Methanosarcina Acetivorans Research Articles

Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review.

Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0′ = −420 mV) with NADH (−320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (−10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms—in which they have been found—are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered.

Enzymatic reconstitution of ribosomal peptide backbone thioamidation

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.

Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.

Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO2, C1 compounds (like methanol or methylated amines), or acetate as terminal electron acceptors. Methanogens are an untapped resource for biotechnological advances related to methane production as well as methane consumption. However, key biological features of these organisms remain poorly understood. One such feature is the enzyme methyl-coenzyme M reductase (referred to as MCR), which catalyzes the last step in the methanogenic pathway and results in methane formation. Gene essentiality has limited genetic analyses of MCR thus far. Therefore, studies of this important enzyme have been limited to biochemical and biophysical techniques that are especially laborious and often reliant on sophisticated instrumentation that is not commonly available. In this chapter, we outline our recently developed CRISPR-Cas9-based genome editing tools and describe how these tools have been used for the introduction of a tandem affinity purification tag at the chromosomal mcr locus in the model methanogen, Methanosarcina acetivorans C2A. We also report a protocol for rapid affinity purification of MCR from M. acetivorans C2A that will enable high-throughput studies of this enzyme in the future.

Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea.

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.

Computational study of the binding mechanism of medium chain acyl-CoA synthetase with substrate in Methanosarcina acetivorans

The acyl-AMP forming family of adenylating enzymes catalyzes the formation of acyl-CoA from an acyl substrate, ATP, and CoA, which is a metabolite of many catabolic and anabolic processes. The medium-chain acyl-CoA synthetase from Methanosarcina acetivorans, designated MacsMa, uses 2-methylbutyrate as its preferred substrate. It is reported that the interaction between the sidechain of Cys298 and Lys256 of this enzyme is important for the catalytic activity. The mutation of these residues resulted in the changes of the structure stability and the reduced or absence catalytic activity. In the present study, the binding mechanism between the substrate 2-methylbutyrate- AMP (2MeBA) and MacsMa were explored by integrating multiple computational methods including molecular docking, molecular dynamics simulations, binding free energy calculation, active site access channel analysis and principal component analysis. The binding free energy between WT, mutated Macs and substrate was calculated by MM-GBSA method, which indicated that the binding affinity between this enzyme and substrate was stronger in the WT than that in the mutated forms (K256L, K256T and C298Y). Per-residue binding free energy decomposition identified some residues, such as Gly327, Phe350, Gly351, Gln352 and Lys461, which are important for the enzyme and substrate binding affinity. The access channels of the mutant system (MacsK256L, MacsK256T and MacsC298Y) were found to be different from those in the wild-type systems. It suggested that K256L and C298Y induced larger flexibility to the overall protein compared with the WT, whereas K256T induced larger flexibility to the partial protein compared with the WT by PCA vector porcupines. This study provides novel insight to understand the substrate binding mechanism of Macs and useful information for the rational enzyme design.

Aged-engineered nanoparticles effect on sludge anaerobic digestion performance and associated microbial communities

To investigate the potential effect of aged engineered nanoparticles (a-ENPs) on sludge digestion performance, 150L pilot anaerobic digesters (AD) were fed with a blend of primary and waste activated sludge spiked either with a mixture of silver oxide, titanium dioxide and zinc oxide or a mixture of their equivalent bulk metal salts to achieve a target concentration of 250, 2000, and 2800mgkg−1 dry weight, respectively. Volatile fatty acids (VFA) were 1.2 times higher in the spiked digesters and significantly different (p=0.05) from the control conditions. Specifically, isovaleric acid concentration was 2 times lower in the control digester compared to the spiked digesters, whereas hydrogen sulfide was 2 times lower in the ENPs spiked digester indicating inhibitory effect on sulfate reducing microorganisms. Based on the ether-linked isoprenoids concentration, the total abundance of methanogens was 1.4 times lower in the ENPs spiked digester than in the control and metal salt spiked digesters. Pyrosequencing indicated 80% decrease in abundance and diversity of methanogens in ENPs spiked digester compared to the control digester. Methanosarcina acetivorans and Methanosarcina barkeri were identified as nano-tolerant as their relative abundance increased by a factor of 6 and 11, respectively, compared to the other digesters. The results further provide compelling evidence on the resilience of Fusobacteria, Actinobacteria and the Trojan horse-like effect of ENPs which offered a competitive advantage to some organisms while reducing microbial abundance and diversity.

