title: A kinetic approach to the dependence of dissimilatory metal reduction by Shewanella oneidensis MR-1 on the outer membrane cytochromes c OmcA and OmcB.
abstract: The Gram-negative bacterium Shewanella oneidensis MR-1 shows a remarkably versatile anaerobic respiratory metabolism. One of its hallmarks is its ability to grow and survive through the reduction of metallic compounds. Among other proteins, outer membrane decaheme cytochromes c OmcA and OmcB have been identified as key players in metal reduction. In fact, both of these cytochromes have been proposed to be terminal Fe(III) and Mn(IV) reductases, although their role in the reduction of other metals is less well understood. To obtain more insight into this, we constructed and analyzed omcA, omcB and omcA/omcB insertion mutants of S. oneidensis MR-1. Anaerobic growth on Fe(III), V(V), Se(VI) and U(VI) revealed a requirement for both OmcA and OmcB in Fe(III) reduction, a redundant function in V(V) reduction, and no apparent involvement in Se(VI) and U(VI) reduction. Growth of the omcB(-) mutant on Fe(III) was more affected than growth of the omcA(-) mutant, suggesting OmcB to be the principal Fe(III) reductase. This result was corroborated through the examination of whole cell kinetics of OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction, showing that OmcB is approximately 11.5 and approximately 6.3 times faster than OmcA at saturating and low nonsaturating concentrations of Fe(III)-nitrilotriacetic acid, respectively, whereas the omcA(-) omcB(-) double mutant was devoid of Fe(III)-nitrilotriacetic acid reduction activity. These experiments reveal, for the first time, that OmcA and OmcB are the sole terminal Fe(III) reductases present in S. oneidensis MR-1. Kinetic inhibition experiments further revealed vanadate (V(2)O(5)) to be a competitive and mixed-type inhibitor of OmcA and OmcB, respectively, showing similar affinities relative to Fe(III)-nitrilotriacetic acid. Neither sodium selenate nor uranyl acetate were found to inhibit OmcA- and OmcB-dependent Fe(III)-nitrilotriacetic acid reduction. Taken together with our growth experiments, this suggests that proteins other than OmcA and OmcB play key roles in anaerobic Se(VI) and U(VI) respiration.
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Jimmy
authors.initials: J
authors.lastname: Borloo
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Bjorn
authors.initials: B
authors.lastname: Vergauwen
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Lina
authors.initials: L
authors.lastname: De Smet
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Brigé
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Motte
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Bart
authors.initials: B
authors.lastname: Devreese
authors.affiliation: Laboratory for Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
authors.forename: Jozef
authors.initials: J
authors.lastname: Van Beeumen
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D045304:Cytochromes c / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction / Q000187:drug effects; D012680:Sensitivity and Specificity; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008670:Metals; D006418:Heme; D045304:Cytochromes c