The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate‐inducible expression of Pho regulon genes in Bacillus subtilis

Abstract

SummaryPhoP-PhoR, one of three two-component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli, was autophosphor ylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphor ylation probably occurs on a conserved histidine residue. PhosphoPhoR phosphorylated its cognate response regulator, PhoP in vitro. B. subtilis phoR was placed in the Bacillus chromosome under the control of the P spac promoter, which is IPTG inducible. The wild-type phoR, under either native promoter or P spac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild-type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR sequences or various extended N-terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild-type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C-terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate-limited conditions, and that the regulation for repression of APase under phosphate-replete conditions remains intact.

Autoinduction ofBacillus subtilis phoPROperon Transcription Results from Enhanced Transcription from EσA- and EσE-Responsive Promoters by Phosphorylated PhoP

“…PhoP and *PhoR were purified as previously described (22). *PhoR is a soluble, truncated form of PhoR (38).…”

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

“…By domain architecture, this protein has all features of a classical periplasmic-sensing HK: the N terminus of this sensor kinase contains two TM helices flanking an extracytoplasmic domain of 120 aa. However, strains expressing PhoR derivatives in which the extracytoplasmic domain between the two TM domains, or even the complete N terminus up to the cytoplasmic side of TM helix 2, are deleted (PhoR is expressed as a soluble, cytoplasmic protein in the latter case) respond almost normally to PhoR-mediated phosphate limitation (233). The authors of this and another study on the homologous PhoR sensor kinase from E. coli could demonstrate that an extended cytoplasmic linker region, termed the C 2 region, between TM helix 2 and the kinase domain is necessary and sufficient for sensing phosphate limitation (228,233).…”

Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

“…Hulett and co-workers discovered that PhoRc could induce phoA under phosphate limiting conditions and that, in contrast to E. coli, repression of phoA under phosphate-replete conditions remained intact in the presence of PhoRc (Shi and Hulett, 1999). Although the authors considered it unlikely at the time, they suggested that the real sensor for the low phosphate signal triggering the Pho response may reside in the PhoR kinase domain (i.e., in the cytoplasm).…”