In vivo measurement by FRET of pathway activity in bacterial chemotaxis.

The two-component pathway in Escherichia coli chemotaxis has become a paradigm for bacterial signal processing. Genetics and biochemistry of the pathway as well as physiological responses have been studied in detail. Despite its relative simplicity, the chemotaxis pathway is renowned for its ability to amplify and integrate weak signals and for its robustness against various kinds of perturbations. All this information inspired multiple attempts at mathematical analysis and computer modeling, but a quantitative understanding of the pathway was hampered by our inability to follow the signal processing in vivo. To address this problem, we developed assays based on fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) that enabled us to monitor activity-dependent protein interactions in real time directly in living cells. Here, we describe quantitative applications of these assays in cell populations and on a single-cell level to study the interaction of the phosphorylated response regulator CheY with its phosphatase CheZ. Since this interaction defines the rate of CheY dephosphorylation, which at steady state equals the rate of CheY phosphorylation, it can be used to characterize intracellular kinase activity and thus to analyze properties of the chemotaxis signaling network.

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