Dual Role of the PhoP～P Response Regulator: Bacillus amyloliquefaciens FZB45 Phytase Gene Transcription Is Directed by Positive and Negative Interactions with the phyC Promoter

摘要

Several Bacillus strains secrete phytase, an enzyme catalyzing dephosphorylation of myo-inositol hexakisphosphate (phytate). We identified the phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 as a member of the phosphate starvation-inducible PhoPR regulon. In vivo and in vitro assays revealed that PhoP～P is essential for phyC transcription. The transcriptional start site was identified downstream of a σA-like promoter region located 27 bp upstream of the probable translation ATG start codon. Inspection of the phyC promoter sequence revealed an unusual structure. The? 35 and ?10 regions are separated by a window of 21 bp. A pair of tandemly repeated PhoP TT(T/A/C)ACA binding boxes was located within and upstream of the ?35 consensus promoter region. A single PhoP box was found within the ?10 consensus promoter region. DNase I footprinting experiments performed with isolated PhoP confirmed that PhoP～P binds at two sites overlapping with the phyC ?35 and ?10 consensus promoter region. While binding of dimeric PhoP～P at ?35 is essential for activation of the phyC promoter, binding of PhoP～P at? 10 suppresses promoter activity. A sixfold enhancement of phyC gene expression was registered after T:G substitution of nucleotide ?13 (mutant MUT13), which eliminates PhoP binding at the single PhoP box without impairing the ?10 consensus sequence. Moreover, MUT13 also expressed phyC during phosphate-replete growth, suggesting that the repressing effect due to binding of PhoP～P at ?10 was abolished. A model is presented in which transcription initiation of phyC is positively and negatively affected by the actual concentration of the PhoP～P response regulator.