Chemotactic excitation responses to caged ligand photorelease of rapidly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Escherichia coli and Salmonella typhimurium) have been measured by computer. Mutants were used to assess the effects of abnormal motility behavior upon signal processing times and test feasibility of kinetic analyses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)ethoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl phosphate were synthesized. These compounds are a 'caged' serine and a 'caged' proton and on flash photolysis release serine and protons and attractant and repellent ligands, respectively, for Tsr, the serine receptor. The product quantum yield for serine was 0.65 (+/- 0.05) and the rate of serine release was proportional to [H+] near-neutrality with a rate constant of 17 s-1 at pH 7.0 and 21 degrees C. The product quantum yield for protons was calculated to be 0.095 on 308-nm irradiation but 0.29 (+/- 0.02) on 300-350-nm irradiation, with proton release occurring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro intermediate and bioassays. Receptor deletion mutants did not respond to photorelease of the caged ligands. Population responses occurred without measurable latency. Response times increased with decreased stimulus strength. Physiological or genetic perturbation of motor rotation bias leading to increased tumbling reduced response sensitivity but did not affect response times. Exceptions were found. A CheR-CheB mutant strain had normal motility, but reduced response. A CheZ mutant had tumbly motility, reduced sensitivity, and increased response time to attractant, but a normal repellent response. These observations are consistent with current ideas that motor interactions with a single parameter, namely phosphorylated CheY protein, dictate motor response to both attractant and repellent stimuli. Inverse motility motor mutants with extreme rotation bias exhibited the greatest reduction in response sensitivity but, nevertheless, had normal attractant response times. This implies that control of CheY phosphate concentration rather than motor reactions limits responses to attractants.

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)-carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates D-glucose and its nonmetabolizable analog methyl alpha-D-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 +/- 3.1 s-1. The response threshold was <10 nM for glucose. Responses to methyl alpha-D-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP-CheW-CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.