FLAGELLAR MOTOR-SWITCH FINDING FACE OF CHEY AND THE BIOCHEMICAL BASISOF SUPPRESSION BY CHEY MUTANTS THAT COMPENSATE FOR MOTOR-SWITCH DEFECTS IN ESCHERICHIA-COLI

CheY is a response regulator protein of Escherichia coli that interact s with the flagellar motor-switch complex to modulate flagellar rotati on during chemotaxis. The switch complex is composed of three proteins , FliG, FliM, and FliN, Recent biochemical data suggest a direct inter action of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY wi th limited allele specificity. The protein products of suppressor cheY alleles were purified and assayed for FliM binding. Six out of nine C heY mutants were defective in FliM binding. Suppressor amino acid subs titutions were mapped on the crystal structure of CheY showing cluster ing of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of g enetic suppression, we cloned, purified, and tested FliM mutants for C heY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected i f CheY suppressors were compensatory conformational suppressors. Furth ermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strain s. However, a chemotaxis assay revealed that the readjustment of the c lockwise bias was not sufficient to make cells chemotactic. Although t he suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called ''pseudotaxis.'' Therefore, our genetic selection scheme generated suppressors of pseudotaxis or swit ch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated Che Y, Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compe nsatory conformational changes.