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 PhoPP 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.

Regulators of aerobic and anaerobic respiration in Bacillus subtilis.

Two Bacillus subtilis genes, designated resD and resE, encode proteins that are similar to those of two-component signal transduction systems and play a regulatory role in respiration. The overlapping resD-resE genes are transcribed during vegetative growth from a very weak promoter directly upstream of resD. They are also part of a larger operon that includes three upstream genes, resABC (formerly orfX14, -15, and -16), the expression of which is strongly induced postexponentially. ResD is required for the expression of the following genes: resA, ctaA (required for heme A synthesis), and the petCBD operon (encoding subunits of the cytochrome bf complex). The resABC genes are essential genes which encode products with similarity to cytochrome c biogenesis proteins. resD null mutations are more deleterious to the cell than those of resE. resD mutant phenotypes, directly related to respiratory function, include streptomycin resistance, lack of production of aa3 or caa3 terminal oxidases, acid accumulation when grown with glucose as a carbon source, and loss of ability to grow anaerobically on a medium containing nitrate. A resD mutation also affected sporulation, carbon source utilization, and Pho regulon regulation. The data presented here support an activation role for ResD, and to a lesser extent ResE, in global regulation of aerobic and anaerobic respiration i B.subtilis.

Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis.

Bacillus subtilis has an alkaline phosphatase (APase) gene family composed of at least four genes. All members of this gene family are expressed postexponentially, either in response to phosphate starvation or sporulation induction or, in some cases, in response to both. The phoA gene (formerly called phoAIV) and the phoB gene (formerly called phoAIII) products have both been isolated from phosphate-starved cells, and a mutation in either gene decreased the total APase expressed under phosphate starvation conditions. Data presented here show that a phoA phoB double mutant reduced APase production during phosphate starvation by 98%, indicating that these two genes are responsible for most of the APase activity during phosphate-limited growth. The promoter for phoA was cloned and used, with the phoB promoter, to examine phosphate regulation in B. subtilis. phoA-lacZ reporter gene assays showed that the expression of the phoA gene commences as the culture enters stationary phase as a result of limiting phosphate concentrations in the growth medium, thereby mimicking the pattern of total APase expression. Induction persists for approximately 2 h and is then turned off. phoA is transcribed from a single promoter which initiates transcription 19 bp before the translation initiation codon. PhoP and PhoR are members of the two-component signal transduction system believed to regulate gene expression in response to limiting phosphate. The expression of phoA or phoB in response to phosphate starvation was equally dependent on PhoP and PhoR for induction. lacZ-promoter fusions showed that both phoA and phoB were hyperinduced, or failed to turn off induction after 2 h, in a spo0A strain of B. subtilis. Mutations in genes which are required for phosphorylation of Spo0A, spo0B and spo0F, also resulted in phoA and phoB hyperinduction, suggesting that phosphorylation of Spo0A is required for the repression of both APases in wild-type strains. The hyperinduction of either APase gene in a spo0A strain was dependent on PhoP and PhoR. Analysis of a phoP-lacZ promoter fusion showed that the phoPR operon is hyperinduced in a spo0A mutant strain, suggesting that Spo0A approximately P represses APases by repressing phoPR transcription. We propose a model for PHO regulation in B. subtilis whereby the phoPR operon is transcribed in response to limiting phosphate concentration, resulting in activation of the PHO regulon transcription, including transcription of phoA and phoB. When the phosphate response fails to overcome the nutrient deficiency, signals for phosphorylation of Spo0A result in production of Spo0A approximately P, which represses transcription of phoPR, thereby repressing synthesis of the PHO regulon.

Bacillus subtilis transcription regulator, Spo0A, decreases alkaline phosphatase levels induced by phosphate starvation.

Alkaline phosphatase (APase) is induced as a culture enters stationary phase because of limiting phosphate. The results presented here show that expression of APase is regulated both negatively and positively. PhoP, a homolog of a family of bacterial transcription factors, and PhoR, a homolog of bacterial histidine protein kinases, are required for induction of APases when phosphate becomes limiting. The induction period lasts 2 to 3 h, after which the rate of APase accumulation is decreased. Mutant strains defective in the Spo0A transcription factor failed to decrease APase production. The consequent hyperinduction of APase in a spo0A strain was dependent on phoP and phoR. spo0B and spo0F strains also overexpressed APase, suggesting that phosphorylated Spo0A is required for repression of APase. An abrB mutant allele in the presence of the mutant spo0A allele in these strains did not significantly change the APase hyperinduction phenotype, demonstrating that Spo0A repression of abrB expression is not the mechanism by which Spo0A-P regulates APase expression. Our previous report that spo0A mutants do not express APases is in conflict with the present data. We show here that the previously used mutants and a number of commonly used spo0 strains, all of which have an APase deficiency phenotype, contain a previously unrecognized mutation in phoR.