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

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