MUTATIONS IN THE CHEMOTACTIC RESPONSE REGULATOR, CHEY, THAT CONFER RESISTANCE TO THE PHOSPHATASE-ACTIVITY OF CHEZ

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway, The concentration of phospho-CheY is though t to determine the swimming behaviour of the cell: high levels of phos pho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow co unter-clockwise rotation of the flagella and smooth swimming, The phos phorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphoryl ation of phospho-CheY, The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ pro tein, which accelerates dephosphorylation, We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY protein s. Two point mutations were identified which were 50-fold and 5-fold l ess sensitive to the activity of CheZ than was the wild-type protein, Nonetheless, the phosphorylation and autodephosphorylation rates of th ese mutants, CheY23ND and CheY26KE, were observed to be identical to t hose of wild-type CheY in the absence of CheZ, These are the first exa mples of cheY mutations that reduce sensitivity to the phosphatase act ivity of CheZ without being altered in terms of their intrinsic phosph orylation and autodephosphorylation rates. Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphor ylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.