Effect of Activated Carbon Addition on the Anaerobic Fermentation of Corn Straw in Mesophilic and Thermophilic Conditions

In order to improve the methane production and concentration, effect of activated carbon addition on the anaerobic fermentation of corn straw under the conditions of mesophilic temperature (38℃) and thermophilic temperature(50℃) was investigated in this study. The results showed that the addition of activated carbon could significantly promote methane production. Compared with the control group in mesophilic and thermophilic conditions, cumulative methane production could be increased by 63% and 96% in test groups. By DGGE analysis, the bacterium enriched by addition of activated carbon was mainly Clostridiales bacterium, compared to Bacillus (without adding activated carbon) in the thermophilic system, while the differences in fermentation with adding activated carbon and without adding activated carbon was not significant in the mesophilic system. With addition of activated carbon, the archaea enriched in the fermentation liquid was mainly Methanosaeta concilii in the mesophilic system, whereas the archaea enriched in the fermentation liquid was mainly Methanosarcina acetivorans in the thermophilic system. The archaea enriched on activated carbon was mainly Methanosaeta concilii at mesophilic temperature, while the archaea enriched on activated carbon was mainly Methanosarcina thermophila at thermophilic temperature.

Unique crystal structures of an SmtB/ArsR transcriptional factor from Methanosarcina acetivorans

Unique crystal structures of an SmtB/ArsR transcriptional factor from <i>Methanosarcina acetivorans</i>

Trace Metal Imaging of Sulfate-Reducing Bacteria and Methanogenic Archaea at Single-Cell Resolution by Synchrotron X-Ray Fluorescence Imaging

ABSTRACTMetal cofactors are required for many enzymes in anaerobic microbial respiration. This study examined iron, cobalt, nickel, copper, and zinc in cellular and abiotic phases at the single-cell scale for a sulfate-reducing bacterium (Desulfococcus multivorans) and a methanogenic archaeon (Methanosarcina acetivorans) using synchrotron X-ray fluorescence microscopy. Relative abundances of cellular metals were also measured by inductively coupled plasma mass spectrometry. For both species, zinc and iron were consistently the most abundant cellular metals. M. acetivorans contained higher nickel and cobalt content than D. multivorans, likely due to elevated metal requirements for methylotrophic methanogenesis. Cocultures contained spheroid zinc sulfides and cobalt/copper sulfides.

Finally, Archaea Get Their CRISPR-Cas Toolbox

The majority of archaea encode CRISPR-Cas systems but only a few CRISPR-Cas-based genetic tools have been developed for organisms from this domain. Nayak and Metcalf have harnessed a bacterial Cas9 protein for genome editing in Methanosarcina acetivorans, enabling efficient gene deletion and replacement.

Phylogenomic proximity and metabolic discrepancy of Methanosarcina mazei Go1 across methanosarcinal genomes

Methanosarcina mazei Go1 is a heterotrophic methanogenic archaean contributing a significant role in global methane cycling and biomethanation process. Phylogenomic relatedness and metabolic discrepancy of this genome were described herein by comparing its whole genome sequence, intergenomic distance, genome function, synteny homologs and origin of replication, and marker genes with very closely related genomes, Methanosarcina acetivorans and Methanosarcina barkeri. Phylogenomic analysis of this study revealed that genome functional feature and metabolic core of M. mazei and M. barkeri could be originated from M. acetivorans. The metabolic core of these genomes shares a common evolutionary origin to perform the metabolic activity at different environmental niches. Genome expansion, dynamics and gene collinearity were constrained and restrained the conservation of the metabolic core genes by duplication events occurring across methanosarcinal genomes. The Darwinian positive selection was an evolutionary constraint to purify the function of core metabolic genes. Using genome-wide metabolic survey, we found the existence of four novel putative metabolic pathways such as complete methanogenesis from acetate, indole-3-acetate biosynthesis V, 4-aminobutyrate degradation III, galactosamine biosynthesis I and siroheme biosynthesis. Overall, the present study would provide a stand point to revisit its phylogenomic status in order to understand the origin and evolution history of this organism.

Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans

Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methanogenic archaeon Methanosarcina acetivorans Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the nonhomologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.

Persulfide Formation Mediates Cysteine and Homocysteine Biosynthesis in Methanosarcina acetivorans.

The mechanisms of sulfur uptake and trafficking in methanogens inhabiting sulfidic environments are highly distinctive. In aerobes, sulfur transfers between proteins occur via persulfide relay, but direct evidence for persulfides in methanogens has been lacking. Here, we use mass spectrometry to analyze tryptic peptides of the Methanosarcina acetivorans SepCysS and MA1821 proteins purified anaerobically from methanogen cells. These enzymes insert sulfide into phosphoseryl(Sep)-tRNACys and aspartate semialdehyde, respectively, to form Cys-tRNACys and homocysteine. A high frequency of persulfidation at conserved cysteines of each protein was identified, while the substantial presence of persulfides in peptides from other cellular proteins suggests that this modification plays a general physiological role in the organism. Purified native SepCysS containing persulfide at conserved Cys260 generates Cys-tRNACys in anaerobic single-turnover reactions without exogenously added sulfur, directly linking active-site persulfide formation in vivo with catalytic activity.

A Transition Metal-Binding, Trimeric βγ-Crystallin from Methane-Producing Thermophilic Archaea, Methanosaeta thermophila.

βγ-Crystallins are important constituents of the vertebrate eye lens, whereas in microbes, they are prevalent as Ca2+-binding proteins. In archaea, βγ-crystallins are conspicuously confined to two methanogens, viz., Methanosaeta and Methanosarcina. One of these, i.e., M-crystallin from Methanosarcina acetivorans, has been shown to be a typical Ca2+-binding βγ-crystallin. Here, with the aid of a high-resolution crystal structure and isothermal titration calorimetry, we report that "Methallin", a βγ-crystallin from Methanosaeta thermophila, is a trimeric, transition metal-binding protein. It binds Fe, Ni, Co, or Zn ion with nanomolar affinity, which is consistent even at 55 °C, the optimal temperature for the methanogen's growth. At the center of the protein trimer, the metal ion is coordinated by six histidines, two from each protomer, leading to an octahedral geometry. Small-angle X-ray scattering analysis confirms that the trimer seen in the crystal lattice is a biological assembly; this assembly dissociates to monomers upon removal of the metal ion. The introduction of two histidines (S17H/S19H) into a homologous βγ-crystallin, Clostrillin, allows it to bind nickel at the introduced site, though with micromolar affinity. However, because of the lack of a compatible interface, nickel binding could not induce trimerization, affirming that Methallin is a naturally occurring trimer for high-affinity transition metal binding. While βγ-crystallins are known to bind Ca2+ and form homodimers and oligomers, the transition metal-binding, trimeric Methallin is a new paradigm for βγ-crystallins. The distinct features of Methallin, such as nickel or iron binding, are also possible imprints of biogeochemical changes during the period of its origin.

Methanosarcina acetivorans utilizes a single NADPH-dependent thioredoxin system and contains additional thioredoxin homologues with distinct functions

