Geothrix fermentans Secretes Two Different Redox-Active Compounds To Utilize Electron Acceptors across a Wide Range of Redox Potentials

ABSTRACTThe current understanding of dissimilatory metal reduction is based primarily on isolates from the proteobacterial generaGeobacterandShewanella. However, environments undergoing active Fe(III) reduction often harbor less-well-studied phyla that are equally abundant. In this work, electrochemical techniques were used to analyze respiratory electron transfer by the only known Fe(III)-reducing representative of theAcidobacteria,Geothrix fermentans. In contrast to previously characterized metal-reducing bacteria, which typically reach maximal rates of respiration at electron acceptor potentials of 0 V versus standard hydrogen electrode (SHE),G. fermentansrequired potentials as high as 0.55 V to respire at its maximum rate. In addition,G. fermentanssecreted two different soluble redox-active electron shuttles with separate redox potentials (−0.2 V and 0.3 V). The compound with the lower midpoint potential, responsible for 20 to 30% of electron transfer activity, was riboflavin. The behavior of the higher-potential compound was consistent with hydrophilic UV-fluorescent molecules previously found inG. fermentanssupernatants. Both electron shuttles were also produced when cultures were grown with Fe(III), but not when fumarate was the electron acceptor. This study reveals thatGeothrixis able to take advantage of higher-redox-potential environments, demonstrates that secretion of flavin-based shuttles is not confined toShewanella, and points to the existence of high-potential-redox-active compounds involved in extracellular electron transfer. Based on differences between the respiratory strategies ofGeothrixandGeobacter, these two groups of bacteria could exist in distinctive environmental niches defined by redox potential.

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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.

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A Membrane-Bound Cytochrome Enables Methanosarcina acetivorans To Conserve Energy from Extracellular Electron Transfer

mBio ◽

10.1128/mbio.00789-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 14

Author(s):

Dawn E. Holmes ◽

Toshiyuki Ueki ◽

Hai-Yan Tang ◽

Jinjie Zhou ◽

Jessica A. Smith ◽

...

Keyword(s):

Electron Transfer ◽

Electron Acceptor ◽

Methane Production ◽

Genetic Manipulation ◽

Methanol Conversion ◽

Extracellular Electron Transfer ◽

Methanosarcina Acetivorans ◽

Coenzyme M ◽

Content Type ◽

Expression Of Genes

ABSTRACT Extracellular electron exchange in Methanosarcina species and closely related Archaea plays an important role in the global carbon cycle and enhances the speed and stability of anaerobic digestion by facilitating efficient syntrophic interactions. Here, we grew Methanosarcina acetivorans with methanol provided as the electron donor and the humic analogue, anthraquione-2,6-disulfonate (AQDS), provided as the electron acceptor when methane production was inhibited with bromoethanesulfonate. AQDS was reduced with simultaneous methane production in the absence of bromoethanesulfonate. Transcriptomics revealed that expression of the gene for the transmembrane, multiheme, c-type cytochrome MmcA was higher in AQDS-respiring cells than in cells performing methylotrophic methanogenesis. A strain in which the gene for MmcA was deleted failed to grow via AQDS reduction but grew with the conversion of methanol or acetate to methane, suggesting that MmcA has a specialized role as a conduit for extracellular electron transfer. Enhanced expression of genes for methanol conversion to methyl-coenzyme M and the Rnf complex suggested that methanol is oxidized to carbon dioxide in AQDS-respiring cells through a pathway that is similar to methyl-coenzyme M oxidation in methanogenic cells. However, during AQDS respiration the Rnf complex and reduced methanophenazine probably transfer electrons to MmcA, which functions as the terminal reductase for AQDS reduction. Extracellular electron transfer may enable the survival of methanogens in dynamic environments in which oxidized humic substances and Fe(III) oxides are intermittently available. The availability of tools for genetic manipulation of M. acetivorans makes it an excellent model microbe for evaluating c-type cytochrome-dependent extracellular electron transfer in Archaea. IMPORTANCE The discovery of a methanogen that can conserve energy to support growth solely from the oxidation of organic carbon coupled to the reduction of an extracellular electron acceptor expands the possible environments in which methanogens might thrive. The potential importance of c-type cytochromes for extracellular electron transfer to syntrophic bacterial partners and/or Fe(III) minerals in some Archaea was previously proposed, but these studies with Methanosarcina acetivorans provide the first genetic evidence for cytochrome-based extracellular electron transfer in Archaea. The results suggest parallels with Gram-negative bacteria, such as Shewanella and Geobacter species, in which multiheme outer-surface c-type cytochromes are an essential component for electrical communication with the extracellular environment. M. acetivorans offers an unprecedented opportunity to study mechanisms for energy conservation from the anaerobic oxidation of one-carbon organic compounds coupled to extracellular electron transfer in Archaea with implications not only for methanogens but possibly also for Archaea that anaerobically oxidize methane.

