Browsing by Author "Tabor, Jeffrey J"

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    Continuous monitoring of signal transduction in living bacteria
    (2022-04-20) Butcher, Ryan John; Tabor, Jeffrey J
    Bacteria utilize two-component system (TCS) signal transduction pathways to thrive in dynamic and often harsh environments. These pathways detect diverse inputs ranging from metabolites to light and temperature. In turn, they regulate a wide variety of cellular behaviors from differentiation to pathogenicity. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and binds DNA to produce a transcriptional response. There are not yet general methods to assess this post-translational activity in living bacteria. Existing methods for studying TCSs rely on transcriptional reporters which do not capture the finer details of TCS activity, or require that cells be lysed to analyze the components within. I first present a novel method enabling real-time detection of response regulator dimerization in living cells. In particular, we measure homotypic Förster Resonance Energy Transfer between RR-fused fluorescent probes to find that phosphorylation-induced dimerization begins within seconds of stimulus addition and stabilizes within minutes in several model pathways. We then combine experiments with mathematical modeling to investigate the extrinsic parameters that affect TCS signaling dynamics. Finally, we observe fluctuating TCS activity during active metabolism and replenishment of the target ligand. In the second portion of this work, I describe a dramatic ligand-induced clustering behavior we discovered in a particular family of SHKs. Through mutational analysis, we identify the critical portions of the SHK that enable clustering. In addition, we propose that this effect could be used to localize intracellular cargo bound to either the SHK or cognate RR. Together, these methods provide new insights into TCS function and could be applied to a wide range of TCSs across diverse bacteria.