The thioredoxin system plays a central role in the intracellular redox maintenance in the majority of cells. The canonical system consists of an NADPH-dependent thioredoxin reductase (TrxR) and thioredoxin (Trx), a disulfide reductase. Although Trx is encoded in almost all sequenced genomes of methanogens, its incorporation into their unique physiology is not well understood. Methanosarcina acetivorans contains a single TrxR (MaTrxR) and seven Trx (MaTrx1-MaTrx7) homologues. We previously showed that MaTrxR and at least MaTrx7 compose a functional NADPH-dependent thioredoxin system. Here, we report the characterization of all seven recombinant MaTrxs. MaTrx1, MaTrx3, MaTrx4 and MaTrx5 lack appreciable disulfide reductase activity, unlike previously characterized MaTrx2, MaTrx6 and MaTrx7. Enzyme assays demonstrated that, of the MaTrxs, only the reduction of disulfide-containing MaTrx7 is linked to the oxidation of reduced coenzymes. NADPH is shown to be supplied to the MaTrxR-MaTrx7 system through the oxidation of the primary methanogen electron carriers F420H2 and ferredoxin, indicating that it serves as a primary intracellular reducing system in M. acetivorans. Bioinformatic analyses also indicate that the majority of methanogens likely utilize an NADPH-dependent thioredoxin system. The remaining MaTrxs may have specialized functions. MaTrx1 and MaTrx3 exhibited thiol oxidase activity. MaTrx3 and MaTrx6 are targeted to the membrane of M. acetivorans and likely function in the formation and the reduction of disulfides in membrane and/or extracellular proteins, respectively. This work provides insight into the incorporation of Trx into the metabolism of methanogens, and this reveals that methanogens contain Trx homologues with alternative properties and activities.

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans.

BackgroundWhile a few studies on the variations in mRNA expression and half-lives measured under different growth conditions have been used to predict patterns of regulation in bacterial organisms, the extent to which this information can also play a role in defining metabolic phenotypes has yet to be examined systematically. Here we present the first comprehensive study for a model methanogen.ResultsWe use expression and half-life data for the methanogen Methanosarcina acetivorans growing on fast- and slow-growth substrates to examine the regulation of its genes. Unlike Escherichia coli where only small shifts in half-lives were observed, we found that most mRNA have significantly longer half-lives for slow growth on acetate compared to fast growth on methanol or trimethylamine. Interestingly, half-life shifts are not uniform across functional classes of enzymes, suggesting the existence of a selective stabilization mechanism for mRNAs. Using the transcriptomics data we determined whether transcription or degradation rate controls the change in transcript abundance. Degradation was found to control abundance for about half of the metabolic genes underscoring its role in regulating metabolism. Genes involved in half of the metabolic reactions were found to be differentially expressed among the substrates suggesting the existence of drastically different metabolic phenotypes that extend beyond just the methanogenesis pathways. By integrating expression data with an updated metabolic model of the organism (iST807) significant differences in pathway flux and production of metabolites were predicted for the three growth substrates.ConclusionsThis study provides the first global picture of differential expression and half-lives for a class II methanogen, as well as provides the first evidence in a single organism that drastic genome-wide shifts in RNA half-lives can be modulated by growth substrate. We determined which genes in each metabolic pathway control the flux and classified them as regulated by transcription (e.g. transcription factor) or degradation (e.g. post-transcriptional modification). We found that more than half of genes in metabolism were controlled by degradation. Our results suggest that M. acetivorans employs extensive post-transcriptional regulation to optimize key metabolic steps, and more generally that degradation could play a much greater role in optimizing an organism’s metabolism than previously thought.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3219-8) contains supplementary material, which is available to authorized users.

Metabolic engineering of Methanosarcina acetivorans for lactate production from methane.

We previously demonstrated anaerobic conversion of the greenhouse gas methane into acetate using an engineered archaeon that produces methyl-coenzyme M reductase (Mcr) from unculturable microorganisms from a microbial mat in the Black Sea to create the first culturable prokaryote that reverses methanogenesis and grows anaerobically on methane. In this work, we further engineered the same host with the goal of converting methane into butanol. Instead, we discovered a process for converting methane to a secreted valuable product, L-lactate, with sufficient optical purity for synthesizing the biodegradable plastic poly-lactic acid. We determined that the 3-hydroxybutyryl-CoA dehydrogenase (Hbd) from Clostridium acetobutylicum is responsible for lactate production. This work demonstrates the first metabolic engineering of a methanogen with a synthetic pathway; in effect, we produce a novel product (lactate) from a novel substrate (methane) by cloning the three genes for Mcr and one for Hbd. We further demonstrate the utility of anaerobic methane conversion with an increased lactate yield compared to aerobic methane conversion to lactate. Biotechnol. Bioeng. 2017;114: 852-861. © 2016 Wiley Periodicals, Inc.

