CONICET | Buscador de Institutos y Recursos Humanos

The chemotaxis system in bacteria allows controlled movement in response to chemical gradients. Chemoreceptors transmit ligand-binding information to an associated histidine kinase, CheA, within a ternary complex that also contains the coupling protein CheW. CheA autophosphorylates and donates its phosphoryl group to the response regulator CheY, which then diffuses to the flagellar motors to control their mode of rotation. Attractants inhibit CheA phosphorylation, decreasing the levels of phosphorylated CheY. CheW binding interactions with receptors and with CheA are important for the formation of ternary complexes that respond to chemotactic stimuli. However, it is not clear whether the role of CheW is limited to bridge the two proteins and thus contribute to the assembly of the large chemoreceptor clusters at the cell poles, or whether it plays an active role in signal transmission through stimuli-mediated conformational changes. In this work, we introduced mutational replacements in CheW, located at the contact surface with chemoreceptors, and analyzed the signaling consequences of such changes. We identified five CheW mutant proteins that failed to mediate chemotaxis responses towards serine, as determined in soft agar swimming assays. Two of them coded for proteins that were unable to activate the CheA kinase, in spite of showing expression levels comparable to wild-type CheW. Two other replacements generated CheW proteins that activated the kinase irrespective to the presence of attractant, indicating that the signaling complex in these cells was locked in the ?on? state. Moreover, one of the mutant proteins, that displayed a significant truncation of the protein, also behaved as a ?lock-on? mutant, indicating that the missing portion of the protein was dispensable to form kinase-activating complexes.