Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V V ). The bacterium reduces V V (vanadate ion) to V IV (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V IV -containing solid.Vanadium is a transition metal which, at neutral pH, can exist in two oxidation states, V IV (vanadyl ion, cationic species VO 2ϩ ), and V V (vanadate ion, anionic species, H 2 VO 4 Ϫ ) (10, 11).The environmental chemistry of vanadium is complex. Vanadium is an abundant element that has proven to be a valuable resource for different industrial applications such as vanadium alloys, oxidation catalysis in sulfuric acid manufacturing and automobile catalytic converters, photographic development, textile dyeing, and ceramic coloring. A number of bacteria are able to reduce metal compounds, most commonly iron and manganese, through anaerobic reduction. Some organisms are known to be able to reduce other metals such as arsenic, mercury, selenium, uranium, technetium, chromium, molybdenum, gold, silver, and copper (4,8,13). The microbial reduction of vanadate has also been reported (1,14). To date, two Pseudomonas strains have been described to be capable of reducing vanadium (5,15). In this study we demonstrate that the gram-negative facultative anaerobic nonfermenting bacterium Shewanella oneidensis (6) can also reduce vanadium V V . Vanadium uptake on solid media. Colonies of S. oneidensis were grown on Luria-Bertani 1% agar plates containing 5 mM vanadate at 28°C for 48 h. Plates were then exposed to hydrogen sulfide in a sealed container. Upon formation of metal sulfide, plates were examined for changes in the regions surrounding or inside bacterial colonies. A halo could be seen indicating possible sequestration of the metal. Darkening of the cells indicated accumulation or possible reduction of the metal (data not shown).Vanadate detection. To facilitate the detection and quantification of the reduction of vanadate to V IV , a vanadate detection assay was designed, based on a detection assay for chromium described by Sandel (12). Vanadate was thereby detected on the basis of its reaction with diphenylcarbazide (DPC) in acid. The assay solution was made by addition of 1% (wt/vol) DPC in acetone to an equal volume of 2 M H 2 SO 4 . A volume of 500 l of diluted sample was added to the same volume of assay solution. Absorbance was measured at 320 nm after 15 min. A standard curve was made with vanadium pentoxide dilutions. Vanadyl ions do not react in this assay.Anaerobic vanadium reduction. S. oneidensis was grown aerobically on a rotary shaker (150 rpm) at 28°C in LB medium containing 2 or 10 mM V 2 O 5 and anaerobically with a Coy anaerobic chamber (Coy Laboratories, Grass Lake, Mich.) in phosphate-buffered defined medium (7) containing 10 mM lactate as the electron donor and 2 or 10 mM V 2 O 5 as the electron acceptor. An increase in lag phase could be detected in cultures grown with 10 mM but not with 2 mM V 2 O 5 compared to c...

