Escherichia coli chemotaxis has emerged as a paradigm for the elucidation of the computational strategies employed by intracellular signaling phosphorelays to respond to environmental changes. The chemotactic phosphorelay is part of a large famly of histidyl-aspartyl phosphorelays that mediate diverse sensory transduction schemes in prokaryotes and eukaryotes. The chemotactic response, in common with other sensory systems, exhibits high sensitivity over a large chemoeffector concentration range. This sensitivity may be achieved by utilizing dynamic, multicomponent receptor assemblies to generate and process chemotactic signals. The flash photorelease assay, based on computerized motion analysis of responses to photochemically generated chemoeffector concentration jumps, has been developed and established as a time-resolved method for quantitative analysis of chemotactic signal processing. Sensitivity modulation of chemoattractant (aspartate and glucose) responses by stimulus strength and concentration range has been measured. The data sets constraints to possible signal amplification mechanisms. Planned work will build on these advances. Excitation responses of negative stimuli (i.e. leucine) will be determined, as a prelude to competition assays between positive and negative stimuli, designed to probe for receptor-receptor interactions. Expression of receptor signaling domains restores normal motility, but not chemotaxis, in mutant strains; allowing evaluation of the mutations on signaling properties. Overexpression of the central signaling phosphoprotein, CheY, affects signaling kinetics when it is expressed alone but not with CheZ, a catalyst for CheY dephosphorylation. Green fluorescent protein (GFP) - CheY chimeras have been constructed to visualize dynamics of CheY interactions with receptor and motor assemblies by low-light level fluorescent microscopy techniques; to relate in vivo to in vitro binding parameters, and to time-resolve their modulation during signaling triggered by photoreleased chemoeffectors. The knowledge gained will contribute to fundamental understanding of cellular response/defense mechanisms and aid diagnosis of aberrations.
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