Biology subjects

Nayak, D. D.

Publications and source records attributed to Nayak, D. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Methanol-specific methyltransferase isozymes have large carbon kinetic isotope effects that impact methane isotopic signatures

The stable hydrogen and carbon isotopic composition of methane is widely used to determine its sources. Methanogenic growth on methanol generates methane with significantly lower 13C/12C ratios relative to other substrates, which is often used as a marker for this metabolism in environmental samples. The biochemical basis for the unusual isotope effect associated with methanol growth is currently unknown. Here, we grew Methanosarcina acetivorans on methanol and measured the change in the carbon and hydrogen stable isotopic compositions of the methane. We coupled these results with an inverse modeling approach to calculate the kinetic isotopic effects (KIEs) of the rate-limiting step, catalyzed by the methanol-specific methyltransferase complex (MTA). Through this process, we estimate the carbon KIE of MTA (13{varepsilon}MTA) as -65.5 {per thousand} and the hydrogen KIE of MTA (2{varepsilon}MTA) as -56 {per thousand}. Next, we show that the 13{varepsilon}MTA contributes substantially to the large isotopic effect observed for methylotrophic methanogenesis on methanol. We also show that mutant strains that express only a single copy of the MTA complex (either MtaC1B1A1, MtaC2B2A1, or MtaC3B3A1) have 13{varepsilon}MTA and 2{varepsilon}MTA that are indistinguishable from the wild-type strain. Finally, based on a thermodynamic analysis, we propose that methanol activation by MTA will remain rate-limiting, even at low environmental methanol concentrations, and the large 13{varepsilon}MTA would be expressed in situ as well. ImportanceMethane is a potent greenhouse gas, and distinguishing between its biological sources is vital for modeling global carbon cycles. Methylotrophic methanogenesis produces methane with a uniquely depleted carbon isotope signature. However, the biochemical mechanisms driving this fractionation have remained unclear. In this study, we identify the methanol-specific methyltransferase (MTA) complex as the primary driver of these large carbon isotope effects. By utilizing Methanosarcina acetivorans mutants, we demonstrate that these effects are consistent across different MTA isozymes. Our results suggest these signatures are intrinsic to the enzyme complex and persist at low substrate concentrations. These findings provide a critical biochemical foundation for using stable isotopes to track microbial methane production in diverse natural ecosystems.

microbiology↗

bioRxiv2026

Component A2 is a redox-sensitive archaeal ATPase activated by methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) is the primary source of biogenic methane on Earth. In the active site of MCR, a nickel (Ni)-containing porphyrin (F430) must be in the Ni1+ oxidation state to initiate catalysis. The reductive activation of MCR, i.e., reduction of F430 to its Ni1+ state, is an ATP-dependent process, but the underlying ATPase and its precise role remain unknown. Component A2 is an ATP-binding protein that associates with MCR but, since it was reported to lack ATPase activity, its putative function was designated as an ATP-carrier protein. In contrast, recent structural insights into the MCR activation complex suggest that component A2 might hydrolyze ATP to drive conformational changes required for enzyme activation. Here, we provide direct biochemical evidence that component A2 is a bona fide ATPase that hydrolyzes ATP under strictly anaerobic conditions and only upon interaction with MCR. Mutational analyses reveal that component A2 must be bound to ATP prior to association with MCR and that residues involved in ATP hydrolysis do not impact protein-protein interaction. The two nucleotide-binding domains of A2 act cooperatively but display asymmetric contributions to ATP hydrolysis and MCR engagement. In addition, a distinctive N-terminal zinc-binding motif (ZBM) is required for maximal ATPase activity but is dispensable for MCR binding. Phylogenetic analyses reveal that this ZBM distinguishes component A2 from related ABC-type ATPases. Together, these findings identify component A2 as a distinct class of remodeling ATPases that powers conformational changes underlying the reductive activation of MCR. Significance StatementA large fraction of methane on Earth is generated by methanogenic archaea using the enzyme methyl coenzyme-M reductase (MCR). The maturation of MCR is a multi-step ATP-dependent process but the role of ATP and the corresponding ATPase(s) have remained unclear. Here, we show that component A2, a protein that is universally conserved in archaea that encode MCR and related enzymes, hydrolyzes ATP only upon interaction with MCR under anaerobic conditions. Our findings, together with recent structural studies, indicate that component A2-mediated ATP hydrolysis facilitates the reductive activation of MCR during the final step of its maturation. These results clarify a key step in the biogenesis of a central enzyme involved in biological methane production.

