title: Sequential action of two-component genetic switches regulates the PHO regulon in Bacillus subtilis.
abstract: 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.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago 60607-7020.
authors.forename: F M
authors.initials: FM
authors.lastname: Hulett
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: G
authors.initials: G
authors.lastname: Sun
authors.forename: R
authors.initials: R
authors.lastname: Chesnut
authors.forename: E
authors.initials: E
authors.lastname: Sharkova
authors.forename: M F
authors.initials: MF
authors.lastname: Duggan
authors.forename: N
authors.initials: N
authors.lastname: Kapp
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005838:Genotype; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009876:Operon; D010710:Phosphates / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D018085:Regulon; D015183:Restriction Mapping; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D007527:Isoenzymes; D010710:Phosphates; D000469:Alkaline Phosphatase