scholarly journals Disentangling Syntrophic Electron Transfer Mechanisms in Methanogenesis Through Electrochemical Stimulation, Omics, and Machine Learning

2021 ◽  
Author(s):  
Heyang Yuan ◽  
Xuehao Wang ◽  
Tzu-Yu Lin ◽  
Jinha Kim ◽  
Wen-Tso Liu
Keyword(s):  
Machine Learning ◽  
Electron Transfer ◽  
Bayesian Network ◽  
Methane Production ◽  
Extracellular Electron Transfer ◽  
Hydrogen Metabolism ◽  
Electrochemical Reactors ◽  
The Core ◽  
Modeling Approach ◽  
Genes Encoding

Abstract Background: Interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. Results: To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, acetate accumulated in all reactors, while propionate was detected only in the electrochemical reactors. Four dominant fermentative bacteria were identified in the core population, and metatranscriptomics analysis suggested that they were responsible for the degradation of fructose and ethanol to propionate, propanol, acetate, and H2. Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation. The results thus confirmed Ca. G. eutrophica’s EET ability. The high-quality draft genome (completeness 99.4%, contamination 0.6%) further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. Redundancy analysis and a Bayesian network constructed with the core population predicted the importance of Ca. G. eutrophica-related OTU650 to methane production. The Bayesian network modeling approach was also applied to the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis.Conclusions: Ca. G. eutrophica is electroactive and simultaneously performs IHT and DIET. The results from the metatranscriptomic analysis have provided valuable information for enrichment and isolation of Ca. G. eutrophica. IHT is predicted to have a stronger impact on methane production than DIET in the electrochemical reactors. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

scholarly journals Disentangling the syntrophic electron transfer mechanisms of Candidatus geobacter eutrophica through electrochemical stimulation and machine learning

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heyang Yuan ◽  
Xuehao Wang ◽  
Tzu-Yu Lin ◽  
Jinha Kim ◽  
Wen-Tso Liu
Keyword(s):  
Machine Learning ◽  
Electron Transfer ◽  
Hydrogen Transfer ◽  
Draft Genome ◽  
Extracellular Electron Transfer ◽  
Hydrogen Metabolism ◽  
Genetic Potential ◽  
Fermentative Bacteria ◽  
Genes Encoding ◽  
Transfer Mechanisms

AbstractInterspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation, indicating Ca. G. eutrophica’s EET ability. The high-quality draft genome further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. A Bayesian network was trained with the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis from dominant fermentative bacteria, Geobacter, and Methanobacterium. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

scholarly journals Methane-Linked Mechanisms of Electron Uptake from Cathodes byMethanosarcina barkeri

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
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 ◽  
Methanosarcina Barkeri ◽  
Extracellular Electron Transfer ◽  
Hydrogen Electrode ◽  
Content Type ◽  
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 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.

scholarly journals Syntrophic interspecies electron transfer drives carbon fixation and growth by Rhodopseudomonas palustris under dark, anoxic conditions

2021 ◽  
Vol 7 (27) ◽  
pp. eabh1852
Author(s):  
Xing Liu ◽  
Lingyan Huang ◽  
Christopher Rensing ◽  
Jie Ye ◽  
Kenneth H. Nealson ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Carbon Fixation ◽  
Rhodopseudomonas Palustris ◽  
Reducing Power ◽  
Extracellular Electron Transfer ◽  
Geobacter Metallireducens ◽  
Electron Transfer Pathway ◽  
Anoxygenic Phototrophs ◽  
Anoxic Environments ◽  
Genes Encoding

In natural anoxic environments, anoxygenic photosynthetic bacteria fix CO2 by photoheterotrophy, photoautotrophy, or syntrophic anaerobic photosynthesis. Here, we describe electroautotrophy, a previously unidentified dark CO2 fixation mode enabled by the electrosyntrophic interaction between Geobacter metallireducens and Rhodopseudomonas palustris. After an electrosyntrophic coculture is formed, electrons are transferred either directly or indirectly (via electron shuttles) from G. metallireducens to R. palustris, thereby providing reducing power and energy for the dark CO2 fixation. Transcriptomic analyses demonstrated the high expression of genes encoding for the extracellular electron transfer pathway in G. metallireducens and the Calvin-Benson-Bassham carbon fixation cycle in R. palustris. Given that sediments constitute one of the most ubiquitous and abundant niches on Earth and that, at depth, most of the sedimentary niche is both anoxic and dark, dark carbon fixation provides a metabolic window for the survival of anoxygenic phototrophs, as well as an as-yet unappreciated contribution to the global carbon cycle.