microbiology↗

bioRxiv2026

A family of archaeal hibernation factors that bind in tandem and protect ribosomes in dormant cells

Under nutrient limitation or stress, ribosome hibernation factors inactivate and protect ribosomes. Although ribosome hibernation plays an important role in microbes, we lack a complete understanding of this process in archaea. Here, we identify a family of hibernation factors, which we designate as single ribosomal subunit inhibitors (SriA-SriD), from the methanogenic archaeon Methanosarcina acetivorans. All four sri genes are encoded in an operon and each Sri protein inhibits protein synthesis in vitro. Deletion of sri genes in M. acetivorans impaired growth recovery after prolonged stationary phase and also led to depletion of the small ribosomal subunit. Cryo-EM structures show that Sri proteins bind to the ribosome in tandem and form conserved protein-protein interfaces. Sri is broadly distributed across archaeal phyla and sri genes frequently co-occur. Together, these findings establish Sri proteins as a distinct group of hibernation factors that protect ribosomes during dormancy and expand our understanding of ribosome hibernation in archaea.

microbiology↗

bioRxiv2025

Histones and nucleoid-associated proteins in Methanosarcina acetivorans mediate recovery from stress and stasis

Many archaea encode both histones and Nucleoid-Associated Proteins (NAPs), which we refer to collectively as archaeal DNA Binding Proteins (DBPs). Whether these DBPs jointly work to compact the genome or have distinct functions remains unknown. Here, we have developed the methanogen, Methanosarcina acetivorans, as a platform to study the function of archaeal DBPs in vivo. M. acetivorans encodes one archaeal histone, hmaA, and two copies of the archaea-specific NAP, mc1 (mc1a & mc1b). We found that each DBP is individually dispensable, but at least one copy of mc1 appears to be required for growth. The growth of the single and double DBP deletion mutants was, by and large, like the parent strain under optimal growth conditions. However, after exposure to stresses or extended periods of incubation in stationary phase, the DBP deletion strains often recovered growth much faster than the parent strain. Conversely, over-expression of DBPs led to a delay in growth recovery that could be abrogated by introducing point mutations in DNA-binding residues. Together, our data suggest that histone and archaea-specific NAPs have partially overlapping roles in M. acetivorans and likely protect the genome after exposure to stress or during prolonged periods of growth stasis. Our findings emphasize that there is no unified function for histones across the tree of life and instead imply that archaeal histones join the ranks of other archaeal NAPs in having strain-specific functions. ImportanceThough it is known that many archaea encode histones in tandem with archaea-specific Nucleoid-Associated Proteins (NAPs), the interplay between these two classes of DNA-Binding Proteins (DBPs) in vivo is not known. Most studies on archaeal DBPs have focused either on archaea known to form histone-based chromatin, or on strains which lack histones and compact their genomes exclusively with archaea-specific NAPs. Our study, therefore, fills an important gap in the literature by characterizing DBPs in a model methanogen, Methanosarcina acetivorans, which encodes both histones and archaea-specific NAPs. While no DBP is essential, at least one copy of MC1 appears to be needed, likely for genome compaction. In addition, mutants lacking DBPs had a growth advantage after being subject to stresses or long periods of growth stasis, indicating that DBPs likely function in genome maintenance when growth has stalled or stopped.

microbiology↗

bioRxiv2025

Identification of radical SAM enzymes responsible for the methylation and desaturation of archaeal lipids and an AttH hydratase mediating hydroxy-GDGT biosynthesis

