Phosphotransfer in Rhodobacter sphaeroides Chemotaxis

The two-component sensing system controlling bacterial chemotaxis is one of the best studied in biology. Rhodobacter sphaeroides has a complex chemosensory pathway comprising two histidine protein kinases (CheAs) and eight downstream response regulators (six CheYs and two CheBs) rather than the single copies of each as in Escherichia coli. We used in vitro analysis of phosphotransfer to start to determine why R. sphaeroides has these multiple homologues. CheA1 and CheA2 contain all the key motifs identified in the histidine protein kinase family, except for conservative substitutions (F-L and F-I) within the F box of CheA2, and both are capable of ATP-dependent autophosphorylation. While the Km values for ATP of CheA1 and CheA2 were similar to that of E. coli, the kcat value was three times lower, but similar to that measured for the related Sinorhizobium meliloti CheA. However, the two CheAs differed both in their ability to phosphorylate the various response regulators and the rates of phosphotransfer. CheA2 phosphorylated all of the CheYs and both CheBs, whilst CheA1 did not phosphorylate either CheB and phosphorylated only the response regulators encoded within its own genetic locus (CheY1, CheY2, and CheY5) and CheY3. The dephosphorylation rates of the R. sphaeroides CheBs were much slower than the E. coli CheB. The dephosphorylation rate of CheY6, encoded by the third chemosensory locus, was ten times faster than that of the E. coli CheY. However, the dephosphorylation rates of the remaining R. sphaeroides CheYs were comparable to that of E. coli CheY.