Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.Bacterial electron transfer to sparingly soluble electron acceptors is a critical component of a wide variety of environmental and energy-generating processes, including biogeochemical cycling of metals, degradation of natural and contaminant organic matter, weathering of clays and minerals, biomineralization of Fe-bearing minerals, reductive precipitation of toxic metals and radionuclides, and electricity generation in microbial fuel cells (17, 33, 34). Anaerobic and facultatively anaerobic bacteria capable of respiring sparingly soluble (<10⁻²⁵ M at pH 7) Fe(III) oxides are ubiquitous in nature and may be found in marine, freshwater, and terrestrial environments, including metal- and radionuclide-contaminated subsurface aquifers (25, 34). Fe(III)-respiring prokaryotes are also deeply rooted and scattered throughout the domains Bacteria and Archaea (possibly indicating an ancient metabolic process) and include hyperthermophiles, psychrophiles, acidophiles, and extreme barophiles (34). Despite their potential environmental, energy-generating, and evolutionary significance, the molecular details of microbial Fe(III) respiration remain unclear.Fe(III)-respiring, neutrophilic bacteria are presented with a unique physiological challenge: they are required to respire anaerobically on electron acceptors found largely as sparingly soluble Fe(III) oxides presumably unable to contact periplasm- or inner membrane (IM)-localized electron transport systems. To overcome this problem, Fe(III)-respiring bacteria are postulated to employ novel respiratory strategies not found in other bacteria (e.g., aerobes, denitrifiers, sulfate-reducing bacteria, and methanogens) that respire soluble electron acceptors (17, 38). The novel respiratory strategies include (i) a direct-contact pathway in which terminal Fe(III) reductases are secreted to the cell outer membrane (OM), where they contact and deliver electrons directly to external Fe(III) oxides (18, 23, 40, 42, 48, 57, 64, 67), (ii) a two-step electron shuttling pathway in which bacterially reduced endogenous or exogenous electron shuttles deliver electrons to external Fe(III) oxides in a second (abiotic) electron transfer reaction (11, 26, 39, 45), and (iii) a two-step Fe(III) chelation (solubilization) pathway in which Fe(III) oxides are first nonreductively dissolved by endogenously synthesized organic ligands prior to reduction of the resulting soluble organic Fe(III) [Fe(III) bound to an organic molecule] complexes (36, 59).Candidate organic ligands for production of soluble organic Fe(III) during anaerobic Fe(III) oxide respiration include siderophores, the Fe(III)-chelating compounds synthesized and secreted by a wide variety of bacteria and fungi for solubilization and subsequent assimilation of otherwise inaccessible Fe(III) substrates (12, 44, 49, 63). Hydroxamate-type siderophores are produced via N⁶ hydroxylation and N⁶ acylation of l-ornithine and, in some cases, cyclization to macrocyclic ring structures (13). The macrocyclic siderophores bisucaberin and putrebactin, for example, are two structural analogs of the cyclic bis(hydroxamate) siderophore alcaligin, synthesized by Aliivibrio salmonicida and Shewanella putrefaciens strain 200, respectively (27, 32, 65). After transport across the cell envelope via a TonB-dependent pathway, Fe(III) is subsequently released from the Fe(III)-siderophore complex by ligand exchange reactions promoted by siderophore ligand hydrolysis and/or protonation or by Fe(III)-siderophore reduction and release of Fe(II) to acceptor ligands (9, 66).The main objectives of the present study were to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are synthesized by Fe(III)-siderophore biosynthesis systems and (ii) if Fe(III)-siderophore reductases are required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. The experimental strategy for this study included (i) identification of genes encoding the siderophore biosynthesis and Fe(III)-siderophore reductase systems in the S. oneidensis genome, (ii) generation of in-frame deletions in the corresponding siderophore biosynthesis and Fe(III)-siderophore reductase genes, (iii) tests of the resulting siderophore biosynthesis mutants for production of siderophores and soluble organic Fe(III) during aerobic and anaerobic Fe(III) oxide respiration, and (iv) tests of the resulting Fe(III)-siderophore reductase mutants for respiration of soluble organic Fe(III) as an anaerobic electron acceptor.     

