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Georgellis, D; Alvarez, AF (2010)

IN VITRO AND IN VIVO ANALYSIS OF THE ARCB/A REDOX SIGNALING PATHWAY

METHOD ENZYMOL 471():205-228
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The Arc (anoxic redox control) two-component system (TCS) is a complex signal transduction system that plays an important role in regulating energy metabolism at the level of transcription in bacteria. This system comprises the ArcB protein, a hybrid membrane-associated sensor kinase, and the ArcA protein, a typical response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphosphorylates ArcA via a His -> Asp -> His -> Asp phosphorelay. Under aerobic conditions, the ArcB kinase activity is silenced by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Under such conditions, ArcB acts as a phosphatase that catalyzes the dephosphorylation of ArcA-P and thereby releasing its transcriptional regulation. This chapter describes general in vitro and in vivo assays and strategies that have been used to characterize the ArcB/A two-component signal transduction system, which could, also, be applied to most other TCS.