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James G Ferry - One of the best experts on this subject based on the ideXlab platform.

  • Methanosarcina Acetivorans: A Model for Mechanistic Understanding of Aceticlastic and Reverse Methanogenesis.
    Frontiers in microbiology, 2020
    Co-Authors: James G Ferry
    Abstract:

    Acetate-utilizing methanogens are responsible for approximately two-thirds of the one billion metric tons of methane produced annually in Earth's anaerobic environments. Methanosarcina Acetivorans has emerged as a model organism for the mechanistic understanding of aceticlastic methanogenesis and reverse methanogenesis applicable to understanding the methane and carbon cycles in nature. It has the largest genome in the Archaea, supporting a metabolic complexity that enables a remarkable ability for adapting to environmental opportunities and challenges. Biochemical investigations have revealed an aceticlastic pathway capable of fermentative and respiratory energy conservation that explains how Ms. Acetivorans is able to grow and compete in the environment. The mechanism of respiratory energy conservation also plays a role in overcoming endothermic reactions that are key to reversing methanogenesis.

  • functional role of mrpa in the mrpabcdefg na h antiporter complex from the archaeon Methanosarcina Acetivorans
    Journal of Bacteriology, 2017
    Co-Authors: Ricardo Jassochavez, Jose S Rodriguezzavala, Cesar Diazperez, James G Ferry
    Abstract:

    The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina Acetivorans was produced in antiporter-deficient Escherichia coli strains EP432 and KNabc and biochemically characterized to determine the role of MrpA in the complex. Both strains containing MrpA grew in the presence of up to 500 mM NaCl and pH values up to 11.0 with no added NaCl. Everted vesicles from the strains containing MrpA were able to generate a NADH-dependent pH gradient (ΔpH), which was abated by the addition of monovalent cations. The apparent Km values for Na+ and Li+ were similar and ranged from 31 to 63 mM, whereas activity was too low to determine the apparent Km for K+. Optimum activity was obtained between pH 7.0 and 8.0. Homology molecular modeling identified two half-closed symmetry-related ion translocation channels that are linked, forming a continuous path from the cytoplasm to the periplasm, analogous to the NuoL subunit of complex I. Bioinformatics analyses revealed genes encoding homologs of MrpABCDEFG in metabolically diverse methane-producing species. Overall, the results advance the biochemical, evolutionary, and physiological understanding of Mrp complexes that extends to the domain Archaea. IMPORTANCE The work is the first reported characterization of an Mrp complex from the domain Archaea, specifically methanogens, for which Mrp is important for acetotrophic growth. The results show that the MrpA subunit is essential for antiport activity and, importantly, that not all seven subunits are required, which challenges current dogma for Mrp complexes from the domain Bacteria. A mechanism is proposed in which an MrpAD subcomplex catalyzes Na+/H+ antiport independent of an MrpBCEFG subcomplex, although the activity of the former is modulated by the latter. Properties of MrpA strengthen proposals that the Mrp complex is of ancient origin and that subunits were recruited to evolve the ancestral complex I. Finally, bioinformatics analyses indicate that Mrp complexes function in diverse methanogenic pathways.

  • assessing methanotrophy and carbon fixation for biofuel production by Methanosarcina Acetivorans
    Microbial Cell Factories, 2016
    Co-Authors: Hadi Nazembokaee, James G Ferry, Saratram Gopalakrishnan, Thomas K Wood, Costas D Maranas
    Abstract:

    Background Methanosarcina Acetivorans is a model archaeon with renewed interest due to its unique reversible methane production pathways. However, the mechanism and relevant pathways implicated in (co)utilizing novel carbon substrates in this organism are still not fully understood. This paper provides a comprehensive inventory of thermodynamically feasible routes for anaerobic methane oxidation, co-reactant utilization, and maximum carbon yields of major biofuel candidates by M. Acetivorans.