The biosynthetic pathway of coenzyme F430 in methanogenic and methanotrophic archaea.

Methyl-coenzyme M reductase (MCR) is the key enzyme of methanogenesis and anaerobic methane oxidation. The activity of MCR is dependent on the unique nickel-containing tetrapyrrole known as coenzyme F430. We used comparative genomics to identify the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes from Methanosarcina acetivorans C2A. The pathway involves nickelochelation by a nickel-specific chelatase, followed by amidation to form Ni-sirohydrochlorin a,c-diamide. Next, a primitive homolog of nitrogenase mediates a six-electron reduction and γ-lactamization reaction before a Mur ligase homolog forms the six-membered carbocyclic ring in the final step of the pathway. These data show that coenzyme F430 can be synthesized from sirohydrochlorin using Cfb enzymes produced heterologously in a nonmethanogen host and identify several targets for inhibitors of biological methane formation.

High-throughput mutation, selection, and phenotype screening of mutant methanogenic archaea

Bacterial and archaeal genomes can contain 30% or more hypothetical genes with no predicted function. Phylogenetically deep-branching microbes, such as methane-producing archaea (methanogens), contain up to 50% genes with unknown function. In order to formulate hypotheses about the function of hypothetical gene functions in the strict anaerobe, Methanosarcina acetivorans, we have developed high-throughput anaerobic techniques to UV mutagenize, screen, and select for mutant strains in 96-well plates. Using these approaches we have isolated 10 mutant strains that exhibit a variety of physiological changes including increased or decreased growth rate relative to the parent strain when cells use methanol and/or acetate as carbon and energy sources. This method provides an avenue for the first step in identifying new gene functions: associating a genetic mutation with a reproducible phenotype. Mutations in bona fide methanogenesis genes such as corrinoid methyltransferases and proton-translocating F420H2:methanophenazine oxidoreductase (Fpo) were also generated, opening the door to in vivo functional complementation experiments. Irradiation-based mutagenesis such as from ultraviolet (UV) light, combined with modern genome sequencing, is a useful procedure to discern systems-level gene function in prokaryote taxa that can be axenically cultured but which may be resistant to chemical mutagens.

The [4Fe-4S] clusters of Rpo3 are key determinants in the post Rpo3/Rpo11 heterodimer formation of RNA polymerase in Methanosarcina acetivorans.

Subunits Rpo3 and Rpb3/AC40 of RNA polymerase (RNAP) from many archaea and some eukaryotes, respectively, contain a ferredoxin‐like domain (FLD) predicted to bind one or two [4Fe‐4S] clusters postulated to play a role in regulating the assembly of RNAP. To test this hypothesis, the two [4Fe‐4S] cluster Rpo3 from Methanosarcina acetivorans was modified to generate variants that lack the FLD or each [4Fe‐4S] cluster. Viability of gene replacement mutants revealed that neither the FLD nor the ability of the FLD to bind either [4Fe‐4S] cluster is essential. Nevertheless, each mutant demonstrated impaired growth due to significantly lower RNAP activity when compared to wild type. Affinity purification of tagged Rpo3 variants from M. acetivorans strains revealed that neither the FLD nor each [4Fe‐4S] cluster is required for the formation of a Rpo3/11 heterodimer, the first step in the assembly of RNAP. However, the association of the Rpo3/11 heterodimer with catalytic subunits Rpo2′ and Rpo1″ was diminished by the removal of the FLD and each cluster, with the loss of cluster 1 having a more substantial effect than the loss of cluster 2. These results reveal that the FLD and [4Fe‐4S] clusters, particularly cluster 1, are key determinants in the post Rpo3/11 heterodimer assembly of RNAP in M. acetivorans.