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Cys303 in the Histidine Kinase PhoR Is Crucial for the Phosphotransfer Reaction in the PhoPR Two-Component System in Bacillus subtilis

Amr Eldakak and F. Marion Hulett^*

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

Received 2 August 2006/ Accepted 23 October 2006

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 PhoRP, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoRP or PhoR_C303AP and PhoP occurred rapidly; 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 difference in 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, which is stabilization that is required for efficient production of PhoPP.

Published ahead of print on 3 November 2006.

Present address: Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110.