  • assessing methanotrophy and carbon fixation for biofuel production by Methanosarcina Acetivorans
    Microbial Cell Factories, 2016
    Co-Authors: Hadi Nazembokaee, James G Ferry, Saratram Gopalakrishnan, Thomas K Wood, Costas D Maranas
    Abstract:

    Methanosarcina Acetivorans is a model archaeon with renewed interest due to its unique reversible methane production pathways. However, the mechanism and relevant pathways implicated in (co)utilizing novel carbon substrates in this organism are still not fully understood. This paper provides a comprehensive inventory of thermodynamically feasible routes for anaerobic methane oxidation, co-reactant utilization, and maximum carbon yields of major biofuel candidates by M. Acetivorans. Here, an updated genome-scale metabolic model of M. Acetivorans is introduced (iMAC868 containing 868 genes, 845 reactions, and 718 metabolites) by integrating information from two previously reconstructed metabolic models (i.e., iVS941 and iMB745), modifying 17 reactions, adding 24 new reactions, and revising 64 gene-protein-reaction associations based on newly available information. The new model establishes improved predictions of growth yields on native substrates and is capable of correctly predicting the knockout outcomes for 27 out of 28 gene deletion mutants. By tracing a bifurcated electron flow mechanism, the iMAC868 model predicts thermodynamically feasible (co)utilization pathway of methane and bicarbonate using various terminal electron acceptors through the reversal of the aceticlastic pathway. This effort paves the way in informing the search for thermodynamically feasible ways of (co)utilizing novel carbon substrates in the domain Archaea.

  • Structural and Biochemical Characterizations of Methanoredoxin from Methanosarcina Acetivorans, a Glutaredoxin-Like Enzyme with Coenzyme M-Dependent Protein Disulfide Reductase Activity
    Biochemistry, 2015
    Co-Authors: Deepa Yenugudhati, R. Siva Sai Kumar, Adepu K. Kumar, Neela H. Yennawar, Hemant P. Yennawar, Divya Prakash, James G Ferry
    Abstract:

    Glutaredoxins (GRXs) are thiol–disulfide oxidoreductases abundant in prokaryotes, although little is understood of these enzymes from the domain Archaea. The numerous characterized GRXs from the domain Bacteria utilize a diversity of low-molecular-weight thiols in addition to glutathione as reductants. We report here the biochemical and structural properties of a GRX-like protein named methanoredoxin (MRX) from Methanosarcina Acetivorans of the domain Archaea. MRX utilizes coenzyme M (CoMSH) as reductant for insulin disulfide reductase activity, which adds to the diversity of thiol protectants in prokaryotes. Cell-free extracts of M. Acetivorans displayed CoMS-SCoM reductase activity that complements the CoMSH-dependent activity of MRX. The crystal structure exhibits a classic thioredoxin-glutaredoxin fold comprising three α-helices surrounding four antiparallel β-sheets. A pocket on the surface contains a CVWC motif, identifying the active site with architecture similar to GRXs. Although it is a monomer ...

Daniel J. Lessner - One of the best experts on this subject based on the ideXlab platform.

  • Methanosarcina Acetivorans contains a functional isc system for iron sulfur cluster biogenesis
    BMC Microbiology, 2020
    Co-Authors: Thomas M Deere, Faith H. Lessner, Divya Prakash, Evert C Duin, Daniel J. Lessner
    Abstract:

    The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina Acetivorans contains three iscSU and two sufCB gene clusters. Here, we report genetic and biochemical characterization of M. Acetivorans iscSU2. Purified IscS2 exhibited pyridoxal 5′- phosphate-dependent release of sulfur from L-cysteine. Incubation of purified IscU2 with IscS2, cysteine, and iron (Fe2+) resulted in the formation of [4Fe-4S] clusters in IscU2. IscU2 transferred a [4Fe-4S] cluster to purified M. Acetivorans apo-aconitase. IscU2 also restored the aconitase activity in air-exposed M. Acetivorans cell lysate. These biochemical results demonstrate that IscS2 is a cysteine desulfurase and that IscU2 is a Fe-S cluster scaffold. M. Acetivorans strain DJL60 deleted of iscSU2 was generated to ascertain the in vivo importance of IscSU2. Strain DJL60 had Fe-S cluster content and growth similar to the parent strain but lower cysteine desulfurase activity. Strain DJL60 also had lower intracellular persulfide content compared to the parent strain when cysteine was an exogenous sulfur source, linking IscSU2 to sulfur metabolism. This study establishes that M. Acetivorans contains functional IscS and IscU, the core components of the ISC Fe-S cluster biogenesis system and provides the first evidence that ISC operates in methanogens.

  • a crispri dcas9 system for archaea and its use to examine gene function during nitrogen fixation by Methanosarcina Acetivorans
    Applied and Environmental Microbiology, 2020
    Co-Authors: Ahmed E Dhamad, Daniel J. Lessner
    Abstract:

    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.

