Isolation and Characterization of Nonchemotactic CheZ Mutants of Escherichia coli

Two-component regulatory systems allow bacteria to sense environmental changes and modify their behavior and gene expression patterns accordingly to optimally exploit available resources or survive adverse conditions (16, 31). This type of signal transduction pathway is minimally composed of a sensor kinase whose autophosphorylation state reflects environmental conditions and a response regulator whose activity is controlled by phosphoryl groups received from the sensor kinase. A fundamental prerequisite for any signal transduction pathway to provide current information is that signaling molecules must turn over on a time scale faster than that on which changes occur in the conditions being monitored. For two-component regulatory systems, there are four known classes of mechanisms by which response regulators may be dephosphorylated. First, response regulator phosphorylation occurs on aspartic acid residues and acyl phosphates are inherently labile to hydrolysis on the time scale of hours (20). Second, some response regulators possess intrinsic autodephosphorylation catalytic activities (13, 19, 26, 47). Third, some sensor kinases, either alone (1, 18) or in conjunction with ancillary proteins (19, 29), catalyze the removal of phosphoryl groups from response regulators in addition to their usual role of providing phosphoryl groups. Fourth, in some systems auxiliary proteins catalyze response regulator dephosphorylation (13, 30, 32). The signal transduction pathway governing chemotaxis in Escherichia coli is one well-characterized example of a two-component regulatory system (43). This information-processing network enables E. coli to move in the direction of increasing attractant or decreasing repellant concentrations by controlling the phosphorylation state of the response regulator protein CheY. Transmembrane receptors detect environmental signals and suitably adjust the autophosphorylation rate of the sensor kinase CheA; CheA-P in turn acts as a phosphodonor to generate CheY-P from CheY (7, 15, 51). CheY-P loses its phosphoryl group via either an intrinsic autodephosphorylation capability or an accelerated dephosphorylation reaction in the presence of CheZ (13, 15). The balance between the rates of phosphorylation and dephosphorylation of CheY defines the intracellular concentration of CheY-P, which dictates the swimming behavior of the cell. CheY-P binds to the flagellar switch protein FliM (48, 49), which changes the direction of flagellar rotation from counterclockwise (CCW) to clockwise (CW) to produce the biased random walk which is necessary for chemotaxis. The phosphatase activity of CheZ, therefore, is critical in determining the intracellular concentration of CheY-P, and CheZ is essential for chemotaxis, as assayed by swarm formation in semisolid agar. CheZ has been the subject of multiple investigations. The central portion of the CheZ amino acid sequence appears to be important for oligomerization (4), and the C-terminal portion binds to CheY-P (3, 27). Important questions concerning the mechanism and regulation of the CheZ-mediated dephosphorylation reaction remain. CheZ requires a Mg2+ cofactor, as does autodephosphorylation (23), but it is not known whether CheZ enhances the intrinsic autodephosphorylation activity of CheY or possesses an independent phosphatase activity. The possibility that CheZ may be regulated by other components of the chemotaxis pathway is attractive, as it could help account for the magnitude of signal amplification (or “gain”) observed in chemotaxis (9). Two interactions that affect CheZ activity have been reported, binding to a short version of CheA, CheAS (44, 45), and oligomerization with CheY-P (4–6), but the molecular basis for either potential regulatory mechanism has not been elucidated. Most previously published studies of CheZ function have primarily employed biochemical or biophysical techniques. In order to address outstanding questions concerning the CheZ reaction mechanism, regulation, or even the potential existence of as yet unrecognized activities, a new experimental approach could be useful. CheZ does not have significant amino acid sequence similarity to any proteins currently in the sequence databases, so sequence comparison furnishes few clues concerning function. In this study, we pursued a genetic approach in which we isolated a large number of nonchemotactic CheZ mutants and mapped the corresponding cheZ mutations. The clustering of the resultant mutations suggested potential regions of functional importance in CheZ. The ability of specific pairs of different nonchemotactic cheZ mutations to complement one another and restore swarming behavior suggested the presence of at least two independent functional domains in CheZ. Finally, an investigation of the ability of selected mutant proteins to stimulate dephosphorylation of CheY and to undergo oligomerization gave information about specific regions or residues in CheZ which are important for function.