Metabolomic analyses show that electron donor and acceptor ratios control anaerobic electron transfer pathways in Shewanella oneidensis

This study investigated the physiological impact of changing electron donor–acceptor ratios on electron transfer pathways in the metabolically flexible subsurface bacterium Shewanella oneidensis, using batch and chemostat cultures, with an azo dye (ramazol black B) as the model electron acceptor. Altering the growth rate did result in changes in biomass yield, but not in other key physiological parameters including the total cytochrome content of the cells, the production of extracellular flavin redox shuttles or the potential of the organism to reduce the azo dye. Dramatic increases in the ability to reduce the dye were noted when cells were grown under conditions of electron acceptor (fumarate) limitation, although the yields of extracellular redox mediators (flavins) were similar under conditions of electron donor (lactate) or acceptor limitation. FT-IR spectroscopy confirmed shifts in the metabolic fingerprints of cells grown under these contrasting conditions, while spectrophotometric analyses supported a critical role for c-type cytochromes, expressed at maximal concentrations under conditions of electron acceptor limitation. Finally, key intracellular metabolites were quantified in batch experiments at various electron donor and acceptor ratios and analysed using discriminant analysis and a Bayesian network to construct a central metabolic pathway model for cells grown under conditions of electron donor or acceptor limitation. These results have identified key mechanisms involved in controlling electron transfer in Shewanella species, and have highlighted strategies to maximise reductive activity for a range of bioprocesses.     

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.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

Impacts of Nitrate and Nitrite on Physiology of Shewanella oneidensis

Shewanella oneidensis exhibits a remarkable versatility in anaerobic respiration, which largely relies on its diverse respiratory pathways. Some of these are expressed in response to the existence of their corresponding electron acceptors (EAs) under aerobic conditions. However, little is known about respiration and the impact of non-oxygen EAs on the physiology of the microorganism when oxygen is present. Here we undertook a study to elucidate the basis for nitrate and nitrite inhibition of growth under aerobic conditions. We discovered that nitrate in the form of NaNO₃ exerts its inhibitory effects as a precursor to nitrite at low concentrations and as an osmotic-stress provider (Na⁺) at high concentrations. In contrast, nitrite is extremely toxic, with 25 mM abolishing growth completely. We subsequently found that oxygen represses utilization of all EAs but nitrate. To order to utilize EAs with less positive redox potential, such as nitrite and fumarate, S. oneidensis must enter the stationary phase, when oxygen respiration becomes unfavorable. In addition, we demonstrated that during aerobic respiration the cytochrome bd oxidase confers S. oneidensis resistance to nitrite, which likely functions via nitric oxide (NO).     

Reduction of Soluble and Insoluble Iron Forms by Membrane Fractions of Shewanella oneidensis Grown under Aerobic and Anaerobic Conditions

The effect of iron substrates and growth conditions on in vitro dissimilatory iron reduction by membrane fractions of Shewanella oneidensis MR-1 was characterized. Membrane fractions were separated by sucrose density gradients from cultures grown with O₂, fumarate, and aqueous ferric citrate as the terminal electron acceptor. Marker enzyme assays and two-dimensional gel electrophoresis demonstrated the high degree of separation between the outer and cytosolic membrane. Protein expression pattern was similar between chelated iron- and fumarate-grown cultures, but dissimilar for oxygen-grown cultures. Formate-dependent ferric reductase activity was assayed with citrate-Fe³⁺, ferrozine-Fe³⁺, and insoluble goethite as electron acceptors. No activity was detected in aerobic cultures. For fumarate and chelated iron-grown cells, the specific activity for the reduction of soluble iron was highest in the cytosolic membrane. The reduction of ferrozine-Fe³⁺ was greater than the reduction of citrate-Fe³⁺. With goethite, the specific activity was highest in the total membrane fraction (containing both cytosolic and outer membrane), indicating participation of the outer membrane components in electron flow. Heme protein content and specific activity for iron reduction was highest with chelated iron-grown cultures with no heme proteins in aerobically grown membrane fractions. Western blots showed that CymA, a heme protein involved in iron reduction, expression was also higher in iron-grown cultures compared to fumarate- or aerobic-grown cultures. To study these processes, it is important to use cultures grown with chelated Fe³⁺ as the electron acceptor and to assay ferric reductase activity using goethite as the substrate.     

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.     

A Periplasmic and Extracellular c-Type Cytochrome of Geobacter sulfurreducens Acts as a Ferric Iron Reductase and as an Electron Carrier to Other Acceptors or to Partner Bacteria

An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans. The G. sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)₃), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly. G. sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate. Wolinella succinogenes was able to reduce externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W. succinogenes in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.     

