Dual Role of the PhoP∼P Response Regulator:Bacillus amyloliquefaciensFZB45 Phytase Gene Transcription Is Directed by Positive and Negative Interactions with thephyCPromoter
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Dual Role of the PhoP∼P Response Regulator:Bacillus amyloliquefaciensFZB45 Phytase Gene Transcription Is Directed by Positive and Negative Interactions with thephyCPromoter
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<jats:title>ABSTRACT</jats:title><jats:p>Several<jats:italic>Bacillus</jats:italic>strains secrete phytase, an enzyme catalyzing dephosphorylation of<jats:italic>myo</jats:italic>-inositol hexakisphosphate (phytate). We identified the<jats:italic>phyC</jats:italic>(phytase) gene from environmental<jats:italic>Bacillus amyloliquefaciens</jats:italic>FZB45 as a member of the phosphate starvation-inducible PhoPR regulon. In vivo and in vitro assays revealed that PhoP∼P is essential for<jats:italic>phy</jats:italic>C transcription. The transcriptional start site was identified downstream of a σ<jats:sup>A</jats:sup>-like promoter region located 27 bp upstream of the probable translation ATG start codon. Inspection of the<jats:italic>phyC</jats:italic>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<jats:italic>phy</jats:italic>C −35 and −10 consensus promoter region. While binding of dimeric PhoP∼P at −35 is essential for activation of the<jats:italic>phyC</jats:italic>promoter, binding of PhoP∼P at− 10 suppresses promoter activity. A sixfold enhancement of<jats:italic>phyC</jats:italic>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<jats:italic>phyC</jats:italic>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<jats:italic>phyC</jats:italic>is positively and negatively affected by the actual concentration of the PhoP∼P response regulator.</jats:p>