bifunctional kinase-phosphatase in bacterial chemotaxis

Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA₃, is not only a phosphodonor for the principal CheY protein, CheY₆, but is also is a specific phosphatase for CheY₆-P. This phosphatase activity accelerates CheY₆-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA₃ possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA₃ is phosphorylated by CheA₄, and it subsequently acts as a phosphodonor for the response regulators. A CheA₃ mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA₃ is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA₃ lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases.