Doctor of Philosophy (Ph.D.)
Virginia Institute of Marine Science
Michael E. Bender
This research examined the effects of quinoline and 4-azafluorene on respiratory electron transport rate (ET), outer membrane permeability and topology, oxygen consumption, and viable cell density in Escherichia coli cell suspensions. ET was estimated spectrophotometrically using INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-(phenyl)-2H-tetrazolium chloride, which is reduced in vivo to a red colored formazan (INTF). Both test compounds caused anomalous dose-response behavior in INT assays: in a defined window of doses, ET rates near or above the controls were observed. These doses showed altered INT reduction kinetics, decreased cellular oxygen demand, and decreased viable cell densities. Experiments with E. coli spheroplast preparations, gram(+) cells, and deep rough mutants suggested that the toxicants increased outer membrane permeability and inhibited normal respiratory function. Results of cell-free ET assays and transmission electron microscopy further indicated altered outer membrane structure and inhibition of respiratory ET via, (1) secondary topological effects on the periplasm and inner membrane, (2) redox cycling of electrons in the respiratory chain, or (3) both 1 and 2 together. Quantitative studies of INT chemical structure and aqueous electrochemistry at Hg, C, and Pt electrodes were conducted to address analytical shortcomings in the literature. Data include nuclear magnetic resonance spectra, results from normal and differential pulse polarography, cyclic voltammetry, ring disc electrode, and spectroelectrochemical experiments. The route of INT reduction involves a slow one electron reduction to a tetrazolinyl radical followed by a fast one electron reduction and addition of one proton to yield formazan. Results on C and Pt electrodes indicated interfering reactions involving adsorbed hydrogen species and the possibility of underpotential generation of hydrogen gas.
© The Author
Catallo, W. James III, "Effects of selected nitrogen-containing aromatic compounds (NCACs) on physiological properties in Escherichia coli" (1989). Dissertations, Theses, and Masters Projects. William & Mary. Paper 1539616602.