Simultaneous Disruption of Phosphate and Carbon Signaling Regulators Enables Adaptive Gene Expression Through Non-Cognate Phosphorylation of PhoP

The PhoP–PhoR two-component system (TCS) controls transcription of the Pho regulon in response to inorganic phosphate limitation in Bacillus subtilis. In addition to its role in phosphate homeostasis, increasing evidence suggests that central metabolic pathways influence Pho regulon activity. Here, we investigated the mechanism underlying phosphate-independent activation of the Pho regulon in a ccpA mutant lacking the global regulator of CCR. Gene expression analyses demonstrated strong glucose-dependent induction of Pho regulon genes in the absence of both CcpA and the cognate histidine kinase PhoR. Using PhosTag gel electrophoresis analysis and a phoasphoablative mutation in phoP (D53A), we detected early PhoP phosphorylation and determined that Pho regulon activation requires a phosphorylatable form of PhoP, even when the cognate histidine kinase phoR gene was deleted. Because PhoR can phosphorylate non-cognate response regulator YycF, we examined whether the essential YycFG signaling system contributes to PhoP activation. Co-immunoprecipitation and in vitro phosphorylation assays confirmed a direct interaction between PhoP and YycG and demonstrated in vitro PhoP phosphorylation by YycG. Taken together, we propose a model in which CcpA limits excessive PhoP accumulation to preserve signaling specificity and prevent aberrant activation through non-cognate kinases. Furthermore, our study reveals a previously unrecognized link between carbon catabolite repression and phosphate starvation signaling and demonstrates another crosstalk between two-component signal transduction systems in the Gram-positive bacterium, B. subtilis.