Fine-Tuning Control of phoBR Expression in Vibrio cholerae by Binding of PhoB to Multiple Pho Boxes

The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at -35 (box 1), -60 (box 2), and -80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under inorganic phosphate (P-i) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low P-i, suggesting a repressive role for PhoB similar to box2 (PhoB bound to box 2) over the transcriptional activity induced by PhoB similar to box1. The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoB similar to box3 eliminated the repressive effect imposed by PhoB similar to box2 on phoBR transcription. Complementation assays with a phoBR- containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon expression in Vibrio cholerae -phoBR cells. This was observed under Pi abundance, when phoBR was expressed at a basal level and, also in low Pi conditions, when Pho regulon genes were fully expressed. Thus, under Pi limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and precise control of phoBR expression in V. cholerae cells.