Bacillus subtilisPhosphorylated PhoP: Direct Activation of the Eσ^A- and Repression of the Eσ^E-ResponsivephoB-P_S+VPromoters during Pho Response

Abstract

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 (PhoPϳP) repressed P S in vitro via direct binding to the promoter, the first example of an E E -responsive promoter that is repressed by PhoPϳP. Whereas either PhoP or PhoPϳP 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 PhoPϳP 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.

CcpA Causes Repression of the phoPR Promoter through a Novel Transcription Start Site, P A6

“…When additional PhoP-PhoR is not required, repression of P A6 by CcpA is not only energy efficient, it also avoids placing the cells at a selective disadvantage as has been observed when cellular concentrations of PhoP were increased inappropriately by expression from multicopy plasmids (34,42) or in spo0 mutants (23). The poor growth phenotype and/or rapid accumulation of spontaneous phoP or phoR mutations in cells overexpressing phoPR or phoP may be the result of inappropriate gene activation or inhibition by higher concentrations of PhoP or PhoPϳP, respectively, as observed in vitro (1).…”

Direct Regulation ofBacillus subtilis phoPRTranscription by Transition State Regulator ScoC

“…Further, not all APase expression in B. subtilis is PhoPϳP activated. phoB (formerly phoAIII) encoding APase B (formerly APase III) (5, 9) is expressed from two differentially regulated promoters, P S (E E -responsive promoter) and P v (E A -responsive promoter), which are PhoPϳP repressed and activated during PSI, respectively (2,5,9). The study by Caldwell et al (7) found a 9.3-fold increase in phoB expression in an scoC mutant compared to the WT strain at the last time point assayed (310 min after inoculation), an increase which represented the greatest fold increase in a group of sporulation genes dependent on Spo0A and F .…”

Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis

“…Which of these phosphatases gets expressed is dependent on the physiological conditions. Their cellular localization varies from secreted to membrane-associated, and it has been shown that the main members of this family, PhoA and PhoB, can complement each other (18,19). Although it has been proposed that the main biological function of the Pho enzymes is the provision of phosphate during phosphate starvation and that phoB expression is accordingly controlled, we were able to purify substantial amounts of PhoB from B. subtilis cells grown in rich medium, and therefore, we assume that PhoB is among the most abundant phosphatases even under those conditions.…”