A Hybrid Extracellular Electron Transfer Pathway Enhances the Survival of Vibrio natriegens

ABSTRACT Vibrio natriegens is the fastest-growing microorganism discovered to date, making it a useful model for biotechnology and basic research. While it is recognized for its rapid aerobic metabolism, less is known about anaerobic adaptations in V. natriegens or how the organism survives when oxygen is limited. Here, we describe and characterize extracellular electron transfer (EET) in V. natriegens, a metabolism that requires movement of electrons across protective cellular barriers to reach the extracellular space. V. natriegens performs extracellular electron transfer under fermentative conditions with gluconate, glucosamine, and pyruvate. We characterized a pathway in V. natriegens that requires CymA, PdsA, and MtrCAB for Fe(III) citrate and Fe(III) oxide reduction, which represents a hybrid of strategies previously discovered in Shewanella and Aeromonas. Expression of these V. natriegens genes functionally complemented Shewanella oneidensis mutants. Phylogenetic analysis of the inner membrane quinol dehydrogenases CymA and NapC in gammaproteobacteria suggests that CymA from Shewanella diverged from Vibrionaceae CymA and NapC. Analysis of sequenced Vibrionaceae revealed that the genetic potential to perform EET is conserved in some members of the Harveyi and Vulnificus clades but is more variable in other clades. We provide evidence that EET enhances anaerobic survival of V. natriegens, which may be the primary physiological function for EET in Vibrionaceae. IMPORTANCE Bacteria from the genus Vibrio occupy a variety of marine and brackish niches with fluctuating nutrient and energy sources. When oxygen is limited, fermentation or alternative respiration pathways must be used to conserve energy. In sedimentary environments, insoluble oxide minerals (primarily iron and manganese) are able to serve as electron acceptors for anaerobic respiration by microorganisms capable of extracellular electron transfer, a metabolism that enables the use of these insoluble substrates. Here, we identify the mechanism for extracellular electron transfer in Vibrio natriegens, which uses a combination of strategies previously identified in Shewanella and Aeromonas. We show that extracellular electron transfer enhanced survival of V. natriegens under fermentative conditions, which may be a generalized strategy among Vibrio spp. predicted to have this metabolism.

Download Full-text

Pyruvate accelerates palladium reduction by regulating catabolism and electron transfer pathway in Shewanella oneidensis

Applied and Environmental Microbiology ◽

10.1128/aem.02716-20 ◽

2021 ◽

Author(s):

Yuan-Yuan Cheng ◽

Wen-Jing Wang ◽

Shi-Ting Ding ◽

Ming-Xing Zhang ◽

Ai-Guo Tang ◽

...

Keyword(s):

Electron Transfer ◽

Carbon Source ◽

Time Window ◽

Carbon Sources ◽

Shewanella Oneidensis ◽

Underlying Mechanism ◽

Extracellular Electron Transfer ◽

Resting Cells ◽

Electron Transfer Pathway ◽

Specific Regulation

Shewanella oneidensis is a model strain of the electrochemical active bacteria (EAB) because of its strong capability of extracellular electron transfer (EET) and genetic tractability. In this study, we investigated the effect of carbon sources on EET in S. oneidensis by using reduction of palladium ions (Pd(II)) as a model and found that pyruvate greatly accelerated the Pd(II) reduction compared with lactate by resting cells. Both Mtr pathway and hydrogenases played a role in Pd(II) reduction when pyruvate was used as a carbon source. Furthermore, in comparison with lactate-feeding S. oneidensis, the transcriptional levels of formate dehydrogenases involving in pyruvate catabolism, Mtr pathway, and hydrogenases in pyruvate-feeding S. oneidensis were up-regulated. Mechanistically, the enhancement of electron generation from pyruvate catabolism and electron transfer to Pd(II) explains the pyruvate effect on Pd(II) reduction. Interestingly, a 2-h time window is required for pyruvate to regulate transcription of these genes and profoundly improve Pd(II) reduction capability, suggesting a hierarchical regulation for pyruvate sensing and response in S. oneidensis. IMPORTANCE The unique respiration of EET is crucial for the biogeochemical cycling of metal elements and diverse applications of EAB. Although a carbon source is a determinant factor of bacterial metabolism, the research into the regulation of carbon source on EET is rare. In this work, we reported the pyruvate-specific regulation and improvement of EET in S. oneidensis and revealed the underlying mechanism, which suggests potential targets to engineer and improve the EET efficiency of this bacterium. This study sheds light on the regulatory role of carbon sources in anaerobic respiration in EAB, providing a way to regulate EET for diverse applications from a novel perspective.

Download Full-text

Flavin Electron Shuttles Dominate Extracellular Electron Transfer by Shewanella oneidensis

mBio ◽

10.1128/mbio.00553-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 237

Author(s):

Nicholas J. Kotloski ◽

Jeffrey A. Gralnick

Keyword(s):

Electron Transfer ◽

Direct Electron Transfer ◽

Shewanella Oneidensis ◽

Phenotypic Characterization ◽

Extracellular Electron Transfer ◽

Wild Type ◽

Content Type ◽

Electron Shuttling ◽

Electron Shuttles ◽

Set Up

ABSTRACT Shewanella 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 Δbfe 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. IMPORTANCE Extracellular 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.

Download Full-text

Extracellular Electron Transfer: Respiratory or Nutrient Homeostasis?

Journal of Bacteriology ◽

10.1128/jb.00029-20 ◽

2020 ◽

Vol 202 (7) ◽

Cited By ~ 2

Author(s):

Lars J. C. Jeuken ◽

Kiel Hards ◽

Yoshio Nakatani

Keyword(s):

Electron Transfer ◽

Iron Reduction ◽

Ferric Iron ◽

Shewanella Oneidensis ◽

Geobacter Sulfurreducens ◽

Extracellular Electron Transfer ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria ◽

Nutrient Homeostasis

ABSTRACT Exoelectrogens are able to transfer electrons extracellularly, enabling them to respire on insoluble terminal electron acceptors. Extensively studied exoelectrogens, such as Geobacter sulfurreducens and Shewanella oneidensis, are Gram negative. More recently, it has been reported that Gram-positive bacteria, such as Listeria monocytogenes and Enterococcus faecalis, also exhibit the ability to transfer electrons extracellularly, although it is still unclear whether this has a function in respiration or in redox control of the environment, for instance, by reducing ferric iron for iron uptake. In this issue of Journal of Bacteriology, Hederstedt and colleagues report on experiments that directly compare extracellular electron transfer (EET) pathways for ferric iron reduction and respiration and find a clear difference (L. Hederstedt, L. Gorton, and G. Pankratova, J Bacteriol 202:e00725-19, 2020, https://doi.org/10.1128/JB.00725-19), providing further insights and new questions into the function and metabolic pathways of EET in Gram-positive bacteria.

Download Full-text

Mtr extracellular electron-transfer pathways in Fe(III)-reducing or Fe(II)-oxidizing bacteria: a genomic perspective

Biochemical Society Transactions ◽

10.1042/bst20120098 ◽

2012 ◽

Vol 40 (6) ◽

pp. 1261-1267 ◽

Cited By ~ 78

Author(s):

Liang Shi ◽

Kevin M. Rosso ◽

John M. Zachara ◽

James K. Fredrickson

Keyword(s):

