Cys303 in the histidine kinase PhoR is crucial for the phosphotransfer reaction in the PhoPR two-component system in Bacillus subtilis.

The PhoPR two-component system activates or represses Pho regulon genes to overcome a phosphate deficiency. The Pho signal transduction network is comprised of three two-component systems, PhoPR, ResDE, and Spo0A. Activated PhoP is required for expression of ResDE from the resA promoter, while ResD is essential for 80% of Pho induction, establishing a positive feedback loop between these two-component systems to amplify the signal received by the Pho system. The role of ResD in the Pho response is via production of terminal oxidases. Reduced quinones inhibit PhoR autophosphorylation in vitro, and it was proposed that the expression of terminal oxidases leads to oxidation of the quinone pool, thereby relieving the inhibition. We show here that the reducing environment generated by dithiothreitol (DTT) in vivo inhibited Pho induction in a PhoR-dependent manner, which is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR(C303A), exhibited reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR(C303A) and DTT are different. PhoR and PhoR(C303A) were similar with regard to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoR approximately P, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoR approximately P or PhoR(C303A) approximately P and PhoP occurred rapidly; most label from PhoR approximately P was transferred to PhoP, but only 10% of the label from PhoR(C303A) approximately P was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoP approximately P or PhoR autophosphatase activity was observed between PhoR and PhoR(C303A) that would explain the release of inorganic phosphate. Our data are consistent with a role for PhoR(C303) in PhoR activity via stabilization of the phosphoryl-protein intermediate(s) during phosphotransfer from PhoR approximately P to PhoP, which is stabilization that is required for efficient production of PhoP approximately P.