Lipid biosynthesis in archaea is highly dependent upon radical (S)-adenosyl methionine (rSAM) enzymes. The formation of their membrane-spanning lipids, known as glycerol dibiphytanyl glycerol tetraethers (GDGTs), is catalyzed by the rSAM tetraether synthase and the subsequent cyclization of their lipid tails is performed by the B12-binding rSAM (B12-rSAM) GDGT ring synthase. Further, GDGT tails are cross-linked by the rSAM GMGT synthase and then methylated by the B12-rSAM GMGT methylase. Here, utilizing three archaeal model organisms - Sulfolobus acidocaldarius, Thermococcus kodakarensis, and Methanosarcina acetivorans - we expand this repertoire of rSAM enzymes further, identifying two B12-rSAM GDGT methylases and a novel B12-rSAM GDGT desaturase. We also extend beyond the rSAM enzyme superfamily, identifying an AttH-like hydratase mediating hydroxy-GDGT (OH-GDGT) biosynthesis. Specifically, we identify two GDGT methylases in S. acidocaldarius and Thermococcus aggregans which possess different substrate specificities. While GDGT methylation is generally low in archaea, we observe high levels of lipid methylation in response to penicillin G and hexanoic acid amphiphile exposure, suggesting a role for lipid methylation in response to membrane-destabilizing chemical agents. Utilizing heterologous expression in M. acetivorans and T. kodakarensis, we uncover a novel B12-rSAM enzyme from Candidatus Bathyarchaeota B1_G15 with unusual tetraether desaturase (Ted) activity that seemingly reverses previous saturation by geranylgeranyl reductase, anaerobically forming double bonds in the inert hydrocarbon tails of a GDGT. Finally, we show that a non-rSAM AttH-like hydratase, genomically associated with Ted, is a hydroxy-GDGT synthase (Hgs), putatively hydrating the double bond(s) introduced by Ted to form OH-GDGTs. These results highlight the value of exploring the rSAM enzyme landscape of archaea - revealing diverse and novel rSAM enzyme activities amongst homologous proteins that invoke new paradigms such as reversibility in the archaeal lipid biosynthesis pathway.

microbiology↗

bioRxiv2025

Genus-specific remodeling of carbon and energy metabolism facilitates acetoclastic methanogenesis in Methanosarcina spp. and Methanothrix spp.

Methanogenic archaea (methanogens) are microorganisms that obligately produce methane as a byproduct of their energy metabolism. While most methanogens grow on CO2+H2, isolates of the Genus Methanosarcina and Methanothrix can use acetate as the sole substrate for methanogenesis. Methanogenic growth on acetate, i.e., acetoclastic methanogenesis, is hypothesized to require two distinct genetic modules: one for the activation of acetate to acetyl-CoA and the other for producing a chemiosmotic gradient using electrons derived from ferredoxin. In Methanosarcina spp., the activation of acetate to acetyl-CoA is mediated by acetate kinase (Ack) and phosphotransacetylase (Pta) whereas Methanothrix spp. encode AMP-forming acetyl-CoA synthetases (Acs). The Rhodobacter nitrogen fixation complex (Rnf) or Energy converting hydrogenase (Ech) are critical for energy conservation in Methanosarcina spp. during growth on acetate, and a F420:phenazine oxidoreductase-like complex (Fpo) likely plays an analogous role in Methanothrix spp. Here, we tested the proposed modularity of these pathways to facilitate acetoclastic methanogenesis. First, we surveyed over a hundred genomes within the Class Methanosarcinia to show that the genomic potential for acetoclastic methanogenesis using distinct combinations of modules is widespread. We then used the genetically tractable strain, Methanosarcina acetivorans, to build all modular combinations for acetoclastic methanogenesis. Our results indicate that Acs, while functional, cannot replace Ack+Pta to rescue acetate growth in M. acetivorans. Similarly, the Fpo bioenergetic complex cannot replace Rnf. As such, our work suggests that, in addition to horizontal gene transfer of core catabolic modules, acetoclastic metabolism in methanogens requires changes in energetic modules too. IMPORTANCEA large fraction of biogenic methane is derived from acetate, yet acetoclastic methanogens, i.e., methanogens that grow on acetate, remain poorly characterized due to their slow growth. Two groups of methanogens, Methanosarcina spp. and Methanothrix spp., perform acetoclastic methanogenesis using distinct sets of genes for acetate activation and energy conservation. It is widely hypothesized that these genetic modules from Methanosarcina spp. and Methanothrix spp. are functionally analogous and would thus be interchangeable. To test this hypothesis, we engineered different combinations of modules for acetoclastic growth in Methanosarcina acetivorans. Our results challenge this hypothesized paradigm of modularity, and we posit that other changes to the carbon and electron transfer pathways were crucial for the emergence of acetoclastic methanogenesis.