Electron Transfer ◽

Cell Envelope ◽

Shewanella Oneidensis ◽

Extracellular Electron Transfer ◽

Bacterial Cells ◽

Electron Transfer Pathway ◽

Protein Components ◽

Micro Organisms ◽

Reducing Bacteria ◽

Mediate Electron Transfer

Originally discovered in the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), key components of the Mtr (i.e. metal-reducing) pathway exist in all strains of metal-reducing Shewanella characterized. The protein components identified to date for the Mtr pathway of MR-1 include four multihaem c-Cyts (c-type cytochromes), CymA, MtrA, MtrC and OmcA, and a porin-like outer membrane protein MtrB. They are strategically positioned along the width of the MR-1 cell envelope to mediate electron transfer from the quinone/quinol pool in the inner membrane to Fe(III)-containing minerals external to the bacterial cells. A survey of microbial genomes has identified homologues of the Mtr pathway in other dissimilatory Fe(III)-reducing bacteria, including Aeromonas hydrophila, Ferrimonas balearica and Rhodoferax ferrireducens, and in the Fe(II)-oxidizing bacteria Dechloromonas aromatica RCB, Gallionella capsiferriformans ES-2 and Sideroxydans lithotrophicus ES-1. The apparent widespread distribution of Mtr pathways in both Fe(III)-reducing and Fe(II)-oxidizing bacteria suggests a bidirectional electron transfer role, and emphasizes the importance of this type of extracellular electron-transfer pathway in microbial redox transformation of iron. The organizational and electron-transfer characteristics of the Mtr pathways may be shared by other pathways used by micro-organisms for exchanging electrons with their extracellular environments.

Download Full-text

Reversing an extracellular electron transfer pathway for electrode-driven NADH generation

10.1101/476481 ◽

2018 ◽

Author(s):

Nicholas M. Tefft ◽

Michaela A. TerAvest

Keyword(s):

Electron Transfer ◽

Shewanella Oneidensis ◽

Electrical Energy ◽

Genetically Engineered ◽

Extracellular Electron Transfer ◽

Microbial Electrosynthesis ◽

Electron Transfer Pathway ◽

Background Strain ◽

Nadh Dehydrogenases ◽

Electron Sink

AbstractMicrobial electrosynthesis is an emerging technology with the potential to simultaneously store renewably generated energy, fix carbon dioxide, and produce high-value organic compounds. However, limited understanding of the route of electrons into the cell remains an obstacle to developing a robust microbial electrosynthesis platform. To address this challenge, we engineered an inward electron transfer pathway inShewanella oneidensisMR-1. The pathway uses native Mtr proteins to transfer electrons from an electrode to the inner membrane quinone pool. Subsequently, electrons are transferred from quinones to NAD+by native NADH dehydrogenases. This reverse functioning of NADH dehydrogenases is thermodynamically unfavorable, therefore we have added a light-driven proton pump (proteorhodopsin) to generate proton-motive force to drive this activity. Finally, we use reduction of acetoin to 2,3-butanediol via a heterologous butanediol dehydrogenase (Bdh) as an electron sink. Bdh is an NADH-dependent enzyme, therefore, observation of acetoin reduction supports our hypothesis that cathodic electrons are transferred to intracellular NAD+. Multiple lines of evidence indicate proper functioning of the engineered electrosynthesis system: electron flux from the cathode is influenced by both light and acetoin availability; and 2,3-butanediol production is highest when both light and a poised electrode are present. Using a hydrogenase-deficientS. oneidensisbackground strain resulted in a stronger correlation between electron transfer and 2,3-butanediol production, suggesting that hydrogen production is an off-target electron sink in the wild-type background. This system represents a promising genetically engineered microbial electrosynthesis platform and will enable a new focus on synthesis of specific compounds using electrical energy.

Download Full-text

Extracellular Electron Transfer Enables Cellular Control of Cu(I)-catalyzed Alkyne-Azide Cycloaddition

10.1101/2021.09.28.462180 ◽

2021 ◽

Author(s):

Gina Partipilo ◽

Austin J. Graham ◽

Brian Belardi ◽

Benjamin K. Keitz

Keyword(s):

