EXCITATORY SIGNALING IN BACTERIA PROBED BY CAGED CHEMOEFFECTORS

Chemotactic excitation responses to caged ligand photorelease of rapid ly swimming bacteria that reverse (Vibrio alginolyticus) or tumble (Es cherichia coli and Salmonella typhimurium) have been measured by compu ter. Mutants were used to assess the effects of abnormal motility beha vior upon signal processing times and test feasibility of kinetic anal yses of the signaling pathway in intact bacteria. N-1-(2-Nitrophenyl)e thoxycarbonyl-L-serine and 2-hydroxyphenyl 1-(2-nitrophenyl) ethyl pho sphate were synthesized. These compounds are a 'caged' serine and a 'c aged' proton and on flash photolysis release serine and protons and at tractant and repellent ligands, respectively, for Tsr, the serine rece ptor. 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 quan tum 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 oc curring at > 10(5) s-1. The pH jumps produced were estimated using pH indicators, the pH-dependent decay of the chromophoric aci-nitro inter mediate and bioassays. Receptor deletion mutants did not respond to ph otorelease 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 no t affect response times. Exceptions were found. A CheR-CheB mutant str ain had normal motility, but reduced response. A CheZ mutant had tumbl y motility, reduced sensitivity, and increased response time to attrac tant, but a normal repellent response. These observations are consiste nt with current ideas that motor interactions with a single parameter, namely phosphorylated CheY.protein, dictate motor response to both at tractant and repellent stimuli. Inverse motility motor mutants with ex treme rotation bias exhibited the greatest reduction in response sensi tivity but, nevertheless, had normal attractant response times. This i mplies that control of CheY-phosphate concentration rather than motor reactions limits responses to attractants.