Determinants of chemotactic signal amplification in Escherichia coli

A well-characterized protein phosphorelay mediates Escherichia coli chemota xis towards the amino acid attractant aspartate. The protein CheY shuttles between flagellar motors and methyl-accepting chemoreceptor (MCP) complexes containing the linker CheW and the kinase CheA. CheA-CheY phosphotransfer generates phospho-CheY, CheY-P. Aspartate triggers smooth swim responses by inactivation of the CheA bound to the target MCP, Tar; but this mechanism alone cannot explain the observed response sensitivity. Here, we used behav ioral analysis of mutants deleted for CheZ, a catalyst of CheY-P dephosphor ylation, or the methyltransferase CheR and/or the methylesterase CheB to ex amine the roles of accelerated CheY-P dephosphorylation and MCP methylation in enhancement of die chemotactic response. The extreme motile bias of the mutants was adjusted towards wild-type values, while preserving much of th e aspartate response sensitivity by expressing fragments of the MCP, Tsr, t hat either activate or inhibit CheA. We then measured responses to small ju mps of aspartate, generated by flash photolysis of photo-labile precursors. The stimulus-response relation fur Delta cheZ mutants overlapped that for the host strains, Delta cheZ excitation response times increased with stimu lus size consistent with formation of an occluded CheA state. Thus, neither CheZ-dependent or independent increases in CheY-P dephosphorylation contri bute to the excitation response. In Delta cheB Delta cheR or Delta cheR mut ants, the dose for a half-maximal response, [Asp](50), was ca 10 muM; but w as elevated to 100 muM in Delta cheB mutants. In addition, the stimulus-res ponse relation for these mutants was linear, consistent with stoichiometric inactivation, in contrast to the non-linear relation for wild-type E. coli . These data suggest that response sensitivity is controlled by differentia l binding of CheR and/or CheB to distinct MCP signaling conformations. (C) 2001 Academic Press.