BME PhD Dissertation Defense - Arion Stettner

  • Starts: 1:00 pm on Friday, September 1, 2017
Title: "Imaging of a Metabolic Flux in Single E. coli Cells" Committee: Daniel Segre, Biology/Bioinformatics (Advisor) John Ngo, BME (Chair) Mary Dunlop, BME Mo Khalil, BME Karen Allen, Chemistry Abstract: For over a hundred years, enzymes, or the proteins that catalyze metabolic reactions, have been characterized in vitro, even though the aqueous solution of a test tube little resembles the crowded intracellular milieu. Since few metabolites show unique fluorescent signatures, metabolism is all but invisible, greatly complicating efforts to describe fluxes in vivo. Here we introduce a new technique called IFI (Intracellular Flux Imaging) for visualizing the flux through a reaction inside single E. coli cells, using a substrate that undergoes an enzyme-catalyzed loss of fluorescence. IFI would not only further our quantitative understanding of metabolism, but enable us to promptly detect enzymes that confer clinically meaningful states, such as antibiotic resistance. We present a particular instance of IFI that couples nfsA, the major nitroreductase of E. coli responsible for its antibiotic sensitivity to nitrofurantoin, to 2-NBDG, a glucose derivative that loses fluorescence upon being reduced by nfsA with NADPH. We correlate the flux through the reaction with the concentration of a fluorescently tagged nfsA and quantify the “flux noise” across a population of E. coli cells. Given that nfsA abolishes 2-NBDG fluorescence by the same molecular mechanism that it activates nitrofurantoin, IFI could serve as a means to rapidly infer the antibiotic resistance of single pathogenic E. coli cells from clinical samples.
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