| Bacillus subtilis phosphate-starvation response (PHO) regulator, PhoP, activates or represses different sets of genes important for survival under such harsh condition. In order for PhoP to exert these function, it must be phosphorylated on a specific aspartate residue residing in its receiver domain. The histidine kinase PhoR phosphorylates PhoP allowing PHO-induction. Here we observed that PhoP whether in the phosphorylated or unphosphorylated form could activate transcription of target genes, at least in vitro. In vivo analysis suggested that when cellular PhoP concentrations were higher than normal, PhoP activated target gene expression in the absence of PhoR. Surprisingly, a null mutation in phoR allowed expression of one of the PHO-regulon genes, phoB, encoding alkaline phosphatase III, in Pi-replete condition and with a second mutation in ccpA, encoding catabolite control protein A that is involved in carbon catabolite regulation (CCR), PHO-induction was Pi-independent. However, glucose supplementation in the culture media was essential for the PhoP-dependent PHO-induction in both wild-type and mutant strains. The data suggested a possible role of PhoR in PHO-regulon inactivation during growth in Pi-replete condition. In a second project where we investigated the role of PhoP∼P in transcription of resA, encoding a thiol-disulfide oxidoreductase required for cytochrome c550 maturation, a direct connection between PHO, RES, and CCR was uncovered. CcpA repressed resA transcription in vitro and PhoP∼P was required to relieve such repression plausibly via competitive binding to target DNA. In addition, ResD, the aerobic/anaerobic pleitropic regulator, was unable to activate transcription of the [ resA.CcpA] complex unless ResD was phosphorylated by its cognate histidine kinase ResE. The ResE auto-kinas activity was inhibited by di-nucleotides such as NADH suggesting that maximal ResD phosphorylation occurred after initial resA transcription. The Michael-Menten kinetic analysis of the ResE histidine kinase indicated that ResE auto-kinase activity has two Km values, one at [ATP] = 50 nM and the second at [ATP] = 3 muM. At high ATP concentrations above 1 muM, less ResD was phosphorylated, whereas at low ATP concentrations less than 50 nM, more ResD was phosphorylated. Interestingly, incubation of ResD with ATP alone in the absence of ResE allowed ResD activation and transcription of the target promoter, resA, at least in vitro. Data suggested a possible two active forms of ResD, the [ResD∼P] form activating anaerobic gene transcription, and a [ResD.ATP] form activating aerobic gene transcription. |
