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

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 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 PhoRϳP, and inhibition of autophosphorylation by DTT. Phosphotransfer between PhoRϳP or PhoR C303A ϳP and PhoP occurred rapidly; most label from PhoRϳP was transferred to PhoP, but only 10% of the label from PhoR C303A ϳP was associated with PhoP, while 90% was released as inorganic phosphate. No difference in PhoPϳP 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 PhoRϳP to PhoP, which is stabilization that is required for efficient production of PhoPϳP.

An integrated network approach identifies the isobutanol response network of Escherichia coli

“…Three other two-component systems are known to have quinone-related activation mechanisms. These include EvgAS of E. coli (ubiquinone mediated), BvgAS of Bordetella pertussis (ubiquinone mediated), and PhoPR of Bacillus subtillis (menaquinol mediated) ( Bock and Gross, 2002 ; Schau et al , 2004 ; Eldakak and Hulett, 2007 ). EvgAS is homologous to BvgAS in structure, and the PhoBR system of E. coli is homologous to the PhoPR system of B. subtillis in structure and function (BLAST results presented in Supplementary Table VI ).…”

DosS Responds to a Reduced Electron Transport System To Induce the Mycobacterium tuberculosis DosR Regulon

“…Rhodobacter sphaeroides uses PrrBA to sense and respond to electron flow through the cbb 3 oxidase (33,34). PhoR in Bacillus subtilis responds to the reduced quinones, and several other bacterial sensors containing redox-active cysteine residues have been reported (12). Thus, it is apparent that monitoring the state of the ETS is an important function in many bacteria.…”

Prokaryotic regulatory systems biology: Common principles governing the functional architectures of Bacillus subtilis and Escherichia coli unveiled by the natural decomposition approach

“…The latter provides further support to the fact that it is not safe to apply the same-level strategy to deal with FBLs. A dual FBL comprising phoP and resD was found because of PhoP might activate or repress resD expression (Eldakak and Hulett, 2007). Both FBLs were enumerated because their dynamic behaviors are completely different.…”