SEQUENTIAL ACTION OF 2-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 spoOA strain of B. subtilis. Mutations in genes which are required for phosphorylation of SpoOA, spoOB and spoOF, also resulted in phoA and phoB hyperinduction, suggesting that phosphorylation of SpoOA is required for the repression of both APases in wild-type strains. The hyperinduction of either APase gene in a spoOA strain was dependent on PhoP and PhoR. Analysis of a phoP-lacZ promoter fusion showed that the phoPR operon is hyperinduced in a spoOA mutant strain, suggesting that SpoOA similar to 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 SpoOA result in production of SpoOA similar to P, which represses transcription of phoPR, thereby repressing synthesis of the PHO regulon.