Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade

Rapid restoration of prestimulus levels of the chemotactic response regulator, CheY- P, is important for preparing bacteria and archaea to respond sensitively to new stimuli. In an extension of previous work ( Szurmant, H., Bunn, M. W., Cannistraro, V. J., and Ordal, G. W. ( 2003) J. Biol. Chem. 278, 48611 - 48616), we describe a new family of CheY- P phosphatases, the CYX family, that is widespread among the bacteria and archaea. These proteins provide another pathway, in addition to the ones involving CheZ of the gamma- and beta- proteobacteria ( e. g. Escherichia coli) or the alternative CheY that serves as a " phosphate sink" among the alpha- proteobacteria ( e. g. Sinorhizobium meliloti), for dephosphorylating CheY- P. In particular, we identify CheC, known previously to be involved in adaptation to stimuli in Bacillus subtilis, as a CheY- P phosphatase. Using an in vitro assay used previously to demonstrate that the switch protein FliY is a CheY- P phosphatase, we have shown that increasing amounts of CheC accelerate the hydrolysis of CheY- P. In vivo, a double mutant lacking cheC and the region of fliY that encodes the CheY- P binding domain is almost completely smooth swimming, implying that these cells contain very high levels of CheY- P. CheC appears to be primarily involved in restoring normal CheY- P levels following the addition of attractant, whereas FliY seems to act on CheY- P constitutively. The activity of CheC is relatively low compared to that of FliY, but we have shown that the chemotaxis protein CheD enhances the activity of CheC 5- fold. We suggest a model for how FliY, CheC, and CheD work together to regulate CheY- P levels in the bacterium.