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

Shewanella oneidensis MR-1 is a Gram-negative c-proteobacterium with an extremely versatile anaerobic respiratory metabolism. Under anaerobic conditions, this organism reduces a variety of organic and inorganic substrates, including fumarate, nitrate, trimethylamine N-oxide, dimethylsulfoxide, sulfite and thiosulfate, as well as various polyvalent metal ions and radionuclides, including iron(III), manganese(IV), chromium(VI), vanadium(V), selenium(VI), uranium(VI), and tellurium(VI) [1][2][3][4][5][6][7]. Bacterial dissimilatory metal 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 OmcBdependent Fe(III)-nitrilotriacetic acid reduction, showing that OmcB is $ 11.5 and $ 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.Abbreviation FR, fumarate reductase.

The Mtr Respiratory Pathway Is Essential for Reducing Flavins and Electrodes in Shewanella oneidensis

“…4A) and manganese oxide (36) but not for the reduction of soluble substrates like flavins (Fig. 3), cobalt(III)-EDTA (21), and ferric citrate (8,36). Another observation made in this study is that strains lacking MtrC attach to electrodes but produce only 10% of the wild-type current when expressed per unit of attached protein (Fig.…”

“…MtrC mutants are able to reduce soluble substrates such as ferric citrate and cobalt(III)-EDTA much more efficiently than mutants lacking MtrA, MtrB, or both OmcA and MtrC (5,6,8,21). If the same trend follows for electrode reduction, cells lacking MtrC may be able to respire enough to begin the colonization of the electrode, whereas ⌬mtrA, ⌬mtrB, and ⌬omcA ⌬mtrC cannot.…”

“…Thus, if any of the components are missing, the entire complex could be inactivated or mislocalized. Overall, this model could explain the observation that while mutant strains lacking OmcA are deficient only in solid substrate reduction, MtrC mutants are defective in the reduction of both soluble and insoluble substrates (8,9,35,36).…”

“…Our data suggest that MtrC is responsible for most of the electron transfer to carbon electrodes, while OmcA is involved in attachment and has a lesser role in electron transfer. These observations could have broader implications regarding the role of OmcA in the reduction of soluble and insoluble substrates (8,9,36).…”

“…These electrons then are transferred to a periplasmic c-type cytochrome, MtrA, and eventually to outer membrane (OM)-anchored c-type cytochromes MtrC and OmcA, which interact with an integral OM scaffolding protein, MtrB (32,33,43). These OM cytochromes then can reduce various substrates, including iron oxides and electrodes (8,9,12,36,47). Since the Mtr system is required by MR-1 to reduce many different substrates (18), it also could be capable of reducing extracellular flavins.…”