In vitro and in vivo analysis of the ArcB/A redox signaling pathway

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.