Constraint-Based Modeling of Carbon Fixation and the Energetics of Electron Transfer in Geobacter metallireducens

Geobacter species are of great interest for environmental and biotechnology applications as they can carry out direct electron transfer to insoluble metals or other microorganisms and have the ability to assimilate inorganic carbon. Here, we report on the capability and key enabling metabolic machinery of Geobacter metallireducens GS-15 to carry out CO₂ fixation and direct electron transfer to iron. An updated metabolic reconstruction was generated, growth screens on targeted conditions of interest were performed, and constraint-based analysis was utilized to characterize and evaluate critical pathways and reactions in G. metallireducens. The novel capability of G. metallireducens to grow autotrophically with formate and Fe(III) was predicted and subsequently validated in vivo. Additionally, the energetic cost of transferring electrons to an external electron acceptor was determined through analysis of growth experiments carried out using three different electron acceptors (Fe(III), nitrate, and fumarate) by systematically isolating and examining different parts of the electron transport chain. The updated reconstruction will serve as a knowledgebase for understanding and engineering Geobacter and similar species.     

Mechanism and Consequences of Anaerobic Respiration of Cobalt by Shewanella oneidensis Strain MR-1

Bacteria from the genus Shewanella are the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability of Shewanella oneidensis to catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA]⁻ reduction by S. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA]²⁻, the product of [Co(III)-EDTA]⁻ respiration, inhibited the growth of S. oneidensis strain MR-1 and that this toxicity was partially abolished by the addition of MgSO₄. We demonstrate that [Co(III)-EDTA]⁻ reduction by S. oneidensis requires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (the c-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developing S. oneidensis strains as a tool for bioremediation.     

Shewanella oneidensis MR‐1 mutants selected for their inability to produce soluble organic‐Fe(III) complexes are unable to respire Fe(III) as anaerobic electron acceptor

Summary Recent voltammetric analyses indicate that Shewanella putrefaciens strain 200 produces soluble organic‐Fe(III) complexes during anaerobic respiration of sparingly soluble Fe(III) oxides. Results of the present study expand the range of Shewanella species capable of producing soluble organic‐Fe(III) complexes to include Shewanella oneidensis MR‐1. Soluble organic‐Fe(III) was produced by S. oneidensis cultures incubated anaerobically with Fe(III) oxides, or with Fe(III) oxides and the alternate electron acceptor fumarate, but not in the presence of O₂, nitrate or trimethylamine‐N‐oxide. Chemical mutagenesis procedures were combined with a novel MicroElectrode Screening Array (MESA) to identify four (designated Sol) mutants with impaired ability to produce soluble organic‐Fe(III) during anaerobic respiration of Fe(III) oxides. Two of the Sol mutants were deficient in anaerobic growth on both soluble Fe(III)‐citrate and Fe(III) oxide, yet retained the ability to grow on a suite of seven alternate electron acceptors. The rates of soluble organic‐Fe(III) production were proportional to the rates of iron reduction by the S. oneidensis wild‐type and Sol mutant strains, and all four Sol mutants retained wild‐type siderophore production capability. Results of this study indicate that the production of soluble organic‐Fe(III) may be an important intermediate step in the anaerobic respiration of both soluble and sparingly soluble forms of Fe(III) by S. oneidensis.     

Effect of NAD+-dependent formate dehydrogenase on anaerobic respiration of Shewanella oneidensis MR-1

An expression plasmid was constructed in order to carry out heterologous expression of the gene of the NAD⁺-dependent formate dehydrogenase (FDH) from methylotrophic bacterium Moraxella sp. in the cells of Shewanella oneidensis MR-1 under aerobic and anaerobic conditions. In both modes of cell cultivation, recombinant FDH activity was revealed in the cell lysate of the transformants. In the medium with la? tate as a carbon source, the rate of anaerobic respiration determined as the rate of conversion of fumarate (the electron acceptor) to succinate was higher in the transformant with recombinant FDH. Anaerobic cultivation of the FDH-containing transformant of S. oneidensis MR-1 in a microbial fuel cell (MFC) revealed increased current density.     

Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella

Many enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c₃ of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron–two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella.     

奥奈达希瓦氏菌CctA介导周质甲基橙还原的电子传递机理

电活性微生物奥奈达希瓦氏菌的胞外电子传递（extracellular electron transfer,EET）在污染物降解、环境修复、生物电化学传感、能源利用等方面具有广泛的应用潜力;四血红素细胞色素CctA （small tetraheme cytochrome）是希瓦氏菌周质空间中最丰富的蛋白质之一,能够参与多种氧化还原过程,但目前对CctA在EET中的行为和机理认识仍然有限。【目的】研究阐明CctA蛋白在希瓦氏菌模式菌株MR-1周质空间以偶氮染料作为电子受体的EET中的作用,补充和拓展希瓦氏菌的厌氧呼吸产能机制。【方法】以周质还原型偶氮染料甲基橙（methyl orange,MO）作为电子受体,在mteal reduction （Mtr）蛋白缺失菌株Δmtr中研究MO的周质还原特点,并通过基因敲除和回补表达研究CctA蛋白在周质电子传递中的作用。【结果】在缺失Mtr通道的情况下,细胞色素CctA可以介导周质空间的电子传递而还原MO。重组表达CctA在低水平时,MO在周质空间中的还原速率与其表达水平呈正相关,更高水平的CctA表达无助于进一步提高MO的还原速率。蛋白膜伏安结果展示了CctA与周质空间内其他高电位氧化还原蛋白的显著区别,可能参与构成一条低电位的MO还原通道。【结论】从分子动力学层面揭示了CctA在周质MO还原中的独特电子传递行为,为进一步推进对细菌周质电子传递机制的理解,以及通过合成生物学设计或改造胞外氧化还原系统、强化生物电化学在污染物降解中的应用提供了重要信息。     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: III. Effects of Orthophenanthroline and Other Chemicals

