Cys303 and redox state of quinone regulate the activity of the histidine kinase PhoR in Bacillus subtilis

• Main Author: Eldakak, Amr Hussein
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2006
• Subjects: Microbiology; Molecular biology
• ISBN: 9780542700347; 0542700344

In B. subtilis, the complex Pho signal transduction network mediates the adaptive response to phosphate starvation conditions. This network encompasses the two-component systems PhoPR, ResDE and the sporulation phosphorelay. The Pho signal transduction network includes a positive feedback loop between PhoPR and ResDE TCS. ResD is responsible for 80% of the Pho response, while PhoP is required for ResD expression from the resA-promoter. A suppressor mutation in ydiH could bypass the requirement for ResD in a ΔresD mutant, for full Pho induction by allowing expression of bd oxidase under phosphate starvation conditions. In this study, a ctaA mutant strain, deficient in the production of heme A, has the same Pho induction phenotype as a ΔresD mutant. This implies that ResD affects Pho induction through the production of terminal oxidases. In vitro data revealed that the reduced state of quinones inhibits the PhoR autophosphorylation. These in vitro studies were supported by the finding that the reduced environment generated by DTT in vivo inhibited the Pho response, as expressed by lacZ expression from Pho-regulated promoters, and this inhibition is PhoR-dependent. These data suggest a requirement for terminal oxidases in vivo for full Pho induction, to relieve the inhibition by reduced quinone on the autophosphorylation on PhoR. To check if the redox state of quinones regulates PhoR through a redox active cysteine residue, Cys303 residue was mutated to alanine. In a PhoRC303A Variant, Pho induction was repressed about 60% in vivo and it was inhibited by the addition of DTT. In vitro, PhoRC303A as active as to PhoRWT. The autophosphorylation of purified PhoR C303A was also inhibited by the reduced form of quinones, which suggests that Cys303 regulates PhoR through a different mechanism. PhoRC303A∼P failed to phosphorylate its cognate response regulator PhoP and the phosphotransfer rate was reduced about 90% compared to the wild-type levels. The inhibition of the phosphotransfer rate in the case of PhoRC303A∼P correlates with the in vivo phenotype, which implies that Cys303 is crucial for the phosphotransfer reaction between PhoR and its cognate RR PhoP.