Function of a potential two-component system protein in Gluconobacter oxydans

Abstract:

The genome of Gluconobacter oxydans encodes a large number of sensor kinases and response regulators, potential members of two-component signal transduction systems (TCSs), by which bacteria recognize and respond to a variety of environmental stimuli. However, little is known about the structures and functions of these proteins in G. oxydans. [Objective] To study the autophosphorylation activity of a potential TCS protein encoded by GOX0645 in G. oxydans 621H, and to reveal the role of this protein in bacterial chemotaxis.[Methods] We performed sequence analysis on GOX0645 from G. oxydans 621H that might encode a TCS member, detected the autophosphorylation activity of the encoded protein by in vitro luminescent kinase assay, and then identified the amino acid residues critical to the autophosphorylation by site-directed mutagenesis. We employed differential centrifugation to study the subcellular localization of the protein, and BiFC assay and Octet^® system to explore the interaction between the protein and a flagellar motor protein of G. oxydans. [Results] The protein encoded by GOX0645 contained the conserved domains of both histidine kinases and response regulators, with a conserved Asp and three His residues associated with autophosphorylation activity. Furthermore, the protein, as a hybrid protein, showed the activity of autophosphorylation which was affected by the concentration of cAMP molecule. The protein was localized in the cytoplasm and had a moderate affinity with the flagellar motor protein, which implied its regulatory role in bacterial chemotaxis. [Conclusion] This study analyzed a potential TCS that might be involved in the regulation of chemotaxis in G. oxydans 621H. This finding contributes to gaining insight into the molecular mechanism of two-component systems in G.oxydans.