microbiology↗

bioRxiv2025

The Ambiguous Genetic Code of Methanogenic Archaea that Grow on Methylamines

Natural genetic code expansion is a phenomenon wherein an additional amino acid is encoded by a stop codon. These non-standard amino acids are beneficial as they facilitate novel biochemical reactions. However, code expansion leads to ambiguity at the recoded stop codon, which can either be read through or terminated. Pyrrolysine (Pyl) is encoded by the amber codon (TAG/UAG) and is widespread in archaea, where it is required for methylamine-mediated methanogenesis, an environmentally important metabolism. Mechanisms to conditionally suppress the amber stop codon for Pyl installation during protein synthesis have not been identified. Using the model methanogen, Methanosarcina acetivorans, we demonstrate that Pyl-encoding archaea maintain an ambiguous genetic code wherein UAG encodes dual meaning as stop and Pyl. Our data suggest that expression of Pyl biosynthesis and incorporation genes is tuned to the cellular demand for Pyl, which allows these archaea to navigate ambiguous stop decoding in response to environmental cues.

microbiology↗

bioRxiv2025

An Archaeal Transcription Factor Bridges Prokaryotic and Eukaryotic Regulatory Paradigms

Archaeal transcription is a hybrid of eukaryotic and bacterial features: a RNAP II-like polymerase transcribes genes organized in circular chromosomes within cells devoid of a nucleus. Consequently, archaeal genomes are depleted in canonical transcription factors (TFs) found in other domains of life. Here we outline the discovery of a cryptic archaea-specific family of ligand-binding TFs, called AmzR (Archaeal Metabolite-sensing Zipper-like Regulators). We identify AmzR through an evolution-based genetic screen and show that it is a negative regulator of methanogenic growth on methylamines in the model archaeon, Methanosarcina acetivorans. AmzR binds its target promoters as an oligomer using paired basic -helices akin to eukaryotic leucine zippers. AmzR also binds methylamines, which reduces its DNA-binding affinity, and allows it to function as a one-component system commonly found in prokaryotes, while containing a eukaryotic-like DNA-binding motif. The AmzR family of TFs are widespread in archaea and broadens the scope of innovations at the prokaryote-eukaryote interface.

microbiology↗

bioRxiv2025

Circular 23S rRNA within archaeal ribosomes

The ribosomal RNAs (rRNAs) that form the core of ribosomes are believed to occur as linear molecules. Here, we investigated rRNAs from diverse and mostly uncultivated archaea and found evidence that, in at least eight phylum-level archaeal lineages, the 23S rRNAs within mature ribosomes are circular. Sequencing of total cellular RNA indicates that the transcriptional abundances of archaeal circular 23S rRNAs vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggested that the circular molecules are within the ribosomes. Thus, we directly sequenced RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirmed that the assembled 23S rRNAs are circular. Structural modeling places the 5 and 3 ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their further circularization within ribosomes. We confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups. Overall, the results uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

microbiology↗

bioRxiv2025

Methanogenesis marker 16 metalloprotein is the primary coenzyme M synthase in Methanosarcina acetivorans

