Methane-Linked Mechanisms of Electron Uptake from Cathodes byMethanosarcina barkeri

ABSTRACTTheMethanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes byMethanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here, we confirm the ability ofM. barkerito perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g., hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g., hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower-potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production inM. barkeri. Our electrochemical measurements of wild-type and mutant strains point to a novel and hydrogenase-free mode of electron uptake with a potential near −484 mV versus standard hydrogen electrode (SHE) (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest thatM. barkerican perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent modes) of electrode interactions on cathodes, including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.IMPORTANCEMethanogenic archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens have been well studied with respect to soluble substrates, a mechanistic understanding of their interaction with solid-phase redox-active compounds is limited. This work provides insight into solid-phase redox interactions inMethanosarcinaspp. using electrochemical methods. We highlight a previously undescribed mode of electron uptake from cathodes that is potentially informative of direct interspecies electron transfer interactions in theMethanosarcinales.

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Methane-linked mechanisms of electron uptake from cathodes byMethanosarcina barkeri

10.1101/415653 ◽

2018 ◽

Author(s):

Annette R. Rowe ◽

Shuai Xu ◽

Emily Gardel ◽

Arpita Bose ◽

Peter Girguis ◽

...

Keyword(s):

Electron Transfer ◽

Methane Production ◽

Extracellular Enzymes ◽

Solid Phase ◽

Electrochemical Techniques ◽

Extracellular Enzyme ◽

Methanosarcina Barkeri ◽

Extracellular Electron Transfer ◽

Cathodic Current ◽

Wide Range

AbstractTheMethanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes byMethanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here we confirm the ability ofM. barkerito perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g. hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g. hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production inM. barkeri. Our electrochemical measurements of wild-type and mutant strains point to a novel and extracellular-enzyme-free mode of electron uptake able to take up electrons at potentials lower than - 498 mV vs. SHE (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest thatM. barkerican perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent) of electrode interactions on cathodes including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.ImportanceMethanogenic Archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens has been well studied with respect to soluble substrates, mechanistic understanding of their interaction with solid phase redox active compounds is limited. This work provides insight into solid phase redox interactions inMethanosarcinausing electrochemical methods. We highlight a previously undescribed mode of electron uptake from cathodes, that is potentially informative of direct interspecies electron transfer interactions in theMethanosarcinales.

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Single-Cell Electrophysiological Measurements Reveal Bacterial Membrane Potential Dynamics during Extracellular Electron Transfer

10.1101/2020.01.15.908145 ◽

2020 ◽

Author(s):

Sahand Pirbadian ◽

Marko S. Chavez ◽

Mohamed Y. El-Naggar

Keyword(s):

Electron Transfer ◽

Membrane Potential ◽

Single Cell ◽

Solid Phase ◽

Electrochemical Techniques ◽

Model Organisms ◽

Extracellular Electron Transfer ◽

Single Cell Level ◽

Cell Level ◽

Wide Range

AbstractExtracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with the electrode potential, produced current, and position of cells relative to the electrodes. The high spatial and temporal resolution of the reported technique can be used to study the single-cell level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

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Spatiotemporal mapping of bacterial membrane potential responses to extracellular electron transfer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2000802117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 20171-20179 ◽

Cited By ~ 1

Author(s):

Sahand Pirbadian ◽

Marko S. Chavez ◽

Mohamed Y. El-Naggar

Keyword(s):

Electron Transfer ◽

Membrane Potential ◽

Single Cell ◽

Electrochemical Techniques ◽

Model Organisms ◽

Extracellular Electron Transfer ◽

Single Cell Level ◽

Cell Level ◽

Membrane Hyperpolarization ◽

Wide Range

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such asShewanella oneidensisMR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET byS. oneidensisMR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 μm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

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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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Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

Applied and Environmental Microbiology ◽

10.1128/aem.00895-14 ◽

2014 ◽

Vol 80 (15) ◽

pp. 4599-4605 ◽

Cited By ~ 398

Author(s):

Amelia-Elena Rotaru ◽

Pravin Malla Shrestha ◽

Fanghua Liu ◽

Beatrice Markovaite ◽

Shanshan Chen ◽

...