Fe(III) Oxide Reduction by Anaerobic Biofilm Formation-DeficientS-Ribosylhomocysteine Lyase (LuxS) Mutant of Shewanella oneidensis

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nanowire-localized c-type cytochromes) and indirect (electron shuttling and Fe(III) solubilization) pathways. In the present study, the influence of anaerobic biofilm formation on Fe(III) oxide reduction by S. oneidensis was determined. The gene encoding the activated methyl cycle (AMC) enzyme S-ribosylhomocysteine lyase (LuxS) was deleted in-frame to generate the corresponding mutant ΔluxS. Conventional biofilm assays and visual inspection via confocal laser scanning microscopy indicated that the wild-type strain formed anaerobic biofilms on Fe(III) oxide-coated silica surfaces, while the ΔluxS mutant was severely impaired in anaerobic biofilm formation on such surfaces. Cell-hematite attachment isotherms demonstrated that the ΔluxS mutant was also severely impaired in attachment to hematite surfaces under anaerobic conditions. The S. oneidensis ΔluxS mutant, however, reduced Fe(III) at wild-type rates during anaerobic incubation with Fe(III) oxide-coated silica surfaces or in batch cultures with Fe(III) oxide or hematite as a terminal electron acceptor. Anaerobic biofilm formation by the ΔluxS mutant was restored to wild-type rates by providing a wild-type copy of luxS in trans or by the addition of AMC or transsulfurylation pathway metabolites involved in organic sulfur metabolism. LuxS is thus required for wild-type anaerobic biofilm formation on Fe(III) oxide surfaces, yet the inability to form wild-type anaerobic biofilms on Fe(III) oxide surfaces does not alter Fe(III) oxide reduction activity.     

Shewanella putrefaciens produces an Fe(III)-solubilizing organic ligand during anaerobic respiration on insoluble Fe(III) oxides

The mechanism of Fe(III) reduction was investigated using voltammetric techniques in anaerobic incubations of Shewanella putrefaciens strain 200 supplemented with Fe(III) citrate or a suite of Fe(III) oxides as terminal electron acceptor. Results indicate that organic complexes of Fe(III) are produced during the reduction of Fe(III) at rates that correlate with the reactivity of the Fe(III) phase and bacterial cell density. Anaerobic Fe(III) solubilization activity is detected with either Fe(III) oxides or Fe(III) citrate, suggesting that the organic ligand produced is strong enough to destabilize Fe(III) from soluble or solid Fe(III) substrates. Results also demonstrate that Fe(III) oxide dissolution is not controlled by the intrinsic chemical reactivity of the Fe(III) oxides. Instead, the chemical reaction between the endogenous organic ligand is only affected by the number of reactive surface sites available to S. putrefaciens. This report describes the first application of voltammetric techniques to demonstrate production of soluble organic-Fe(III) complexes by any Fe(III)-reducing microorganism and is the first report of a Fe(III)-solubilizing ligand generated by a metal-reducing member of the genus Shewanella.     

Characterisation of the dissimilatory reduction of Fe(III)-oxyhydroxide at the microbe-mineral interface: the application of STXM-XMCD

A combination of scanning transmission X‐ray microscopy and X‐ray magnetic circular dichroism was used to spatially resolve the distribution of different carbon and iron species associated with Shewanella oneidensis MR‐1 cells. S. oneidensis MR‐1 couples the reduction of Fe(III)‐oxyhydroxides to the oxidation of organic matter in order to conserve energy for growth. Several potential mechanisms may be used by S. oneidensis MR‐1 to facilitate Fe(III)‐reduction. These include direct contact between the cell and mineral surface, secretion of either exogenous electron shuttles or Fe‐chelating agents and the production of conductive ‘nanowires’. In this study, the protein/lipid signature of the bacterial cells was associated with areas of magnetite (Fe₃O₄), the product of dissimilatory Fe(III) reduction, which was oversaturated with Fe(II) (compared to stoichiometric magnetite). However, areas of the sample rich in polysaccharides, most likely associated with extracellular polymeric matrix and not in direct contact with the cell surface, were undersaturated with Fe(II), forming maghemite‐like (γ‐Fe₂O₃) phases compared to stoichiometric magnetite. The reduced form of magnetite will be much more effective in environmental remediation such as the immobilisation of toxic metals. These findings suggest a dominant role for surface contact‐mediated electron transfer in this study and also the inhomogeneity of magnetite species on the submicron scale present in microbial reactions. This study also illustrates the applicability of this new synchrotron‐based technique for high‐resolution characterisation of the microbe–mineral interface, which is pivotal in controlling the chemistry of the Earth’s critical zone.     

The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate,and fumarate by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1

Background  

Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.     