2-mercaptoethanesulfonate (Coenzyme M, CoM) is an organic sulfur-containing cofactor used for hydrocarbon metabolism in Archaea and Bacteria. In Archaea, CoM serves as an alkyl group carrier for enzymes belonging to the alkyl-CoM reductase family, including methyl-CoM reductase, which catalyzes methane formation in methanogens. Two pathways for the biosynthesis of CoM are present in methanogenic archaea. The initial steps of these pathways are distinct but the last two reactions, leading up to CoM formation, are universally conserved. The final step is proposed to be mediated by methanogenesis marker metalloprotein 16 (MMP16), a putative sulfurtransferase, that replaces the aldehyde group of sulfoacetaldehyde with a thiol to generate CoM. The assignment of MMP16 as CoM synthase (ComF) is not widely accepted as deletion mutants have been shown to grow without any CoM dependence. Here, we investigate the role of MMP16 in the model methanogen, Methanosarcina acetivorans. We show that a mutant lacking MMP16 has a CoM-dependent growth phenotype and a global transcriptomic profile reflective of CoM-starvation. Additionally, the {Delta}MMP16 mutant is a CoM auxotroph in sulfide-free medium. These data reinforce prior claims that MMP16 is a bona fide ComF but point to backup pathway(s) that can conditionally compensate for its absence. We found that L- aspartate semialdehyde sulfurtransferase (L-ASST), catalyzing a sulfurtransferase reaction during homocysteine biosynthesis in methanogens, is potentially involved in genetic compensation of the MMP16 deletion. Even though, both, L-ASST and MMP16 are members of the COG1900 family, site-directed mutagenesis of conserved cysteine residues implicated in catalysis reveal that the underlying reaction mechanisms may be distinct. Altogether, we have provided concrete evidence that MMP16 is the primary ComF in methanogenic archaea. Author SummaryMethane is a high energy renewable fuel that is the primary constituent of natural gas and a potent greenhouse gas. A significant fraction of global methane emissions is generated by the activity of methanogenic archaea. These microorganisms use a cofactor called Coenzyme M (CoM) as a methyl carrier for methane production mediated by the enzyme Methyl-Coenzyme M reductase. Since methane production is essential for energy conservation in methanogens, they need to synthesize or import CoM. Accordingly, most methanogens encode either one of two CoM biosynthesis pathways. Methanogenesis marker 16 metalloprotein (MMP16) is proposed to catalyze the last step of CoM biosynthesis in both pathways however experimental evidence to this effect is lacking. Here we demonstrate that MMP16 is, indeed, the primary CoM synthase (ComF) in the model methanogen, Methanosarcina acetivorans. Since MMP16 is uniquely and widely distributed in methanogens, it can serve as an ideal candidate for the design of anti- methanogenic chemical inhibitors.

microbiology↗

bioRxiv2024

Structure of an Archaeal Ribosome with a Divergent Active Site

The ribosome is the universal translator of the genetic code and is shared across all life. Despite divergence in ribosome structure over the course of evolution, the peptidyl transferase center (PTC), the catalytic site of the ribosome, has been thought to be nearly universally conserved. Here, we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the Pyrobaculum calidifontis ribosome. We find that sequence variation leads to the rearrangement of key PTC base triples and differences between archaeal and bacterial ribosomal proteins also enable sequence variation in archaeal PTCs. Finally, we identify a novel archaeal ribosome hibernation factor that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome.

evolutionary biology↗

bioRxiv2024

CRISPR-enabled control of gene expression sets the isotopic composition of microbial methane

The stable isotopic composition of biogenic methane varies substantially in the environment and is routinely used to fingerprint its source. However, the underlying cause of this variation is debated. Here, we experimentally manipulate the growth rate of the model methanogen, Methanosarcina acetivorans, using CRISPR mutagenesis to generate a tunable version of the key and final enzyme in methanogenesis, methyl-coenzyme M reductase (MCR). We demonstrate that the carbon and hydrogen isotopic composition of methane change as a function of MCR expression and growth rate. Using an isotope enabled metabolic model we show that these changes stem from a substrate-independent increase in reversibility of methanogenic enzymes. Overall, these data provide a novel framework for calibrating growth coupled changes in the isotopic composition of biogenic methane.

microbiology↗

bioRxiv2023

Physiological and transcriptomic response to methyl-coenzyme M reductase limitation in Methanosarcina acetivorans