Keyword(s):

Electron Transfer ◽

Model Organism ◽

Methanosarcina Barkeri ◽

Physical Contact ◽

Acetate Metabolism ◽

Geobacter Metallireducens ◽

Content Type ◽

Direct Interspecies Electron Transfer ◽

Interspecies Electron Transfer ◽

Effective Form

ABSTRACTDirect interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability ofMethanosarcina barkerito participate in DIET was evaluated in coculture withGeobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficientG. metallireducensstrain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficientG. metallireducensisolates to share electrons withM. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. WhenM. barkeriwas grown in coculture with the H2-producingPelobacter carbinolicus, incapable of DIET,M. barkeriutilized H2as an electron donor but metabolized little of the acetate thatP. carbinolicusproduced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism.P. carbinolicus-M. barkericocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2transfer.M. barkeriis the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2or electrons derived from DIET for CO2reduction. Furthermore,M. barkeriis genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

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Putative Extracellular Electron Transfer in Methanogenic Archaea

Frontiers in Microbiology ◽

10.3389/fmicb.2021.611739 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kailin Gao ◽

Yahai Lu

Keyword(s):

Electron Transfer ◽

Methanogenic Archaea ◽

Methanosarcina Barkeri ◽

Extracellular Electron Transfer ◽

Future Research ◽

Methanosarcina Acetivorans ◽

Geobacter Metallireducens ◽

Methanosarcina Mazei ◽

Current State ◽

Electron Transfers

It has been suggested that a few methanogens are capable of extracellular electron transfers. For instance, Methanosarcina barkeri can directly capture electrons from the coexisting microbial cells of other species. Methanothrix harundinacea and Methanosarcina horonobensis retrieve electrons from Geobacter metallireducens via direct interspecies electron transfer (DIET). Recently, Methanobacterium, designated strain YSL, has been found to grow via DIET in the co-culture with Geobacter metallireducens. Methanosarcina acetivorans can perform anaerobic methane oxidation and respiratory growth relying on Fe(III) reduction through the extracellular electron transfer. Methanosarcina mazei is capable of electromethanogenesis under the conditions where electron-transfer mediators like H2 or formate are limited. The membrane-bound multiheme c-type cytochromes (MHC) and electrically-conductive cellular appendages have been assumed to mediate the extracellular electron transfer in bacteria like Geobacter and Shewanella species. These molecules or structures are rare but have been recently identified in a few methanogens. Here, we review the current state of knowledge for the putative extracellular electron transfers in methanogens and highlight the opportunities and challenges for future research.

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A Previously Uncharacterized, Nonphotosynthetic Member of the Chromatiaceae Is the Primary CO2-Fixing Constituent in a Self-Regenerating Biocathode

Applied and Environmental Microbiology ◽

10.1128/aem.02947-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 699-712 ◽

Cited By ~ 56

Author(s):

Zheng Wang ◽

Dagmar H. Leary ◽

Anthony P. Malanoski ◽

Robert W. Li ◽

W. Judson Hervey ◽

...

Keyword(s):

Microbial Fuel Cells ◽

Iron Oxidation ◽

Extracellular Electron Transfer ◽

Hydrogen Electrode ◽

Content Type ◽

Iron Oxidizing Bacteria ◽

Distinct Cluster ◽

The Family

ABSTRACTBiocathode extracellular electron transfer (EET) may be exploited for biotechnology applications, including microbially mediated O2reduction in microbial fuel cells and microbial electrosynthesis. However, biocathode mechanistic studies needed to improve or engineer functionality have been limited to a few select species that form sparse, homogeneous biofilms characterized by little or no growth. Attempts to cultivate isolates from biocathode environmental enrichments often fail due to a lack of some advantage provided by life in a consortium, highlighting the need to study and understand biocathode consortiain situ. Here, we present metagenomic and metaproteomic characterization of a previously described biocathode biofilm (+310 mV versus a standard hydrogen electrode [SHE]) enriched from seawater, reducing O2, and presumably fixing CO2for biomass generation. Metagenomics identified 16 distinct cluster genomes, 15 of which could be assigned at the family or genus level and whose abundance was roughly divided betweenAlpha- andGammaproteobacteria. A total of 644 proteins were identified from shotgun metaproteomics and have been deposited in the the ProteomeXchange with identifier PXD001045. Cluster genomes were used to assign the taxonomic identities of 599 proteins, withMarinobacter,Chromatiaceae, andLabrenziathe most represented. RubisCO and phosphoribulokinase, along with 9 other Calvin-Benson-Bassham cycle proteins, were identified fromChromatiaceae. In addition, proteins similar to those predicted for iron oxidation pathways of known iron-oxidizing bacteria were observed forChromatiaceae. These findings represent the first description of putative EET and CO2fixation mechanisms for a self-regenerating, self-sustaining multispecies biocathode, providing potential targets for functional engineering, as well as new insights into biocathode EET pathways using proteomics.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01253-20 ◽

2020 ◽

Vol 86 (19) ◽

Cited By ~ 1

Author(s):

Bridget E. Conley ◽

Matthew T. Weinstock ◽

Daniel R. Bond ◽

Jeffrey A. Gralnick

Keyword(s):

Electron Transfer ◽

Shewanella Oneidensis ◽

Anaerobic Respiration ◽

Basic Research ◽

Extracellular Electron Transfer ◽

Content Type ◽

Electron Transfer Pathway ◽

Sedimentary Environments ◽

Genetic Potential ◽

Iron And Manganese

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.

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