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 proteins, regulate the phosphate starvation response. Here, we used two other pho regulon promoters, the phoA and pstS promoters, to examine the 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 TTAACA-like sequences upstream of these two promoters, while phosphorylated PhoP extended the binding region in both the 5' and the 3' direction and, additionally, protected sequences internal to the coding region of these two genes. The PhoP binding sites in the coding region were necessary for full induction from either promoter during phosphate starvation. Deletion of these sites eliminated approximately 75% and 45% of the induced promoter activity of the phoA and pstS promoters respectively. In vitro transcription assays using the phoA promoters with various 3' ends confirmed the requirement of the PhoP-P binding to the coding region for full promoter activity. The multiple TTAACA-like sequences in the phoA and pstS promoters were essential for promoter activity, and deletion of one or more of these sequences in either promoter eliminated the promoter activity. Two pairs of TTAACA-like sequences were required for efficient PhoP binding and were suggested 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.