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J. Bacteriol. doi:10.1128/JB.01205-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

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

^* To whom correspondence should be addressed. Email: Hulett{at}uic.edu.

	Abstract

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 was required for expression of ResDE from the resA-promoter while ResD was 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 on the Pho response is via production of terminal oxidases. Reduced quinones inhibited 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 DTT in vivo inhibited Pho induction in a PhoR dependent manner that is in agreement with our previous in vitro data. A strain containing a PhoR variant, PhoR_C303A had reduced Pho induction and remained sensitive to inhibition by DTT, suggesting that the mechanisms for Pho reduction via PhoR_C303A and DTT differed. PhoR and PhoR_C303A were similar with regards to cellular concentration, limited proteolysis patterns, rate of autophosphorylation, stability of PhoRP and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoRP or PhoR_C303AP and PhoP occurred rapidly and most label from PhoRP was transferred to PhoP but only 10% of the label from PhoR_C303AP was associated with PhoP while 90% was released as inorganic phosphate. No change in phosphatase activity for PhoPP 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 PhoRP to PhoP, stabilization required for efficient production of PhoPP.