Rerouting Cellular Electron Flux To Increase the Rate of Biological Methane Production

ABSTRACTMethanogens are anaerobic archaea that grow by producing methane, a gas that is both an efficient renewable fuel and a potent greenhouse gas. We observed that overexpression of the cytoplasmic heterodisulfide reductase enzyme HdrABC increased the rate of methane production from methanol by 30% without affecting the growth rate relative to the parent strain. Hdr enzymes are essential in all known methane-producing archaea. They function as the terminal oxidases in the methanogen electron transport system by reducing the coenzyme M (2-mercaptoethane sulfonate) and coenzyme B (7-mercaptoheptanoylthreonine sulfonate) heterodisulfide, CoM-S-S-CoB, to regenerate the thiol-coenzymes for reuse. InMethanosarcina acetivorans, HdrABC expression caused an increased rate of methanogenesis and a decrease in metabolic efficiency on methylotrophic substrates. When acetate was the sole carbon and energy source, neither deletion nor overexpression of HdrABC had an effect on growth or methane production rates. These results suggest that in cells grown on methylated substrates, the cell compensates for energy losses due to expression of HdrABC with an increased rate of substrate turnover and that HdrABC lacks the appropriate electron donor in acetate-grown cells.

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Physiological Evidence for Isopotential Tunneling in the Electron Transport Chain of Methane-Producing Archaea

Applied and Environmental Microbiology ◽

10.1128/aem.00950-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 5

Author(s):

Nikolas Duszenko ◽

Nicole R. Buan

Keyword(s):

Escherichia Coli ◽

Electron Transport ◽

Stationary Phase ◽

Exponential Growth ◽

Electron Transport Chain ◽

Methanosarcina Barkeri ◽

Metabolic Efficiency ◽

Methanosarcina Acetivorans ◽

Content Type ◽

Transport Chain

ABSTRACT Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri. The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans. Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh. IMPORTANCE Can we grow more from less? The ability to optimize and manipulate metabolic efficiency in cells is the difference between commercially viable and nonviable renewable technologies. Much can be learned from methane-producing archaea (methanogens) which evolved a successful metabolic lifestyle under extreme thermodynamic constraints. Methanogens use highly efficient electron transport systems and supramolecular complexes to optimize electron and carbon flow to control biomass synthesis and the production of methane. Worldwide, methanogens are used to generate renewable methane for heat, electricity, and transportation. Our observations suggest Methanosarcina acetivorans, but not Methanosarcina barkeri, has electrically quantized membranes. Escherichia coli, a model facultative anaerobe, has optimal electron transport at the stationary phase but not during exponential growth. This study also suggests the metabolic efficiency of bacteria and archaea can be improved using exogenously supplied lipophilic electron carriers. The enhancement of methanogen electron transport through methanophenazine has the potential to increase renewable methane production at an industrial scale.

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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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Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria inIn VitroRuminal Cultures

Applied and Environmental Microbiology ◽

10.1128/aem.02779-10 ◽

2011 ◽

Vol 77 (8) ◽

pp. 2634-2639 ◽

Cited By ~ 82

Author(s):

Zhenming Zhou ◽

Qingxiang Meng ◽

Zhongtang Yu

Keyword(s):

Methane Production ◽

Volatile Fatty Acids ◽

Denaturing Gradient Gel Electrophoresis ◽

Cellulolytic Bacteria ◽

Bacterial Populations ◽

Content Type ◽

Specific Pcr ◽

Methanogen Population ◽

Propynoic Acid

ABSTRACTThe objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens usingin vitrocultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyltrans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce theFibrobacter succinogenespopulation. SN reduced theRuminococcus albuspopulation, while PA and 2NEOH increased the populations of bothR. albusandRuminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide futureinvivostudies to develop effective mitigation of methane emission from ruminants.