Shewanella oneidensis MR-1 is a free-living gram-negative ␥-proteobacterium that is able to use a large number of oxidizing molecules, including fumarate, nitrate, dimethyl sulfoxide, trimethylamine N-oxide, nitrite, and insoluble iron and manganese oxides, to drive anaerobic respiration. Here we show that S. oneidensis MR-1 is able to grow on vanadate as the sole electron acceptor. Oxidant pulse experiments demonstrated that proton translocation across the cytoplasmic membrane occurs during vanadate reduction. Proton translocation is abolished in the presence of protonophores and the inhibitors 2-heptyl-4-hydroxyquinoline N-oxide and antimycin A. Redox difference spectra indicated the involvement of membrane-bound menaquinone and cytochromes c, which was confirmed by transposon mutagenesis and screening for a vanadate reduction-deficient phenotype. Two mutants which are deficient in menaquinone synthesis were isolated. Another mutant with disruption in the cytochrome c maturation gene ccmA was unable to produce any cytochrome c and to grow on vanadate. This phenotype could be restored by complementation with the pEC86 plasmid expressing ccm genes from Escherichia coli. To our knowledge, this is the first report of E. coli ccm genes being functional in another organism. Analysis of an mtrB-deficient mutant confirmed the results of a previous paper indicating that OmcB may function as a vanadate reductase or may be part of a vanadate reductase complex.One of the primary tasks of a microorganism is to catalyze chemical reactions in order to obtain energy for metabolic growth from its environment. The most well-known electron acceptor is O 2 . However, in the absence of oxygen, some microorganisms can grow by coupling the oxidation of simple organic acids, alcohols, H 2 , or aromatic compounds to the reduction of Fe(III) or Mn(IV) (21). Fe(III) and Mn(IV) reduction has an important impact on the organic and inorganic geochemistry of anaerobic aquatic sediments and groundwater (20). Many other metals, including Fe(III), Mn(IV), Mn(III), Cr(VI), Hg(II), Au(III), Ag(I), Mo(VI), Co(III), Pd(II), As(V), Se(VI), Se(IV), U(VI), Tc(VII), Te(IV), V(V), can be enzymatically reduced, but microorganisms are not necessarily able to conserve energy from the reduction process (20,43). A distinction is made between respiratory metal reduction, in which the electron flow is coupled to a proton-translocating complex to allow ATP generation, and dissimilatory reduction, in which electrons are transferred without the generation of a proton motive force (24). Vanadate is a known electron acceptor for anaerobic respiration (9, 23, 35, 52), but only limited data are available on the biological vanadium reduction process, concomitant precipitation effects, and the geochemical implications. Although vanadium is not abundant, it is omnipresent in nature (51). Of importance is the comparatively large amount of vanadium in seawater, which contains vanadate at an average concentration of 30 nM (7). This makes vanadium the second most common tr...

Shewanella oneidensis contains four genes that encode proteins that have high sequence identity with yeast OYE (Old Yellow Enzyme, an NADPH oxidoreductase), the well-studied archetype of the OYE protein family. The present paper describes the first comparative study of OYEs that are present in a single bacterial species, performed to gain insight into their biochemical properties and physiological importance. The four proteins [named SYE1-SYE4 (Shewanella Yellow Enzyme 1-4)] were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The yield of SYE2, however, was too low for further characterization, even after expression attempts in S. oneidensis. The SYE1, SYE3 and SYE4 proteins were found to have characteristics similar to those of other OYE family members. They were identified as flavoproteins that catalyse the reduction of different alpha,beta-unsaturated carbonyl compounds and form charge transfer complexes with a range of phenolic compounds. Whereas the properties of SYE1 and SYE3 were very similar, those of SYE4 were clearly different in terms of ligand binding, catalytic efficiency and substrate specificity. Also, the activity of SYE4 was found to be NADPH-dependent, whereas SYE1 and SYE3 had a preference for NADH. It has been suggested that yeast OYE protects the actin cytoskeleton from oxidative stress. There are indications that bacterial OYEs are also involved in the oxidative stress response, but their exact role is unclear. Induction studies in S. oneidensis revealed that yeast and bacterial OYEs may share a common physiological role, i.e. the protection of cellular components against oxidative damage. As only SYE4 was induced under oxidative stress conditions, however, a functional divergence between bacterial OYEs is likely to exist.

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.

SummaryMany studies have reported microorganisms as efficient biocatalysts for colour removal of dye‐containing industrial wastewaters. We present the first comprehensive study to identify all molecular components involved in decolorization by bacterial cells. Mutants from the model organism Shewanella oneidensis MR‐1, generated by random transposon and targeted insertional mutagenesis, were screened for defects in decolorization of an oxazine and diazo dye. We demonstrate that decolorization is an extracellular reduction process requiring a multicomponent electron transfer pathway that consists of cytoplasmic membrane, periplasmic and outer membrane components. The presence of melanin, a redox‐active molecule excreted by S. oneidensis, was shown to enhance the dye reduction rates. Menaquinones and the cytochrome CymA are the crucial cytoplasmic membrane components of the pathway, which then branches off via a network of periplasmic cytochromes to three outer membrane cytochromes. The key proteins of this network are MtrA and OmcB in the periplasm and outer membrane respectively. A model of the complete dye reduction pathway is proposed in which the dye molecules are reduced by the outer membrane cytochromes either directly or indirectly via melanin.