Identification and analysis of the Shewanella oneidensis major oxygen-independent coproporphyrinogen III oxidase gene

Shewanella oneidenesis MR-1 is a facultative anaerobe that can use a large number of electron acceptors including metal oxides. During anaerobic respiration, S. oneidensis MR-1 synthesizes a large number of c cytochromes that give the organism its characteristic orange color. Using a modified mariner transposon, a number of S. oneidensis mutants deficient in anaerobic respiration were generated. One mutant, BG163, exhibited reduced pigmentation and was deficient in c cytochromes normally synthesized under anaerobic condition. The deficiencies in BG163 were due to insertional inactivation of hemN1, which exhibits a high degree of similarity to genes encoding anaerobic coproporphyrinogen III oxidases that are involved in heme biosynthesis. The ability of BG163 to synthesize c cytochromes under anaerobic conditions, and to grow anaerobically with different electron acceptors was restored by the introduction of hemN1 on a plasmid. Complementation of the mutant was also achieved by the addition of hemin to the growth medium. The genome sequence of S. oneidensis contains three putative anaerobic coproporphyrinogen III oxidase genes. The protein encoded by hemN1 appears to be the major enzyme that is involved in anaerobic heme synthesis of S. oneidensis. The other two putative anaerobic coproporphyrinogen III oxidase genes may play a minor role in this process.     

Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides

Shewanella alga BrY adhesion to hydrous ferric oxide, goethite, and hematite was examined. Adhesion to each oxide followed the Langmuir adsorption model. No correlation between adhesion and Fe(III) oxide surface area or crystallinity was observed. Zeta potential measurements suggested that electrostatic interactions do not influence S. alga BrY adhesion to these minerals. Cell adhesion does not appear to explain the recalcitrance of crystalline Fe(III) oxides to bacterial reduction. Received: 12 May 2000 / Accepted: 19 June 2000     

Current Production and Metal Oxide Reduction by Shewanella oneidensis MR-1 Wild Type and Mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.     

Melanin Production and Use as a Soluble Electron Shuttle for Fe(III) Oxide Reduction and as a Terminal Electron Acceptor by Shewanella algae BrY

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.     

Fe(III) reduction during pyruvate fermentation by Desulfotomaculum reducens strain MI‐1

Desulfotomaculum reducens MI‐1 is a Gram‐positive, sulfate‐reducing bacterium also capable of reducing several metals, among which is Fe(III). Very limited knowledge is available on the potential mechanism(s) of metal reduction among Gram‐positive bacteria, despite their preponderance in the microbial communities that inhabit some inhospitable environments (e.g., thermal or hyperthermal ecosystems, extreme pH or salinity environments, heavy metal or radionuclide contaminated sediments). Here, we show that in the presence of pyruvate, this micro‐organism is capable of reducing both soluble Fe(III)‐citrate and solid‐phase hydrous ferric oxide, although growth is sustained by pyruvate fermentation rather than Fe(III) respiration. Despite the fact that Fe(III) reduction does not support direct energy conservation, D. reducens uses it as a complementary means of discarding excess reducing equivalent after H₂ accumulation in the culture headspace renders proton reduction unfavorable. Thus, Fe(III) reduction permits the oxidation of greater amounts of pyruvate than fermentation alone. Fe(III) reduction by D. reducens is mediated by a soluble electron carrier, most likely riboflavin. Additionally, an intracellular electron storage molecule acts as a capacitor and accumulates electrons during pyruvate oxidation for slow release to Fe(III). The reductase responsible for the transfer of electrons from the capacitor to the soluble carrier has not been identified, but data presented here argue against the involvement of c‐type cytochromes.     

Crp‐dependent cytochrome bd oxidase confers nitrite resistance to Shewanella oneidensis

Shewanella oneidensis is able to respire on a variety of organic and inorganic substrates, including nitrate and nitrite. Conversion of nitrate to nitrite and nitrite to ammonium is catalysed by periplasmic nitrate and nitrite reductases (NAP and NRF) respectively. Global regulator Crp (c yclic AMP r eceptor p rotein) is essential for growth of S. oneidensis on both nitrate and nitrite. In this study, we discovered that crp mutants are not only severely deficient in nitrate or nitrite respiration, but are also hypersensitive to nitrite. This hypersusceptibility phenotype is independent of nitrite respiration. Using random transposon mutagenesis, we obtained 73 Δcrp suppressor strains resistant to nitrite. Transposon insertion sites in 24 suppressor strains were exclusively mapped in the region upstream of the cyd operon encoding a cytochrome bd oxidase, resulting in expression of the operon now driven by a Crp‐independent promoter. Further investigation indicated that the promoter in suppressor strains comes from the transposon. Mutational analysis of the cydB gene (encoding the essential subunit II of the bd oxidase) confirmed that the cytochrome bd oxidase confers nitrite resistance to S. oneidensis.     