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Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A

Journal of Bacteriology ◽

10.1128/jb.00947-09 ◽

2009 ◽

Vol 191 (22) ◽

pp. 6928-6935 ◽

Cited By ~ 13

Author(s):

Rina B. Opulencia ◽

Arpita Bose ◽

William W. Metcalf

Keyword(s):

Methane Production ◽

Posttranscriptional Regulation ◽

Promoter Strength ◽

Methanosarcina Acetivorans ◽

Vitro Assay ◽

Phenotypic Differences ◽

Fusion Data ◽

Encoding Genes ◽

Similar Growth

ABSTRACT Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate.

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Methanogens and Methanogenesis in the Rumens and Ceca of Lambs Fed Two Different High-Grain-Content Diets

Applied and Environmental Microbiology ◽

10.1128/aem.03115-12 ◽

2012 ◽

Vol 79 (6) ◽

pp. 1777-1786 ◽

Cited By ~ 35

Author(s):

M. Popova ◽

D. P. Morgavi ◽

C. Martin

Keyword(s):

Metabolic Activity ◽

Methane Production ◽

Direct Evidence ◽

Species Level ◽

Ex Situ ◽

Content Type ◽

Copy Numbers ◽

Dietary Starch ◽

Induced Changes

ABSTRACTThe amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant's digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane productionin vivoandex situwas also monitored.In vivodaily methane emissions (CH4g/day) were 36% (P< 0.05) lower in corn-fed lambs than in wheat-fed lambs.Ex situmethane production (μmol/h) was 4-fold higher for ruminal contents than for cecal contents (P< 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrAcopy numbers;P< 0.01). Clone library analysis indicated thatMethanobrevibacterwas the dominant genus in both sites. Diet induced changes at the species level, as theMethanobrevibacter millerae-M. gottschalkii-M. smithiiclade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affectmcrAexpression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.

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Regulation of the CRISPR-Associated Genes by Rv2837c (CnpB) via an Orn-Like Activity in Tuberculosis Complex Mycobacteria

Journal of Bacteriology ◽

10.1128/jb.00743-17 ◽

2018 ◽

Vol 200 (8) ◽

Cited By ~ 5

Author(s):

Yang Zhang ◽

Jun Yang ◽

Guangchun Bai

Keyword(s):

Transcriptional Regulation ◽

Adaptive Immunity ◽

Parent Strain ◽

Tuberculosis Complex ◽

Content Type ◽

Multifunctional Protein ◽

Type Iii ◽

Associated Proteins ◽

Cyclic Dinucleotides ◽

Insight Into

ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins (Cas) provide bacteria and archaea with adaptive immunity to specific DNA invaders. Mycobacterium tuberculosis encodes a type III CRISPR-Cas system that has not been experimentally explored. In this study, we found that the CRISPR-Cas systems of both M. tuberculosis and Mycobacterium bovis BCG were highly upregulated by deletion of Rv2837c ( cnpB ), which encodes a multifunctional protein that hydrolyzes cyclic di-AMP (c-di-AMP), cyclic di-GMP (c-di-GMP), and nanoRNAs (short oligonucleotides of 5 or fewer residues). By using genetic and biochemical approaches, we demonstrated that the CnpB-controlled transcriptional regulation of the CRISPR-Cas system is mediated by an Orn-like activity rather than by hydrolyzing the cyclic dinucleotides. Additionally, our results revealed that tuberculosis (TB) complex mycobacteria are functional in processing CRISPR RNAs (crRNAs), which are also more abundant in the Δ cnpB strain than in the parent strain. The elevated crRNA levels in the Δ cnpB strain could be partially reduced by expressing Escherichia coli orn . Our findings provide new insight into transcriptional regulation of bacterial CRISPR-Cas systems. IMPORTANCE Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins (Cas) provide adaptive immunity to specific DNA invaders. M. tuberculosis encodes a type III CRISPR-Cas system that has not been experimentally explored. In this study, we first demonstrated that the CRISPR-Cas systems in tuberculosis (TB) complex mycobacteria are functional in processing CRISPR RNAs (crRNAs). We also showed that Rv2837c (CnpB) controls the expression of the CRISPR-Cas systems in TB complex mycobacteria through an oligoribonuclease (Orn)-like activity, which is very likely mediated by nanoRNA. Since little is known about regulation of CRISPR-Cas systems, our findings provide new insight into transcriptional regulation of bacterial CRISPR-Cas systems.

