Autoinduction ofBacillus subtilis phoPROperon Transcription Results from Enhanced Transcription from Eσ^A- and Eσ^E-Responsive Promoters by Phosphorylated PhoP

Abstract

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...

Is PhoR–PhoP partner fidelity strict? PhoR is required for the activation of the pho regulon in Streptomyces coelicolor

“…In B. subtilis, the well characterized phoPR promoter region contains five different starts which respond to 285 different environmental stimuli, some of them constitutive but enhanced by PhoP~P (phosphorylated PhoP) binding (Paul et al 2004). Constitutive low expression of the S. coelicolor phoRP promoter region in a similar manner is very plausible since a basal level of PhoR-PhoP ready to be activated when phosphate is scarce must be available within the cell.…”

Transcription and autoregulation of the Rv3134c-devR-devS operon of Mycobacterium tuberculosis

“…Many two-component systems are transcribed from more than one promoter: for example phoPQ in Salmonella typhimurium (Soncini et al, 1995), bvgAS of Bordetella pertussis (Scarlato et al, 1990) and phoPR in Bacillus subtilis (Paul et al, 2004). This strategy allows for differential usage and regulation of promoters under diverse environmental conditions to facilitate bacterial adaptation.…”

Bacillus subtilisPhosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-ResponsivephoB-PS+VPromoters during Pho Response

“…For genes classified as both Pho and E E regulon genes, the mechanism responsible for inclusion of the phoPR promoter has been explained by PhoPϳP amplification of A -and E -responsive phoPR operon promoters (35). Here we showed that the PhoP-regulated phoB promoter, P V , is a A promoter and that transcription from the second promoter, P S, requires only E E , although the P S transcription was delayed in a phoPR mutant (Fig.…”

“…The putative yycOP and glnQ promoters are interesting in that both contain two putative consensus repeats for a PhoP dimer binding site on the coding strand on either side of the Ϫ35 element of the putative E promoter. It should be interesting to determine if E E -responsive PhoP-activated genes exist (in addition to autoregulation) and if their requirement for PhoP binding is different than the requirement for all PhoP-activated A promoters studied (13) or for the PhoP-enhanced autoregulation E E promoter (35).…”

“…Inclusion of the phoPR promoter among these genes and operons is explained by evidence indicating that three of the six phoPR operon promoters (35; A. Puri and F. M. Hulett, unpublished data) require PhoPϳP for enhanced transcription. One of these promoters is an E Eresponsive promoter, and two are E A promoters (35). phoB promoter deletion analysis was previously performed using a number of nonisogenic B. subtilis strains that contained mutations in important regulatory genes, including abrB, spo0A, spo0B, sigF, spoIIE, sigE, phoR, phoP, and phoS (9,20).…”