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Genomic plasticity enables a secondary electron transport pathway in Shewanella oneidensis.

ABSTRACT: Microbial dissimilatory iron reduction is an important biogeochemical process. It is physiologically challenging because iron occurs in soils and sediments in the form of insoluble minerals such as hematite or ferrihydrite. Shewanella oneidensis MR-1 evolved an extended respiratory chain to the cell surface to reduce iron minerals. Interestingly, the organism evolved a similar strategy for reduction of dimethyl sulfoxide (DMSO), which is reduced at the cell surface as well. It has already been established that electron transfer through the outer membrane is accomplished via a complex in which ?-barrel proteins enable interprotein electron transfer between periplasmic oxidoreductases and cell surface-localized terminal reductases. MtrB is the ?-barrel protein that is necessary for dissimilatory iron reduction. It forms a complex together with the periplasmic decaheme c-type cytochrome MtrA and the outer membrane decaheme c-type cytochrome MtrC. Consequently, mtrB deletion mutants are unable to reduce ferric iron. The data presented here show that this inability can be overcome by a mobile genomic element with the ability to activate the expression of downstream genes and which is inserted within the SO4362 gene of the SO4362-to-SO4357 gene cluster. This cluster carries genes similar to mtrA and mtrB and encoding a putative cell surface DMSO reductase. Expression of SO4359 and SO4360 alone was sufficient to complement not only an mtrB mutant under ferric citrate-reducing conditions but also a mutant that furthermore lacks any outer membrane cytochromes. Hence, the putative complex formed by the SO4359 and SO4360 gene products is capable not only of membrane-spanning electron transfer but also of reducing extracellular electron acceptors.

SUBMITTER: Schicklberger M

PROVIDER: S-EPMC3568626 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Genomic plasticity enables a secondary electron transport pathway in Shewanella oneidensis.

Schicklberger M M Sturm G G Gescher J J

Applied and environmental microbiology 20121207 4

Microbial dissimilatory iron reduction is an important biogeochemical process. It is physiologically challenging because iron occurs in soils and sediments in the form of insoluble minerals such as hematite or ferrihydrite. Shewanella oneidensis MR-1 evolved an extended respiratory chain to the cell surface to reduce iron minerals. Interestingly, the organism evolved a similar strategy for reduction of dimethyl sulfoxide (DMSO), which is reduced at the cell surface as well. It has already been e ...[more]

PMID: 23220953

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Project description:Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO2 and renewable electricity1-6. Theoretically EET could do this with an efficiency comparable to H2-oxidation7,8 but without the need for a volatile intermediate and the problems it causes for scale up9. However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-110-14, though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation15.

Project description:UnlabelledShewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the ?bfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria.ImportanceExtracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.

Project description:Background: Microbial fuel cells (MFCs) are a novel bioelectrochemical devices that can use exoelectrogens as biocatalyst to convert various organic wastes into electricity. Among them, acetate, a major component of industrial biological wastewater and by-product of lignocellulose degradation, could release eight electrons per mole when completely degraded into CO2 and H2O, which has been identified as a promising carbon source and electron donor. However, Shewanella oneidensis MR-1, a famous facultative anaerobic exoelectrogens, only preferentially uses lactate as carbon source and electron donor and could hardly metabolize acetate in MFCs, which greatly limited Coulombic efficiency of MFCs and the capacity of bio-catalysis. Results: Here, to enable acetate as the sole carbon source and electron donor for electricity production in S. oneidensis, we successfully constructed three engineered S. oneidensis (named AceU1, AceU2, and AceU3) by assembling the succinyl-CoA:acetate CoA-transferase (SCACT) metabolism pathways, including acetate coenzyme A transferase encoded by ato1 and ato2 gene from G. sulfurreducens and citrate synthase encoded by the gltA gene from S. oneidensis, which could successfully utilize acetate as carbon source under anaerobic and aerobic conditions. Then, biochemical characterizations showed the engineered strain AceU3 generated a maximum power density of 8.3 ± 1.2 mW/m2 with acetate as the sole electron donor in MFCs. In addition, when further using lactate as the electron donor, the maximum power density obtained by AceU3 was 51.1 ± 3.1 mW/m2, which approximately 2.4-fold higher than that of wild type (WT). Besides, the Coulombic efficiency of AceU3 strain could reach 12.4% increased by 2.0-fold compared that of WT, which demonstrated that the engineered strain AceU3 can further utilize acetate as an electron donor to continuously generate electricity. Conclusion: In the present study, we first rationally designed S. oneidensis for enhancing the electron generation by using acetate as sole carbon source and electron donor. Based on synthetic biology strategies, modular assembly of acetate metabolic pathways could be further extended to other exoelectrogens to improve the Coulombic efficiency and broaden the spectrum of available carbon sources in MFCs for bioelectricity production.

Project description:Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design.

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Genomic plasticity enables a secondary electron transport pathway in Shewanella oneidensis.

Publications

Genomic plasticity enables a secondary electron transport pathway in Shewanella oneidensis.

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