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis, the microbial metabolism responsible for nearly all biological methane emissions to the atmosphere. Decades of biochemical and structural studies have generated detailed insights into MCR function in vitro, yet very little is known about the interplay between MCR and methanogen physiology. For instance, while it is routinely stated that MCR catalyzes the rate-limiting step of methanogenesis, this statement has not been categorically tested. Here, to gain a more direct understanding of MCRs control on the growth of Methanosarcina acetivorans, we generate a strain with an inducible mcr operon on the chromosome, allowing for careful control of MCR expression. We show that MCR is not growth rate limiting in substrate-replete batch cultures. However, through careful titration of MCR expression, growth-limiting state(s) can be obtained. Transcriptomic analysis of M. acetivorans experiencing MCR-limitation reveals a global response with hundreds of differentially expressed genes across diverse functional categories. Notably, MCR limitation leads to a strong induction of methylsulfide methyltransferases, likely due to insufficient recycling of metabolic intermediates. In addition, the mcr operon does not seem to be transcriptionally regulated, i.e., it is constitutively expressed, suggesting that the overabundance of MCR might be beneficial when cells experience nutrient limitation or stressful conditions. Altogether, we show that there is wide range of cellular MCR concentrations that can sustain optimal growth, suggesting that other factors like anabolic reactions might be rate-limiting for methanogenic growth. ImportanceMethane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. In methanogens, methyl-coenzyme M reductase (MCR) catalyzes methane formation. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. Here, we show that Methanosarcina acetivorans grown under standard laboratory conditions produces more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher throughput manner than isolation and biochemical characterization of pure protein.

microbiology↗

bioRxiv2023

MmcA is an electron conduit that facilitates both intracellular and extracellular electron transport in Methanosarcina acetivorans

Methanogens are a diverse group of Archaea that couple energy conservation to the production of methane gas. While most methanogens have no alternate mode of energy conservation, strains like Methanosarcina acetivorans are known to also conserve energy by dissimilatory metal reduction (DSMR) in the presence of soluble ferric iron or iron-containing minerals. The ecological ramifications of energy conservation decoupled from methane production in methanogens are substantial, yet the molecular details are poorly understood. In this work, we conducted in vitro and in vivo studies with a multiheme c-type cytochrome (MHC), called MmcA, to establish its role during methanogenesis and DSMR in M. acetivorans. MmcA purified from M. acetivorans can donate electrons to methanophenazine, a membrane-bound electron carrier, to facilitate methanogenesis. In addition, MmcA can also reduce Fe(III) and the humic acid analog anthraquinone-2,6-disulfonate (AQDS) during DSMR. Furthermore, mutants lacking mmcA have slower Fe(III) reduction rates. The redox reactivities of MmcA are consistent with the electrochemical data where MmcA displays reversible redox features ranging from -100 to -450 mV versus SHE. MmcA is prevalent in members of the Order Methanosarcinales but does not belong to a known family of MHCs linked to extracellular electron transfer, bioinformatically, and instead forms a distinct clade that is closely related to octaheme tetrathionate reductases. Taken together, this study shows that MmcA is widespread in methanogens with cytochromes where it acts as an electron conduit to support a variety of energy conservation strategies that extend beyond methanogenesis.

microbiology↗

bioRxiv2023

McrD binds asymmetrically to methyl-coenzyme M reductase improving active site accessibility during assembly

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane and its activity accounts for nearly all biologically produced methane released into the atmosphere. The assembly of MCR is an intricate process involving the installation of a complex set of post-translational modifications and the unique Ni porphyrin cofactor F430. Despite decades of research, details of MCR assembly remain largely unresolved. Here, we report the structural characterization of MCR in two intermediate states of assembly. These intermediate states lack one or both F430 cofactors and form complexes with the previously uncharacterized McrD protein. McrD is found to bind asymmetrically to MCR, displacing large regions of the alpha subunit and increasing active site accessibility for the installation of F430--shedding light on the assembly of MCR and the role of McrD therein. This work offers crucial information for the expression of MCR in a heterologous host and provides new targets for the design of MCR inhibitors. One-sentence summaryStructural characterization of methyl-coenzyme M reductase assembly intermediates.

