F. Marion Hulett

THE SIGNAL-TRANSDUCTION NETWORK FOR PHO REGULATION IN BACILLUS SUBTILIS

Depletion of nutrients, including phosphate, is a stress often encountered by a bacterial cell, and results in slowed growth, marking the cessation of exponential growth. Genes that are transcriptionally activated during phosphate starvation have been used to examine the signal‐transduction mechanisms governing the Pho regulon in Bacillus subtilis. Alkaline phosphatase, the traditional reporter protein for Pho regulation in prokaryotes, is encoded by a multigene family in B. subtilis. Characterization of the alkaline phosphatase family was a breakthrough in the study of regulation of the Pho regulon, especially the discovery of promoter elements exclusively responsive to phosphate‐starvation regulation. Current data suggest that at least three two‐component signal‐transduction systems interact, forming a regulatory network that controls the phosphate‐deficiency response in B. subtilis. The interconnected pathways involve the PhoP–PhoR system, whose primary role is to mediate the phosphate‐deficiency response; the Spo0 phosphorelay required for the initiation of sporulation; and a newly discovered signal‐transduction system, ResD–ResE, which also has a role in respiratory regulation during late growth. Parallel pathways positively regulate the Pho response via PhoP–PhoR. One pathway includes the ResD–ResE system, while the other involves a transition‐state regulator, AbrB. The Spo0 system represses the Pho response by negatively regulating both pathways. This review will discuss how the characterization of the APase multigene family made possible studies which show that the Pho regulon in B. subtilis is regulated by the integrated action of the Res, Pho and Spo signal‐transduction systems.

PhoP~P and RNA polymerase sigmaA holoenzyme are sufficient for transcription of Pho regulon promoters in Bacillus subtilis: PhoP~P activator sites within the coding region stimulate transcription in vitro

The Bacillus subtilis pstS operon and phoA gene are members of the Pho regulon that is controlled by PhoR, a histidine kinase, and PhoP, a response regulator. Footprinting analysis showed that phosphorylated PhoP extended the PhoP protected region in pstS and phoA promoters, and also bound to a separate site within the coding region of each gene. Our previous in vivo studies have shown that, in contrast to other Pho regulon promoters that are not expressed in either phoP or phoR mutants, a low‐level induction from the pstS promoter (25% of parent strain) can be detected in a phoR mutant. In this study, by using an in vitro transcription system, we demonstrate that (i) only phosphorylated PhoP is a transcriptional activator of the pstS operon and of the phoA gene; (ii) phosphorylated PhoP and RNA polymerase σA holoenzyme are sufficient for in vitro transcription of the pstS promoter and the phoA promoter; (iii) the activation of the pstS promoter requires lower concentrations of phosphorylated PhoP than does the phoA promoter for transcription; and (iv) PhoP binding sites in both the pstS promoter core binding region and in the 5′ coding region of the gene, which have been identified by footprinting analysis, are important for the transcription of the pstS promoter in vitro.

Pho signal transduction network reveals direct transcriptional regulation of one two-component system by another two-component regulator: Bacillus subtilis PhoP directly regulates production of ResD.

The Bacillus subtilis ResD–ResE two‐component system is responsible for the regulation of a number of genes involved in cytochrome c biogenesis and haem A biosynthesis, and it is required for anaerobic respiration in this organism. We reported previously that the operon encoding these regulatory proteins, the resABCDE operon, is induced under several conditions, one of which is phosphate starvation. We report here that this transcription requires the PhoP–PhoR two‐component system, whereas other induction conditions do not. The PhoP∼P response regulator directly binds to and is essential for transcriptional activation of the resABCDE operon as well as being involved in repression of the internal resDE promoter during phosphate‐limited growth. The concentration of ResD in various phoP mutant strains corroborates the role of PhoP in the production of ResD. These interactions result in a regulatory network that ties together the cellular functions of respiration/energy production and phosphate starvation. Significantly, this represents the first evidence for direct involvement of one two‐component system in transcription of a second two‐component system.