  • a crispri dcas9 system for archaea and its use to examine gene function during nitrogen fixation by Methanosarcina Acetivorans
    bioRxiv, 2020
    Co-Authors: Ahmed E Dhamad, Daniel J. Lessner
    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 (gRNA), one targeting the promoter and the other 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 a NrpR1 as 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.

  • Methanosarcina Acetivorans utilizes a single NADPH-dependent thioredoxin system and contains additional thioredoxin homologues with distinct functions.
    Microbiology (Reading England), 2017
    Co-Authors: Addison C. Mccarver, Faith H. Lessner, Jose M. Soroeta, Daniel J. Lessner
    Abstract:

    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.

  • the 4fe 4s clusters of rpo3 are key determinants in the post rpo3 rpo11 heterodimer formation of rna polymerase in Methanosarcina Acetivorans
    MicrobiologyOpen, 2017
    Co-Authors: Matthew E Jennings, Elizabeth A Karr, Faith H. Lessner, Daniel J. Lessner
    Abstract:

    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.

William W Metcalf - One of the best experts on this subject based on the ideXlab platform.

  • functional interactions between posttranslationally modified amino acids of methyl coenzyme m reductase in Methanosarcina Acetivorans
    PLOS Biology, 2020
    Co-Authors: Dipti D Nayak, Roy Rodriguezcarerro, Shihui Dong, Douglas A Mitchell, Satish K Nair, Neha Agrawal, William W Metcalf
    Abstract:

    The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina Acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. Acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

  • functional interactions between post translationally modified amino acids of methyl coenzyme m reductase in Methanosarcina Acetivorans
    bioRxiv, 2019
    Co-Authors: Dipti D Nayak, Roy Rodriguezcarerro, Shihui Dong, Douglas A Mitchell, Satish K Nair, Neha Agrawal, William W Metcalf
    Abstract:

    Abstract Methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six post-translationally modified amino acids near the active site. Recent studies have identified genes that install two of these modifications (thioglycine and 5-(S)-methylarginine), yet little is known about the installation and function of the remaining post-translationally modified residues. Here, we provide in vivo evidence that a dedicated SAM-dependent methyltransferase encoded by a gene we designated mcmA is responsible for formation of S-methylcysteine in Methanosarcina Acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue plays an important role in adaptation to a mesophilic lifestyle. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. Acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. Surprisingly, high-resolution crystal structures of the various unmodified MCRs were indistinguishable from the fully modified enzyme, suggesting that interactions between the post-translationally modified residues do not exert a major influence on the physical structure of the enzyme, but rather serve to fine-tune the activity and efficiency of MCR.

  • biochemical characterization of the methylmercaptopropionate cob i alamin methyltransferase from Methanosarcina Acetivorans
    bioRxiv, 2019
    Co-Authors: He Fu, Michelle N Goettge, William W Metcalf
    Abstract:

    ABSTRACT Methanogenesis from methylated substrates is initiated by substrate specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina Acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate(MMPA):Coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 μM. CoM did not serve as a methyl acceptor, thus a second, unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, could also be demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were co-expressed. Biochemical characterization of this complex was not feasible because the corrinoid cofactor of this complex was in the inactive Co(II) state and could not be reactivated by incubation with strong reductants. The MtsF protein, comprised of both corrinoid and methyltransferase domains, co-purifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism. IMPORTANCE MMPA is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate methyl-CoM. Therefore, additional enzymes required for methanogenic MMPA catabolism await discovery.

  • biochemical characterization of the methylmercaptopropionate cob i alamin methyltransferase from Methanosarcina Acetivorans
    Journal of Bacteriology, 2019
    Co-Authors: He Fu, Michelle N Goettge, William W Metcalf
    Abstract:

    ABSTRACT Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina Acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s−1 and apparent Km for MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism. IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

  • genetic techniques for studies of methyl coenzyme m reductase from Methanosarcina Acetivorans c2a
    Methods in Enzymology, 2018
    Co-Authors: Dipti D Nayak, William W Metcalf
    Abstract:

    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.

Robert P. Gunsalus - One of the best experts on this subject based on the ideXlab platform.

