

The Bacillus subtilis ResDE two-component system plays a positive role in global regulation of genes involved in aerobic and anaerobic respiration. ctaA is one of the several genes involved in aerobic respiration that requires ResD for in vivo expression. The ctaAB-divergent promoter regulatory region has three ResD binding sites; A1, A2, and A3. The A2 site is essential for in vivo promoter activity, while binding sites A2 and A3 are required for full ctaA promoter activity. In this study, we demonstrate the role of ResDϳP in the activation of the ctaA promoter using an in vitro transcription system. The results indicate that the ctaA promoter (binding sites A2 and A3) has two transcriptional start sites. Binding site A2 was sufficient for weak transcription of the upstream promoter (Pv) by E The Bacillus subtilis two-component regulatory pair, designated ResD and ResE, has a positive role in global regulation of both aerobic and anaerobic respiration (18,23). The ResDE system is required for transcription of the following genes and operons involved in aerobic respiration; the resABCDE operon (23), encoding proteins similar to those involved in cytochrome c biogenesis (resABC) (6) and ResD-ResE (resDE) (23); the petCBD operon, encoding subunits of the cytochrome bf complex; the ctaBCDEF operon (12), encoding CtaB, which is required for the synthesis of heme O from heme B (ctaB) (25) and structural genes for cytochrome caa 3 (ctaCDEF) (22) and ctaA (23); and a gene required for heme A biogenesis (24, 25) and hence for the synthesis of the heme A-containing terminal cytochrome oxidases aa 3 and caa 3 . Recognition of phenotypic traits shared by resD and ctaA mutants (15) led to a study that revealed that ResD has an essential role in the activation of in vivo expression of the ctaA promoter (23). Phenotypic similarities shared by resD and ctaA mutants, among others, included a sporulation defect and the absence of the heme A-containing terminal oxidases aa 3 and caa 3 . A recent study has shown that either one of these two terminal oxidases is sufficient for sporulation since a qoxABCD (structural genes for aa 3 ) ctaCD (structural genes for caa 3 ) double mutant is sporulation deficient but a single mutant with either mutation is not (28). Thus, the sporulation defect in a resD mutant may be explained by the role of ResD in ctaA and/or ctaB regulation.A direct role for ResD in ctaA promoter activation was suggested in a recent study which showed that there are three ResD binding sites (A1, A2, and A3) in the intercistronic ctaAB promoter region to which either unphosphorylated or phosphorylated ResD binds (29). A1 and A2 are situated upstream of the Ϫ35 promoter region, and A3 is downstream of the Ϫ10 region of the ctaA promoter previously identified (15). Deletion experiments revealed that binding site A1 did not influence the in vivo expression of the ctaA gene (29), suggesting that site A1 may be involved in the regulation of the divergent ctaB promoter, which also requires ResD for expression (12). ctaA-lacZ...

Induction of the Pho response in Bacillus subtilis occurs when the P i concentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. The phoPR promoter region contains three A -responsive promoters; only promoter P A4 is PhoP autoregulated. Expression of the phoPR operon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of a phoPR promoter-lacZ fusion in a scoC loss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in the scoC strain from a phoP promoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA on phoP expression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the ؊10 and/or ؊35 elements of five (P B1 , P E2 , P A3 , P A4 , and P A6 ) of the six promoters within the phoPR promoter region. P A6 was expressed in vivo from the phoP cre1 promoter fusion in both wild-type and scoC strains. Evidence for ScoC repression in vivo was shown by primer extension for P A4 and P A3 from the wild-type promoter and for P A4 and P E2 from the phoP cre1 promoter. The latter may reflect ScoC repression of sporulation that indirectly affects phoPR transcription. ScoC was shown to repress P A6 , P A4 , P E2 , and P B1 in vitro.