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

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
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.

scholarly journals Physiological roles of short-chain and long-chain menaquinones (vitamin K2) in Lactococcus cremoris

2021 ◽  
Author(s):  
Yue Liu ◽  
Nikolaos Charamis ◽  
Sjef Boeren ◽  
Joost Blok ◽  
Alisha Geraldine Lewis ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Bacterial Species ◽  
Biomass Accumulation ◽  
Vitamin K2 ◽  
Physiological Significance ◽  
Short Chain ◽  
Extracellular Electron Transfer ◽  
Growth And Survival ◽  
Genes Encoding ◽  
Long Chain

Lactococcus cremoris and L. lactis are well-known for their occurrence and applications in dairy fermentations, but their niche extends to a range of natural and food production environments. L. cremoris and L. lactis produce MKs (vitamin K2), mainly as the long-chain forms represented by MK-9 and MK-8, and a detectable amount of short-chain forms represented by MK-3. The physiological significance of the different MK forms in the lifestyle of these bacterial species has not been investigated extensively. In this study, we used L. cremoris MG1363 to construct mutants producing different MK profiles by deletion of genes encoding (i) a menaquinone-specific isochorismate synthase, (ii) a geranyltranstransferase and (iii) a prenyl diphosphate synthase. These gene deletions resulted in (i) a non-MK producer (ΔmenF), (ii) a presumed MK-1 producer (ΔispA) and (iii) a MK-3 producer (Δllmg_0196), respectively. By examining the phenotypes of the MG1363 wildtype strain and respective mutants, including biomass accumulation, stationary phase survival, oxygen consumption, primary metabolites, azo dye/copper reduction, and proteomes, under aerobic, anaerobic and respiration-permissive conditions, we could infer that short-chain MKs like MK-1 and MK-3 are preferred to mediate extracellular electron transfer and reaction with extracellular oxygen, while the long-chain MKs like MK-9 and MK-8 are more efficient in aerobic respiratory electron transport chain. The different electron transfer routes mediated by short-chain and long-chain MKs likely support growth and survival of L. cremoris in a range of (transiently) anaerobic and aerobic niches including food fermentations, highlighting the physiological significance of diverse MKs in L. cremoris.

scholarly journals Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates

2019 ◽  
Author(s):  
Christopher M. Dundas ◽  
Benjamin K. Keitz
Keyword(s):  
Electron Transfer ◽  
Dynamic Range ◽  
Reduction Rate ◽  
Shewanella Oneidensis ◽  
Logic Gates ◽  
Extracellular Electron Transfer ◽  
Response Curves ◽  
Fluorescent Reporters ◽  
Genes Encoding ◽  
Functional Understanding

AbstractExtracellular electron transfer pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging since a functional understanding of its EET pathway proteins and their effect on engineering parameterizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures, that predictably turn on and turn off EET flux, respectively, in response to IPTG. Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs. inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

scholarly journals Phylogenetic and structural diversity of aromatically dense pili from environmental metagenomes

2019 ◽  
Author(s):  
M. S. Bray ◽  
J. Wu ◽  
C.C. Padilla ◽  
F. J. Stewart ◽  
D. A. Fowle ◽  
...  
Keyword(s):  
Amino Acids ◽  
Electron Transfer ◽  
Microbial Respiration ◽  
Aromatic Amino Acids ◽  
Structural Diversity ◽  
Type Iv Pili ◽  
Extracellular Electron Transfer ◽  
Environmental Distribution ◽  
Type Iv ◽  
Genes Encoding

SummaryElectroactive type IV pili, or e-pili, are used by some microbial species for extracellular electron transfer. Recent studies suggest that e-pili may be more phylogenetically and structurally diverse than previously assumed. Here, we used updated aromatic density thresholds (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps, and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57) to search for putative e-pilin genes in metagenomes from diverse ecosystems with active microbial metal cycling. Environmental putative e-pilins were diverse in length and phylogeny, and included truncated e-pilins inGeobacterspp., as well as longer putative e-pilins in Fe(II)-oxidizingBetaproteobacteriaandZetaproteobacteria.Originality and SignificanceElectroactive pili (e-pili) are used by microorganisms to respire solid metals in their environment through extracellular electron transfer. Thus, e-pili enable microbes to occupy specific environmental niches. Additionally, e-pili have important potential for biotechnological applications. Currently the repertoire of known e-pili is small, and their environmental distribution is largely unknown. Using sequence analysis, we identified numerous genes encoding putative e-pili from diverse anoxic, metal-rich ecosystems. Our results expand the diversity of putative e-pili in environments where metal oxides may be important electron acceptors for microbial respiration.

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

2016 ◽  
Vol 82 (17) ◽  
pp. 5428-5443 ◽  
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.

scholarly journals Methane-linked mechanisms of electron uptake from cathodes byMethanosarcina barkeri

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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