SITES INTERNAL TO THE CODING REGIONS OF PHOA AND PSTS BIND PHOP AND ARE REQUIRED FOR FULL PROMOTER ACTIVITY

Bacillus subtilis PhoP and PhoR, a pair of two-component regulatory pr oteins, regulate the phosphate starvation response. Here, we used two other pho regulon promoters, the phoA and pstS promoters, to examine t he mechanism of PhoP-specific activation of its target promoters. Both gel shift and DNase I footprinting assays indicate that PhoP bound to the two promoters. Unphosphorylated PhoP bound only to the multiple T TAACA-like sequences upstream of these two promoters, while phosphoryl ated PhoP extended the binding region in both the 5' and the 3' direct ion and, additionally, protected sequences internal to the coding regi on of these two genes. The PhoP binding sites in the coding region wer e necessary for full induction from either promoter during phosphate s tarvation. Deletion of these sites eliminated approximately 75% and 45 % of the induced promoter activity of the phoA and pstS promoters resp ectively, in vitro transcription assays using the phoA promoters with various 3' ends confirmed the requirement of the PhoP similar to P bin ding to the coding region for full promoter activity. The multiple TTA ACA-like sequences in the phoA and pstS promoters were essential for p romoter activity, and deletion of one or more of these sequences in ei ther promoter eliminated the promoter activity. Two pairs of TTAACA-li ke sequences were required for efficient PhoP binding and were suggest ed to be one B. subtilis Pho box. Based on our data, we have proposed a model for activation of the phoA and the pstS promoter by PhoP.