A soluble reductase which is released by Literia monocytogenes will be isolated from spent culture supernatant fluid using ion-exchange resins and/or affinity chromatography gels. The reductant will be characterized by polyacrylamide gel electrophoresis to determine the molecular weight and the number of subunits present, by molecular sieve chromatography to obtain a second estimation of the molecular weight, by UV-VIS spectroscopy to determine the spectral characteristics of the reductant, and by reaction with phenol-sulfuric acid to determine if the reductase is a glycoprotein. The activity of the reductase, as a function of NADA & MADPH, variable temperature, pH, age and ionic strength will also be assessed. The mechanism of the reductive mobilization of iron from both Fe3+-transferrin-carbonate and ferritin will be determined by a combination of the following two methods: 1) analysis of the family of spectra generated during the reductive removal of iron from these iron proteins, and 2) analysis of the kinetics of the reductive removal of iron from Fe3+-transferrin-carbonate and ferritin as a function of the concentration of the reactants. The role of the reductant in supplying iron to Listeria will be assessed by determining if the reductant has chelating properties and, if so, by studying the interaction of the Fe2+-reductant with the microorganism. One hypothesis, that the reductive mobilization of iron is necessary for the growth of Listeria, will be evaluated by isolating and studying temperature senstive mutants defective in reductase production. Associated with this will be kinetic studies to assess the blocking of reductase activity through the use of different potential inhibitors. The health relatedness of this project includes assessing both the potential and the feasibility of controlling the growth of pathogenic microorganisms through blocking the iron acquisition process.
| Cowart, Richard E (2002) Reduction of iron by extracellular iron reductases: implications for microbial iron acquisition. Arch Biochem Biophys 400:273-81 |
| Vartivarian, S E; Cowart, R E (1999) Extracellular iron reductases: identification of a new class of enzymes by siderophore-producing microorganisms. Arch Biochem Biophys 364:75-82 |
| Barchini, E; Cowart, R E (1996) Extracellular iron reductase activity produced by Listeria monocytogenes. Arch Microbiol 166:51-7 |
| Cowart, R E; Singleton, F L; Hind, J S (1993) A comparison of bathophenanthrolinedisulfonic acid and ferrozine as chelators of iron(II) in reduction reactions. Anal Biochem 211:151-5 |
| Adams, T J; Vartivarian, S; Cowart, R E (1990) Iron acquisition systems of Listeria monocytogenes. Infect Immun 58:2715-8 |
| Cowart, R E; Lashmet, J; McIntosh, M E et al. (1990) Adherence of a virulent strain of Listeria monocytogenes to the surface of a hepatocarcinoma cell line via lectin-substrate interaction. Arch Microbiol 153:282-6 |
