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Journal of Bacteriology, August 2005, p. 5166-5178, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5166-5178.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Bacillus subtilis Phosphorylated PhoP: Direct Activation of the E^A- and Repression of the E^E-Responsive phoB-P_S+V Promoters during Pho Response

Wael R. Abdel-Fattah, Yinghua Chen, Amr Eldakak, and F. Marion Hulett^*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607

Received 6 January 2005/ Accepted 25 April 2005

The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-P_S, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-P_V. E^E was necessary and sufficient for P_S expression in vitro. P_S expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of ^E is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoPP) repressed P_S in vitro via direct binding to the promoter, the first example of an E^E-responsive promoter that is repressed by PhoPP. Whereas either PhoP or PhoPP in the presence of E^A was sufficient to stimulate transcription from the phoB-P_V promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoPP were required for P_V promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P_i-replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

Present address: Department of Medicine, Section of Hematology/Oncology, University of Chicago, Chicago, Ill.

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