biochemistry↗

bioRxiv2022

The dual role of a multi-heme cytochrome in methanogenesis: MmcA is important for energy conservation and carbon metabolism in Methanosarcina acetivorans

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c-type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex ({Delta}rnf) or just the MmcA subunit ({Delta}mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA are required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin regeneration that arise in each strain. Transcriptomic data suggest that the {Delta}mmcA mutant might regenerate ferredoxin by upregulating the cytosolic Wood-Ljundahl pathway for acetyl-CoA synthesis, whereas the {Delta}rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to regenerate ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of Rnf or MmcA leads to some shared and some unique transcriptional changes in methyltransferase genes and regulatory proteins. Overall, our study provides systems-level insights into the non-overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA. ImportanceMethane is a greenhouse gas that is ten times more potent than carbon dioxide and efforts to curb emissions are crucial to meet climate goals. Methane emissions primarily stem from the metabolic activity of microorganisms called methanogenic archaea (methanogens). The electron transport chain (ETC) in methanogens that belong to the Order Methanosarcinales has been the focus of many in vitro studies to date, but the endogenous functions of the bioenergetic complexes that comprise the ETC have rarely been investigated. In this study, we use genetic techniques to functionally characterize the Rnf bioenergetic complex and the associated multi-heme c-type cytochrome MmcA in the model methanogen, Methanosarcina acetivorans. Our results show that MmcA and Rnf have shared and unique roles in the cell, and that, contrary to current knowledge, M. acetivorans has the capacity to induce at least two alternative pathways for ferredoxin regeneration in the absence of a functional Rnf complex.

microbiology↗

bioRxiv2022

An Archaea-Specific c-type Cytochrome Maturation Machinery is Crucial for Methanogenesis in Methanosarcina acetivorans

C-type cytochromes (cyt c) are proteins that covalently bind heme and are integral to electron transport chains. A growing body of evidence suggests that cyt c play a vital role in both intra- and extra-cellular electron transfer processes in Archaea, especially in members that metabolize methane and other short chain alkanes. Elaborate mechanisms for the biogenesis of cyt c are known in Bacteria and Eukarya but this process remains largely uncharacterized in Archaea. Here, we have used the model methanogenic archaeon Methanosarcina acetivorans to characterize a distinct form of the system I cyt c maturation machinery (referred to as the Ccm machinery henceforth) that is broadly distributed in members of the Archaea. Phenotypic analyses of M. acetivorans mutants deficient in essential components of the Ccm machinery reveal that cyt c are broadly important for growth and methanogenesis, but the magnitude of their impact can vary substantially depending on the growth substrate. Heterologous expression of a synthetic operon with the Ccm machinery (CcmABCEF) from M. acetivorans is both necessary and sufficient for cyt c biogenesis in a non-native host (M. barkeri Fusaro) that is incapable of cyt c biogenesis. Even though components of the Ccm machinery are universally conserved across the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this pathway through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we have demonstrated the convergent evolution of a novel Archaea-specific Ccm machinery for cyt c biogenesis and its role in methane metabolism. Significance StatementMicroorganisms belonging to the domain Archaea play an especially important role in regulating atmospheric methane levels. Specifically, methanogens are the primary source of biogenic methane and anaerobic methanotrophic archaea (ANME) consume a substantial proportion of methane released in marine sediments. Genomic studies have implicated a class of electron-transfer proteins called c-type cytochromes as being crucial in mediating archaeal methane metabolism in the environment. However, neither the biogenesis nor the role of c-type cytochromes in methane metabolism has ever been investigated. Here, we have used a model methanogen, Methanosarcina acetivorans, to characterize a distinct pathway for maturation of c-type cytochromes that seems to be uniformly conserved across the Archaea and have also identified substrate-specific functional roles for c-type cytochromes during methanogenesis.

microbiology↗

bioRxiv2020

EfgA is a conserved formaldehyde sensor that halts bacterial translation in response to elevated formaldehyde

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that inhibits translation in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved and may include translational machinery. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and modulates translational activity.

microbiology↗