Electron Transfer ◽

Mammalian Cells ◽

Shewanella Oneidensis ◽

Anaerobic Respiration ◽

Central Carbon Metabolism ◽

Metal Species ◽

Extracellular Electron Transfer ◽

Metal Catalyst ◽

Genetic Circuits ◽

Cellular Control

AbstractExtracellular electron transfer (EET) is an anaerobic respiration process that couples carbon oxidation to the reduction of metal species. In the presence of a suitable metal catalyst, EET allows for cellular metabolism to control a variety of synthetic transformations. Here, we report the use of EET from the model electroactive bacterium Shewanella oneidensis for metabolic and genetic control over Cu(I)-catalyzed Alkyne-Azide Cycloaddition (CuAAC). CuAAC conversion under anaerobic and aerobic conditions was dependent on live, actively respiring S. oneidensis cells. In addition, reaction progress and kinetics could be further manipulated by tailoring the central carbon metabolism of S. oneidensis. Similarly, CuAAC activity was dependent on specific EET pathways and could be manipulated using inducible genetic circuits controlling the expression of EET-relevant proteins including MtrC, MtrA, and CymA. EET-driven CuAAC also exhibited modularity and robustness in ligand tolerance and substrate scope. Furthermore, the living nature of this system could be exploited to perform multiple reaction cycles without requiring regeneration, something inaccessible to traditional chemical reductants. Finally, S. oneidensis enabled bioorthogonal CuAAC membrane labelling on live mammalian cells without affecting cell viability, suggesting that S. oneidensis can act as a dynamically tunable biocatalyst in complex environments. In summary, our results demonstrate how EET can expand the reaction scope available to living systems by enabling cellular control of CuAAC.

Download Full-text

Regulation of Gene Expression in Shewanella oneidensis MR-1 during Electron Acceptor Limitation and Bacterial Nanowire Formation

Applied and Environmental Microbiology ◽

10.1128/aem.01615-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5428-5443 ◽

Cited By ~ 23

Author(s):

Sarah E. Barchinger ◽

Sahand Pirbadian ◽

Christine Sambles ◽

Carol S. Baker ◽

Kar Man Leung ◽

...

Keyword(s):

Gene Expression ◽

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Shewanella Oneidensis ◽

Oxygen Limitation ◽

Extracellular Electron Transfer ◽

Transport Pathways ◽

Content Type ◽

Genes Encoding

ABSTRACTIn limiting oxygen as an electron acceptor, the dissimilatory metal-reducing bacteriumShewanella oneidensisMR-1 rapidly forms nanowires, extensions of its outer membrane containing the cytochromes MtrC and OmcA needed for extracellular electron transfer. RNA sequencing (RNA-Seq) analysis was employed to determine differential gene expression over time from triplicate chemostat cultures that were limited for oxygen. We identified 465 genes with decreased expression and 677 genes with increased expression. The coordinated increased expression of heme biosynthesis, cytochrome maturation, and transport pathways indicates thatS. oneidensisMR-1 increases cytochrome production, including the transcription of genes encoding MtrA, MtrC, and OmcA, and transports these decaheme cytochromes across the cytoplasmic membrane during electron acceptor limitation and nanowire formation. In contrast, the expression of themtrAandmtrChomologsmtrFandmtrDeither remains unaffected or decreases under these conditions. TheompWgene, encoding a small outer membrane porin, has 40-fold higher expression during oxygen limitation, and it is proposed that OmpW plays a role in cation transport to maintain electrical neutrality during electron transfer. The genes encoding the anaerobic respiration regulator cyclic AMP receptor protein (CRP) and the extracytoplasmic function sigma factor RpoE are among the transcription factor genes with increased expression. RpoE might function by signaling the initial response to oxygen limitation. Our results show that RpoE activates transcription from promoters upstream ofmtrCandomcA. The transcriptome and mutant analyses ofS. oneidensisMR-1 nanowire production are consistent with independent regulatory mechanisms for extending the outer membrane into tubular structures and for ensuring the electron transfer function of the nanowires.IMPORTANCEShewanella oneidensisMR-1 has the capacity to transfer electrons to its external surface using extensions of the outer membrane called bacterial nanowires. These bacterial nanowires link the cell's respiratory chain to external surfaces, including oxidized metals important in bioremediation, and explain whyS. oneidensiscan be utilized as a component of microbial fuel cells, a form of renewable energy. In this work, we use differential gene expression analysis to focus on which genes function to produce the nanowires and promote extracellular electron transfer during oxygen limitation. Among the genes that are expressed at high levels are those encoding cytochrome proteins necessary for electron transfer.Shewanellacoordinates the increased expression of regulators, metabolic pathways, and transport pathways to ensure that cytochromes efficiently transfer electrons along the nanowires.