THREE TWO-COMPONENT SIGNAL-TRANSDUCTION SYSTEMS INTERACT FOR PHO REGULATION IN BACILLUS SUBTILIS

The Pho regulon of Bacillus subtilis is controlled by three two‐component signal‐transduction systems: PhoP/PhoR, ResD/ResE, and the phosphorelay leading to the phosphorylation of Spo0A. Two of these systems act as positive regulators, while the third is involved in negative regulation of the Pho regulon. Under phosphate‐starvation‐induction conditions, the response regulator (RR) PhoP, and the histidine protein kinase (HK) PhoR, are involved in the induction of Pho‐regulon genes including the phoPR operon and genes encoding the major vegetative alkaline phosphatases, phoA and phoB. ResD (the RR) and ResE (the HK) are positive regulators of both aerobic and anaerobic respiration in B. subtilis. Current data suggest that they are also positive regulators of the Pho regulon, as is the transition‐state regulatory protein AbrB. Data presented reveal that ResDE and AbrB are involved in activation of the Pho regulon through separate regulatory pathways. Spo0A∼P (RR) exerts a negative effect on the Pho regulon through its repression of AbrB, and possibly through repression of ResDE. Both pathways converge to regulate transcription of the phoPR operon.

Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis.

The two‐component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE‐dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis‐acting sequences required for ResDE‐dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE‐dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.

Sites internal to the coding regions of phoA and pstS bind PhoP and are required for full promoter activity

Bacillus subtilis PhoP and PhoR, a pair of two‐component regulatory proteins, regulate the phosphate starvation response. Here, we used two other pho regulon promoters, the phoA and pstS promoters, to examine the mechanism of PhoP‐specific activation of its target promoters. Both gel shift and DNase I footprinting assays indicate that PhoP bound to the two promoters. Unphosphorylated PhoP bound only to the multiple TTAACA‐like sequences upstream of these two promoters, while phosphorylated PhoP extended the binding region in both the 5′ and the 3′ direction and, additionally, protected sequences internal to the coding region of these two genes. The PhoP binding sites in the coding region were necessary for full induction from either promoter during phosphate starvation. Deletion of these sites eliminated approximately 75% and 45% of the induced promoter activity of the phoA and pstS promoters respectively. In vitro transcription assays using the phoA promoters with various 3′ ends confirmed the requirement of the PhoP∼P binding to the coding region for full promoter activity. The multiple TTAACA‐like sequences in the phoA and pstS promoters were essential for promoter activity, and deletion of one or more of these sequences in either promoter eliminated the promoter activity. Two pairs of TTAACA‐like sequences were required for efficient PhoP binding and were suggested to be one B. subtilis Pho box. Based on our data, we have proposed a model for activation of the phoA and the pstS promoter by PhoP.

ResD signal transduction regulator of aerobic respiration in Bacillus subtilis: ctaA promoter regulation.

A two‐component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration. In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A‐containing cytochrome terminal oxidases. Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD‐controlled genes. Here, we report the overexpression and purification of ResD and ResE and their role in gene activation. ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state. The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays. DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the −35 promoter region and one (A3) downstream of the −10 of the promoter. The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter–lacZ fusions. Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD∼P required to protect site A2 is reduced when site A3 is present. In vivo transcription assays from promoter–lacZ fusion constructs showed that DNA containing ResD‐binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.

The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate‐inducible expression of Pho regulon genes in Bacillus subtilis

PhoP–PhoR, one of three two‐component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli, was autophosphorylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue. Phospho–PhoR phosphorylated its cognate response regulator, PhoP in vitro. B. subtilis phoR was placed in the Bacillus chromosome under the control of the Pspac promoter, which is IPTG inducible. The wild‐type phoR, under either native promoter or Pspac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild‐type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N‐terminal transmembrane PhoR sequences or various extended N‐terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild‐type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C‐terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate‐limited conditions, and that the regulation for repression of APase under phosphate‐replete conditions remains intact.

CcpA Causes Repression of the phoPR Promoter through a Novel Transcription Start Site, PA6

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. Esigma(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 Esigma(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.

Autoinduction of Bacillus subtilis phoPR Operon Transcription Results from Enhanced Transcription from EσA- and EσE-Responsive Promoters by Phosphorylated PhoP

Received 3 February 2004/Accepted 1 April 2004 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 PA4 and PA3 as E A promoters and PE2 as an E E promoter. Phosphorylated PhoP (PhoPP) enhanced transcription from each of these promoters. E B was sufficient for in vitro transcription of the PB1 promoter. P5 was active only in a sigB mutant strain. These studies are the first to report a role for PhoPP in activation of promoters that also have activity in the absence of Pho regulon induction and an activation role for PhoP Pa t an E E promoter. Information concerning PB1 and P5 creates a basis for further exploration of the regulatory coordination or overlap of the PhoPR and SigB regulons during phosphate starvation.