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An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors

mBio ◽

10.1128/mbio.02034-14 ◽

2014 ◽

Vol 5 (6) ◽

Cited By ~ 79

Author(s):

Caleb E. Levar ◽

Chi Ho Chan ◽

Misha G. Mehta-Kolte ◽

Daniel R. Bond

Keyword(s):

Electron Transfer ◽

Electron Acceptors ◽

Geobacter Sulfurreducens ◽

Extracellular Electron Transfer ◽

Redox Potentials ◽

Inner Membrane ◽

Content Type ◽

Transfer Strategies ◽

Reducing Bacteria ◽

Metal Reducing Bacteria

ABSTRACTDissimilatory metal-reducing bacteria, such asGeobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption ofimcH, encoding an inner membranec-type cytochrome, eliminated the ability ofG. sulfurreducensto reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but theimcHmutant retained the ability to reduce Fe(III) oxides with potentials of ≤−0.1 V versus SHE. TheimcHmutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to −0.1 V versus SHE triggered exponential growth. At potentials of ≤−0.1 V versus SHE, both the wild type and theimcHmutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not triggerimcHmutant growth. These results demonstrate thatG. sulfurreducenspossesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments whereGeobacteris found.IMPORTANCEInsoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein inGeobacter sulfurreducenswhich is required only for electron transfer to high-potential compounds, and we show thatG. sulfurreducenshas the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell.

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Redox potentials along the redox-active low-barrier H-bonds in electron transfer pathways

Physical Chemistry Chemical Physics ◽

10.1039/d0cp04265j ◽

2020 ◽

Vol 22 (44) ◽

pp. 25467-25473 ◽

Cited By ~ 1

Author(s):

Keisuke Saito ◽

Manoj Mandal ◽

Hiroshi Ishikita

Keyword(s):

Electron Transfer ◽

Proton Transfer ◽

Redox Potential ◽

Driving Force ◽

Electronic Coupling ◽

Redox Potentials ◽

Redox Active ◽

Electron Transfer Pathways

Local proton transfer along redox-active low-barrier H-bonds can alter the driving force or electronic coupling for electron transfer, as the redox potential values depend on the H+ position in low-barrier H-bonds.

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Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms

10.1101/2019.12.12.872085 ◽

2019 ◽

Cited By ~ 1

Author(s):

Scott H. Saunders ◽

Edmund C.M. Tse ◽

Matthew D. Yates ◽

Fernanda Jiménez Otero ◽

Scott A. Trammell ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Electron Transfer ◽

Redox Reactions ◽

Extracellular Dna ◽

Extracellular Electron Transfer ◽

Bacterial Biofilms ◽

Active Metabolites ◽

Electron Shuttles ◽

Redox Active

SUMMARYExtracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in select organisms, a widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. How these shuttles catalyze electron transfer within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes in P. aeruginosa biofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms.

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Bacterial Microcompartments Coupled with Extracellular Electron Transfer Drive the Anaerobic Utilization of Ethanolamine in Listeria monocytogenes

mSystems ◽

10.1128/msystems.01349-20 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Zhe Zeng ◽

Sjef Boeren ◽

Varaang Bhandula ◽

Samuel H. Light ◽

Eddy J. Smid ◽

...

Keyword(s):

Electron Transfer ◽

Listeria Monocytogenes ◽

Electron Acceptor ◽

Cell Membranes ◽

Extracellular Electron Transfer ◽

Vitamin B ◽

Content Type ◽

Redox Imbalance ◽

Bacterial Microcompartments

ABSTRACT Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from cell membranes. EA catabolism is suggested to occur in a cellular metabolic subsystem called a bacterial microcompartment (BMC), and the activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that the activation of eut is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent in Listeria monocytogenes. Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics, and electron microscopy. First, we show vitamin B12-induced activation of the eut operon in L. monocytogenes coupled to the utilization of EA, thereby enabling growth. Next, we demonstrate BMC formation connected with EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP-generating acetate branch causes an apparent redox imbalance due to the reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMCs to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild-type, BMC mutant, and EET mutant strains, we demonstrate an interaction between BMCs and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of BMC-dependent EA catabolism in L. monocytogenes growth in anaerobic environments like the human gastrointestinal tract, with a crucial role for the flavin-based EET system in redox balancing. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness, and as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires the balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

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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.