Reaction centers from Rhodopseudomonas spheroides mediate the photochemical oxidation of cytochrome c (cyt c), and show a time-varying fluorescence of P870. Analyses of these effects indicate that the reaction centers contain a primary photochemical electron acceptor capable of holding one electron. Native or added ubiquinone (UQ) can act as a secondary electron acceptor. Orthophenanthroline (o-phen) blocks electron transfer from primary to secondary acceptors, and allows the primary acceptor to be exhibited in the foregoing experiments. Other chelators (with the possible exception of 8-hydroxyquinoline) and dichlorophenyldimethylurea (DCMU) are without apparent effect on reaction centers. o-Phen also inhibits the primary photochemical act in reaction centers; this effect is prevented by the presence of UQ. 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) inhibits the primary photochemistry in reaction centers but does not affect secondary electron transfer.     

Growth of Strain SES-3 with Arsenate and Other Diverse Electron Acceptors

The selenate-respiring bacterial strain SES-3 was able to use a variety of inorganic electron acceptors to sustain growth. SES-3 grew with the reduction of arsenate to arsenite, Fe(III) to Fe(II), or thiosulfate to sulfide. It also grew in medium in which elemental sulfur, Mn(IV), nitrite, trimethylamine N-oxide, or fumarate was provided as an electron acceptor. Growth on oxygen was microaerophilic. There was no growth with arsenite or chromate. Washed suspensions of cells grown on selenate or nitrate had a constitutive ability to reduce arsenate but were unable to reduce arsenite. These results suggest that strain SES-3 may occupy a niche as an environmental opportunist by being able to take advantage of a diversity of electron acceptors.     

Secretion of Flavins by Shewanella Species and Their Role in Extracellular Electron Transfer

Fe(III)-respiring bacteria such as Shewanella species play an important role in the global cycle of iron, manganese, and trace metals and are useful for many biotechnological applications, including microbial fuel cells and the bioremediation of waters and sediments contaminated with organics, metals, and radionuclides. Several alternative electron transfer pathways have been postulated for the reduction of insoluble extracellular subsurface minerals, such as Fe(III) oxides, by Shewanella species. One such potential mechanism involves the secretion of an electron shuttle. Here we identify for the first time flavin mononucleotide (FMN) and riboflavin as the extracellular electron shuttles produced by a range of Shewanella species. FMN secretion was strongly correlated with growth and exceeded riboflavin secretion, which was not exclusively growth associated but was maximal in the stationary phase of batch cultures. Flavin adenine dinucleotide was the predominant intracellular flavin but was not released by live cells. The flavin yields were similar under both aerobic and anaerobic conditions, with total flavin concentrations of 2.9 and 2.1 μmol per gram of cellular protein, respectively, after 24 h and were similar under dissimilatory Fe(III)-reducing conditions and when fumarate was supplied as the sole electron acceptor. The flavins were shown to act as electron shuttles and to promote anoxic growth coupled to the accelerated reduction of poorly crystalline Fe(III) oxides. The implications of flavin secretion by Shewanella cells living at redox boundaries, where these mineral phases can be significant electron acceptors for growth, are discussed.     

Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.     

Promotion of Iron Oxide Reduction and Extracellular Electron Transfer in Shewanella oneidensis by DMSO

The dissimilatory metal reducing bacterium Shewanella oneidensis MR-1, known for its capacity of reducing iron and manganese oxides, has great environmental impacts. The iron oxides reducing process is affected by the coexistence of alternative electron acceptors in the environment, while investigation into it is limited so far. In this work, the impact of dimethyl sulphoxide (DMSO), a ubiquitous chemical in marine environment, on the reduction of hydrous ferric oxide (HFO) by S. oneidensis MR-1 was investigated. Results show that DMSO promoted HFO reduction by both wild type and ΔdmsE, but had no effect on the HFO reduction by ΔdmsB, indicating that such a promotion was dependent on the DMSO respiration. With the DMSO dosing, the levels of extracellular flavins and omcA expression were significantly increased in WT and further increased in ΔdmsE. Bioelectrochemical analysis show that DMSO also promoted the extracellular electron transfer of WT and ΔdmsE. These results demonstrate that DMSO could stimulate the HFO reduction through metabolic and genetic regulation in S. oneidensis MR-1, rather than compete for electrons with HFO. This may provide a potential respiratory pathway to enhance the microbial electron flows for environmental and engineering applications.