| Description |
Bacterial chemotaxis is the directed movement of a bacterium in response to environmental chemical stimuli. Cells of E. coli, for example, swim by rotating their flagella and direct their movement by regulating reversals between counterclockwise (CCW) and clockwise (CW) rotation. The switch from CCW to CW rotation occurs when phosphorylated CheY (CheY-P) interacts with the proteins FliM and FliN, present in an assembly within the flagellum called the switch complex (1). Interaction of CheY-P with the switch complex increases the probability of CW rotation, which gives rise to an erratic motion, called tumbling, which causes the cell to change its direction of swimming. In the present work, we examined interactions between CheY and the switch complex by means of a widely-applied method called the Bacterial Two-Hybrid Adenylate Cyclase (BACTH) method, in which a positive interaction results in the reconstitution of a readily measurable enzymatic activity. The results show that CheY binds to the protein FliN in E. coli and to the middle domain of the protein FliM in B. subtilis. Our experiments also showed that significant chemotactic ability is retained when the N-terminus of FliM (FliMN), which functions in the initial capture of CheY-P, is moved from its normal location onto the CheY protein itself. Two-hybrid experiments indicate that both CheY and the engineered FliMN-CheY interact with FliN. We also mapped the regions of FliN involved in the interaction with CheY-P using a collection of point mutations. These findings cast light on the mechanism by which the flagellar motor responds to the chemotactic signaling protein CheY and suggest avenues for further research. Because motility and chemotaxis are factors contributing to the virulence of bacterial pathogens, the results may ultimately find applications in the clinic. |
