TIME RESOLVED STUDIES OF CHEMOTACTIC EXCITATION

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Our long term objective in this work is to understand the signaling process in bacterial chemotaxis. We will develop methods for applying, and monitoring responses to, rapid chemotactic stimuli, and determine properties of the excitation signalling pathway. Given that flagella switch randomly, in order to quantitate perturbations in excitation kinetics it is necessary to time-resolve the behavior of large number of cells in response to stimuli which are precisely defined temporally. Two methodologies will be combined: (1) Flash photolytic jumps of chemotactic stimuli in microsecond times, and (2) computer-assisted motion analysis of the behavioral responses of free-swimming bacteria with 17 millisecond resolution. Such rapid chemoeffector generation will ensure that receptor binding events are not rate limiting and the resolution in the monitoring system is sufficient to register the onset of the response, given known response latencies. Chemoeffector jumps will be accomplished by pulse laser excitation of photoabile caged precursors. Direct measurement of the excitation time of Salmonella typhimurium and Escherichia coli provides a powerful tool for establishing structure/function relationships, especially when combined with the molecular genetic manipulations of the transducers and other chemotaxis components possible with these bacteria. After developement of the motion analysis routines, excitation times in wildtype cells for a number of different transducer-mediated as well as transducer- independent (e.g. protonmotive force sensor) reception systems will be analyzed. The role, if any, of transducer methylation and of the methylation machinery in the kinetics of both transducer- dependent and transducer-independent excitation will be assessed. We will first determine whether signaling is limited by receptor or post-receptor reactions in wildtype cells. Stringently controlled plasmid-altered strains will be used to modulate the levels of the signaling components singly or in combination. This, together with changes of temperature and pH, will allow us to make different reactions in the signaling pathway rate-limiting, providing a strategy for ordering products of the known chemotaxis genes in the pathway. The flash-photolytic technique will also make possible rapid intracellular jumps of ions and small metabolites potentially involved in signaling (e.g. cyclic nucleotides and divalent cations).