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Tuning Redox Potential of Anthraquinone-2-Sulfonate (AQS) by Chemical Modification to Facilitate Electron Transfer From Electrodes in Shewanella oneidensis

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.705414 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ning Xu ◽

Tai-Lin Wang ◽

Wen-Jie Li ◽

Yan Wang ◽

Jie-Jie Chen ◽

...

Keyword(s):

Electron Transfer ◽

Redox Potential ◽

Environmental Remediation ◽

Rational Design ◽

Coulombic Efficiency ◽

Shewanella Oneidensis ◽

Extracellular Electron Transfer ◽

Total Charge ◽

Redox Potentials ◽

Chemical Production

Bioelectrochemical systems (BESs) are emerging as attractive routes for sustainable energy generation, environmental remediation, bio-based chemical production and beyond. Electron shuttles (ESs) can be reversibly oxidized and reduced among multiple redox reactions, thereby assisting extracellular electron transfer (EET) process in BESs. Here, we explored the effects of 14 ESs on EET in Shewanella oneidensis MR-1, and found that anthraquinone-2-sulfonate (AQS) led to the highest cathodic current density, total charge production and reduction product formation. Subsequently, we showed that the introduction of -OH or -NH2 group into AQS at position one obviously affected redox potentials. The AQS-1-NH2 exhibited a lower redox potential and a higher Coulombic efficiency compared to AQS, revealing that the ESs with a more negative potential are conducive to minimize energy losses and improve the reduction of electron acceptor. Additionally, the cytochromes MtrA and MtrB were required for optimal AQS-mediated EET of S. oneidensis MR-1. This study will provide new clues for rational design of efficient ESs in microbial electrosynthesis.

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Insight to Microbial Fe(III) Reduction Mediated by Redox-Active Humic Acids with Varied Redox Potentials

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18136807 ◽

2021 ◽

Vol 18 (13) ◽

pp. 6807

Author(s):

Jingtao Duan ◽

Zhiyuan Xu ◽

Zhen Yang ◽

Jie Jiang

Keyword(s):

Molecular Weight ◽

Electron Transfer ◽

Humic Acids ◽

Weight Fraction ◽

Electrochemical Analysis ◽

Microbial Reduction ◽

Redox Potentials ◽

Groundwater Environment ◽

Redox Active ◽

Different Molecular Weight

Redox-active humic acids (HA) are ubiquitous in terrestrial and aquatic systems and are involved in numerous electron transfer reactions affecting biogeochemical processes and fates of pollutants in soil environments. Redox-active contaminants are trapped in soil micropores (<2 nm) that have limited access to microbes and HA. Therefore, the contaminants whose molecular structure and properties are not damaged accumulate in the soil micropores and become potential pollution sources. Electron transfer capacities (ETC) of HA reflecting redox activities of low molecular weight fraction (LMWF, <2.5) HA can be detected by an electrochemical method, which is related to redox potentials (Eh) in soil and aquatic environments. Nevertheless, electron accepting capacities (EAC) and electron donating capacities (EDC) of these LMWF HA at different Eh are still unknown. EDC and EAC of different molecular weight HA at different Eh were analyzed using electrochemical methods. EAC of LMWF at −0.59 V was 12 times higher than that at −0.49 V, while EAC increased to 2.6 times when the Eh decreased from −0.59 V to −0.69 V. Afterward, LMWF can act as a shuttle to stimulate microbial Fe(III) reduction processes in microbial reduction experiments. Additionally, EAC by electrochemical analysis at a range of −0.49–−0.59 V was comparable to total calculated ETC of different molecular weight fractions of HA by microbial reduction. Therefore, it is indicated that redox-active functional groups that can be reduced at Eh range of −0.49–−0.59 are available to microbial reduction. This finding contributes to a novel perspective in the protection and remediation of the groundwater environment in the biogeochemistry process.

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