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Genetic Analysis of the Methanol- and Methylamine-Specific Methyltransferase 2 Genes of Methanosarcina acetivorans C2A

Journal of Bacteriology ◽

10.1128/jb.00117-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4017-4026 ◽

Cited By ~ 29

Author(s):

Arpita Bose ◽

Matthew A. Pritchett ◽

William W. Metcalf

Keyword(s):

Genetic Analysis ◽

Methane Production ◽

Untranslated Region ◽

Gene Fusions ◽

Methanosarcina Acetivorans ◽

Concerted Effort ◽

Genes Encoding ◽

Low Levels ◽

Reduced Rate

ABSTRACT The entry of methanol into the methylotrophic pathway of methanogenesis is mediated by the concerted effort of two methyltransferases, namely, methyltransferase 1 (MT1) and methyltransferase 2 (MT2). The mtaA1, mtaA2, and mtbA genes of Methanosarcina acetivorans C2A encode putative methanol- or methylamine-specific MT2 enzymes. To address the in vivo roles of these genes in growth and methanogenesis from known substrates, we constructed and characterized mutants with deletions of each of these genes. The mtaA1 gene is required for growth on methanol, whereas mtaA2 was dispensable. However, the mtaA2 mutant had a reduced rate of methane production from methanol. Surprisingly, deletion of mtaA1 in combination with deletions of the genes encoding three methanol-specific MT1 isozymes led to lack of growth on acetate, suggesting that MT1 and MT2 enzymes might play an important role during growth on this substrate. The mtbA gene was required for dimethylamine and monomethylamine (MMA) utilization and was important, but not required, for trimethylamine utilization. Analysis of reporter gene fusions revealed that both mtaA1 and mtbA were expressed on all methanogenic substrates tested. However, mtaA1 expression was induced on methanol, while mtbA expression was down-regulated on MMA and acetate. mtaA2 was expressed at very low levels on all substrates. The mtaA1 transcript had a large 5′ untranslated region (UTR) (275 bp), while the 5′ UTR of the mtbA transcript was only 28 bp long.

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Distinct Physiological Roles of the Three Ferredoxins Encoded in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽

10.1128/mbio.02807-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 7

Author(s):

Brett W. Burkhart ◽

Hallie P. Febvre ◽

Thomas J. Santangelo

Keyword(s):

Electron Flux ◽

Electron Flow ◽

Physiological Role ◽

High Energy ◽

Electron Acceptors ◽

Content Type ◽

Maximal Energy ◽

Thermococcus Kodakarensis ◽

Small Proteins ◽

Energy Gains

ABSTRACT Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins—an abundant class of Fe-S-containing small proteins—are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis. We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology. IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.

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Methane-Linked Mechanisms of Electron Uptake from Cathodes byMethanosarcina barkeri

mBio ◽

10.1128/mbio.02448-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 17

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.

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A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

Applied and Environmental Microbiology ◽

10.1128/aem.01402-20 ◽

2020 ◽

Vol 86 (21) ◽

Cited By ~ 1

Author(s):

Ahmed E. Dhamad ◽

Daniel J. Lessner

Keyword(s):

Nitrogen Fixation ◽

Target Genes ◽

Methanogenic Archaea ◽

Transcript Abundance ◽

Gene Repression ◽

Methanosarcina Acetivorans ◽

Content Type ◽

Crispr Interference ◽

Cellular Processes ◽

Guide Rnas

ABSTRACT CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD. Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2. Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans. IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.

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