E. coli modulates its swimming behavior in response to environmental stimuli by changing the direction of flagellar rotation. The modulation is done by the chemotaxis signal transduction protein CheY. Earlier studies suggested that CheY shifts the direction of flagellar rotation from the default, counterclockwise, to clockwise according to its phosphorylation level. It was further hypothesized that clockwise rotation is generated by the high-affinity binding of phosphorylated CheY (CheY~P) to the N-terminal domain of the motor protein FliM (FliMN) in an ultra-sensitive process. Here, however, we provide evidence that the binding of CheY to FliMN is not required for clockwise generation or for the chemotactic response to stimuli. We show that, instead, binding of CheY, acetylated at a specific site, to the middle domain of FliM generates CW rotation and enables chemotactic response even in the absence of the N terminus of FliM. These results suggest that the function of the high affinity binding of CheY~P to FliMN is to increase the local concentration of CheY at the motor, and that clockwise is generated by an acetylation-dependent mechanism.