Download Full-text

Divergent Nrf Family Proteins and MtrCAB Homologs Facilitate Extracellular Electron Transfer inAeromonas hydrophila

Applied and Environmental Microbiology ◽

10.1128/aem.02134-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 4

Author(s):

Bridget E. Conley ◽

Peter J. Intile ◽

Daniel R. Bond ◽

Jeffrey A. Gralnick

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Aeromonas Hydrophila ◽

Shewanella Oneidensis ◽

Geobacter Sulfurreducens ◽

Model Systems ◽

Extracellular Electron Transfer ◽

Metal Reduction ◽

Inner Membrane ◽

Content Type

ABSTRACTExtracellular electron transfer (EET) is a strategy for respiration in which electrons generated from metabolism are moved outside the cell to a terminal electron acceptor, such as iron or manganese oxide. EET has primarily been studied in two model systems,Shewanella oneidensisandGeobacter sulfurreducens. Metal reduction has also been reported in numerous microorganisms, includingAeromonasspp., which are ubiquitousGammaproteobacteriafound in aquatic ecosystems, with some species capable of pathogenesis in humans and fish. Genomic comparisons ofAeromonasspp. revealed a potential outer membrane conduit homologous toS. oneidensisMtrCAB. While the ability to respire metals and mineral oxides is not widespread in the genusAeromonas, 90% of the sequencedAeromonas hydrophilaisolates contain MtrCAB homologs.A. hydrophilaATCC 7966 mutants lackingmtrAare unable to reduce metals. Expression ofA. hydrophila mtrCABin anS. oneidensismutant lacking homologous components restored metal reduction. Although the outer membrane conduits for metal reduction were similar, homologs of theS. oneidensisinner membrane and periplasmic EET components CymA, FccA, and CctA were not found inA. hydrophila. We characterized a cluster of genes predicted to encode components related to a formate-dependent nitrite reductase (NrfBCD), here named NetBCD (forNrf-likeelectrontransfer), and a predicted diheme periplasmic cytochrome, PdsA (periplasmicdihemeshuttle). We present genetic evidence that proteins encoded by this cluster facilitate electron transfer from the cytoplasmic membrane across the periplasm to the MtrCAB conduit and function independently from an authentic NrfABCD system.A. hydrophilamutants lackingpdsAandnetBCDwere unable to reduce metals, while heterologous expression of these genes could restore metal reduction in anS. oneidensismutant background. EET may therefore allowA. hydrophilaand other species ofAeromonasto persist and thrive in iron- or manganese-rich oxygen-limited environments.IMPORTANCEMetal-reducing microorganisms are used for electricity production, bioremediation of toxic compounds, wastewater treatment, and production of valuable compounds. Despite numerous microorganisms being reported to reduce metals, the molecular mechanism has primarily been studied in two model systems,Shewanella oneidensisandGeobacter sulfurreducens. We have characterized the mechanism of extracellular electron transfer inAeromonas hydrophila, which uses the well-studiedShewanellasystem, MtrCAB, to move electrons across the outer membrane; however, mostAeromonasspp. appear to use a novel mechanism to transfer electrons from the inner membrane through the periplasm and to the outer membrane. The conserved use of MtrCAB inShewanellaspp. andAeromonasspp. for metal reduction and conserved genomic architecture of metal reduction genes inAeromonasspp. may serve as genomic markers for identifying metal-reducing microorganisms from genomic or transcriptomic sequencing. Understanding the variety of pathways used to reduce metals can allow for optimization and more efficient design of microorganisms used for practical applications.

Download Full-text