Bacillus subtilis PhoP binds to the phoB tandem promoter exclusively within the phosphate starvation-inducible promoter.

AUTOR(ES)

Liu, W

RESUMO

Several gene products, including three two-component systems, make up a signal transduction network that controls the phosphate starvation response in Bacillus subtilis. Epistasis experiments indicate that PhoP, a response regulator, is furthest downstream of the known regulators in the signaling pathway that regulates Pho regulon genes. We report the overexpression, purification, and use of PhoP in investigating its role in Pho regulon gene activation. PhoP was a substrate for both the kinase and phosphatase activities of its cognate sensor kinase, PhoR. It was not phosphorylated by acetyl phosphate. Purified phosphorylated PhoP (PhoPP) had a half-life of approximately 2.5 h, which was reduced to about 15 min by addition of the same molar amount of *PhoR (the cytoplasmic region of PhoR). ATP significantly increased phosphatase activity of *PhoR on PhoPP. In gel filtration and cross-linking studies, both PhoP and PhoPP were shown to be dimers. The dimerization domain was located within the 135 amino acids at the N terminus of PhoP. Phosphorylated or unphosphorylated PhoP bound to one of the alkaline phosphatase gene promoters, the phoB promoter. Furthermore, PhoP bound exclusively to the -18 to -73 region (relative to the transcriptional start site +1) of the phosphate starvation-inducible promoter (Pv) but not to the adjacent developmentally regulated promoter (Ps). These data corroborate the genetic data for phoB regulation and suggest that activation of phoB is via direct interaction between PhoP and the phoB promoter. Studies of the phosphorylation, oligomerization, and DNA binding activity of the PhoP protein demonstrate that its N-terminal phosphorylation and dimerization domain and its C-terminal DNA binding domain function independently of one another, distinguishing PhoP from other response regulators, such as PhoB (Escherichia coli) and NtrC.

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