Comparative analysis of membranous proteomics of Shewanella decolorationis S12 grown with azo compound or Fe (III) citrate as sole terminal electron acceptor

Shewanella decolorationis S12 is capable of carrying out anaerobic respiration using azo dyes and Fe (III) citrate as electron acceptors. In the present study, proteomic techniques including two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry were used to analyze the similarity and the dissimilarity of the membrane proteins isolated from strain S12 cells grown in amaranth or Fe (III) citrate with defined inorganic salt medium. The cells of strain S12 grown under a saturated dissolved oxygen condition served as controls. This is the first work that made the comparative analysis of cell membranous proteomics of strain S12 grown with azo compound or Fe (III) citrate as a sole terminal electron acceptor. The results showed that most of the membrane proteins of strain S12 under azo respiration are similar to those under Fe (III) respiration, but dissimilar from those of oxygen-grown cells. FdnH and FrdB were expressed specifically in azo respiration. NqrA-2, DctP, and hypothetical protein SO_4719 showed relative overexpression in azo respiration compared with Fe (III) respiration. OmpA family protein SO_3545 was detected to be specific to Fe (III) respiration. Furthermore, ArgF, SdhA, and HoxK were expressed markedly in both amaranth- and Fe (III) citrate-grown cultures compared with oxygen-grown cultures.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis revealed by Tn‐seq

The availability of increasingly inexpensive sequencing combined with an ever‐expanding molecular biology toolbox has transported classical bacterial genetics into the 21st century. Whole genome genetic fitness analysis using transposon mutagenesis combined with next‐generation high‐throughput sequencing (Tn‐seq) promises to revolutionize systems level analysis of microbial metabolism. Tn‐seq measures the frequency of actual members of a heterogeneous mutant pool undergoing purifying selection to determine the contribution of every non‐essential gene in the genome to the fitness of an organism under a given condition. Here we use Tn‐seq to assess gene function in the Gram negative γ‐proteobacterium Shewanella oneidensis strain MR‐1. In addition to being a model environmental organism, there is considerable interest in using S. oneidensis as a platform organism for bioremediation and biotechnology, necessitating a complete understanding of the metabolic pathways that may be utilized. Our analysis reveals unique aspects of S. oneidensis metabolism overlooked by over 30 years of classical genetic and systems level analysis. We report the utilization of an alternative citrate synthase and describe a dynamic branching of the S. oneidensis anaerobic tricarboxylic acid cycle, unreported in any other organism, which may be a widespread strategy for microbes adept at dissipating reducing equivalents via anaerobic respiration.     

Shewanella oneidensis MR-1-Induced Fe(III) Reduction Facilitates Roxarsone Transformation

Although microbial activity and associated iron (oxy)hydroxides are known in general to affect the environmental dynamics of 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), the mechanistic understanding of the underlying biophysico-chemical processes remains unclear due to limited experimental information. We studied how Shewanella oneidensis MR-1 –a widely distributed metal-reducing bacterium, in the presence of dissolved Fe(III), affects roxarsone transformations and biogeochemical cycling in a model aqueous system. The results showed that the MR-1 strain was able to anaerobically use roxarsone as a terminal electron acceptor and to convert it to a single product, 3-amino-4-hydroxybenzene arsonic acid (AHBAA). The presence of Fe(III) stimulated roxarsone transformation via MR-1-induced Fe(III) reduction, whereby the resulting Fe(II) acted as an efficient reductant for roxarsone transformation. In addition, the subsequent secondary Fe(III)/Fe(II) mineralization created conditions for adsorption of organoarsenic compounds to the yielded precipitates and thereby led to arsenic immobilization. The study provided direct evidence of Shewanella oneidensis MR-1-induced direct and Fe(II)-associated roxarsone transformation. Quantitative estimations revealed a candidate mechanism for the early-stage environmental dynamics of roxarsone in nature, which is essential for understanding the environmental dynamics of roxarsone and successful risk assessment.     

Fe(III)-enhanced Azo Reduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12 is capable of high rates of azo dye decolorization and dissimilatory Fe(III) reduction. Under anaerobic conditions, when Fe(III) and azo dye were copresent in S12 cultures, dissimilatory Fe(III) reduction and azo dye biodecolorization occurred simultaneously. Furthermore, the dye decolorization was enhanced by the presence of Fe(III). When 1 mM Fe(III) was added, the methyl red decolorizing efficiency was 72.1% after cultivation for 3 h, whereas the decolorizing efficiency was only 60.5% in Fe(III)-free medium. The decolorizing efficiencies increased as the concentration of Fe(III) was increased from 0 to 6 mM. Enzyme activities, which mediate the dye decolorization and Fe(III) reduction, were not affected by preadaption of cells to Fe(III) and azo dye nor by the addition of chloramphenicol. Both the Fe(III) reductase and the azo reductase were membrane associated. The respiratory electron transport chain inhibitors metyrapone, dicumarol, and stigmatellin showed significantly different effects on Fe(III) reduction than on azo dye decolorization.     

Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.     

Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.