  • structure of the surface layer of the methanogenic archaean Methanosarcina Acetivorans
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: M A Arbing, Lars Rohlin, S Chan, A Shin, Tung Phan, Robert P. Gunsalus
    Abstract:

    Archaea have a self-assembling proteinaceous surface (S-) layer as the primary and outermost boundary of their cell envelopes. The S-layer maintains structural rigidity, protects the organism from adverse environmental elements, and yet provides access to all essential nutrients. We have determined the crystal structure of one of the two “homologous” tandem polypeptide repeats that comprise the Methanosarcina Acetivorans S-layer protein and propose a high-resolution model for a microbial S-layer. The molecular features of our hexameric S-layer model recapitulate those visualized by medium resolution electron microscopy studies of microbial S-layers and greatly expand our molecular view of S-layer dimensions, porosity, and symmetry. The S-layer model reveals a negatively charged molecular sieve that presents both a charge and size barrier to restrict access to the cell periplasmic-like space. The β-sandwich folds of the S-layer protein are structurally homologous to eukaryotic virus envelope proteins, suggesting that Archaea and viruses have arrived at a common solution for protective envelope structures. These results provide insight into the evolutionary origins of primitive cell envelope structures, of which the S-layer is considered to be among the most primitive: it also provides a platform for the development of self-assembling nanomaterials with diverse functional and structural properties.

  • identification of the major expressed s layer and cell surface layer related proteins in the model methanogenic archaea Methanosarcina barkeri fusaro and Methanosarcina Acetivorans c2a
    Archaea, 2012
    Co-Authors: Lars Rohlin, Deborah R Leon, Robert P. Gunsalus
    Abstract:

    Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina Acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. Acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.

  • apo and ligand bound structures of moda from the archaeon Methanosarcina Acetivorans
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2010
    Co-Authors: Sum Chan, Robert P. Gunsalus, Iulia Giuroiu, Irina Chernishof, Michael R Sawaya, Janet Chiang, Mark A Arbing, Jeanne L Perry
    Abstract:

    The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina Acetivorans (MaModA) have been determined in the apo­protein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed α/β domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the `Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.

  • carbon dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the archaean Methanosarcina Acetivorans strain c2a
    BMC Microbiology, 2010
    Co-Authors: Lars Rohlin, Robert P. Gunsalus
    Abstract:

    Background The archaeon, Methanosarcina Acetivorans strain C2A forms methane, a potent greenhouse gas, from a variety of one-carbon substrates and acetate. Whereas the biochemical pathways leading to methane formation are well understood, little is known about the expression of the many of the genes that encode proteins needed for carbon flow, electron transfer and/or energy conservation. Quantitative transcript analysis was performed on twenty gene clusters encompassing over one hundred genes in M. Acetivorans that encode enzymes/proteins with known or potential roles in substrate conversion to methane.

  • quantitative proteomic and microarray analysis of the archaeon Methanosarcina Acetivorans grown with acetate versus methanol
    Journal of Proteome Research, 2007
    Co-Authors: Lingyun Li, Robert P. Gunsalus, Lars Rohlin, Tomas Rejtar, Barry L. Karger, Qingbo Li, Kirsty Salmon, James G Ferry
    Abstract:

    Methanosarcina Acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs methanol-grown cells metabolically labeled with 14N vs 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel...

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  • Role of a putative tungsten-dependent formylmethanofuran dehydrogenase in Methanosarcina Acetivorans
    Archives of Microbiology, 2015
    Co-Authors: Nicole Matschiavelli, Michael Rother
    Abstract:

    Methanogenesis, the biological production of methane, is the sole means for energy conservation for methanogenic archaea. Among the few methanogens shown to grow on carbon monoxide (CO) is Methanosarcina Acetivorans , which produces, beside methane, acetate and formate in the process. Since CO-dependent methanogenesis proceeds via formation of formylmethanofuran from CO_2 and methanofuran, catalyzed by formylmethanofuran dehydrogenase, we were interested whether this activity could participate in the formate formation from CO. The genome of M. Acetivorans encodes four putative formylmethanofuran dehydrogenases, two annotated as molybdenum-dependent and the remaining two as tungsten-dependent enzymes. A mutant lacking one of the putative tungsten enzymes grew very slowly on CO and only after a prolonged adaptation period, which suggests an important role for this isoform during growth on CO. Methanol- and CO-dependent growth of the mutant required the presence of molybdenum indicating an indispensable function of this metal in the remaining isoforms. CO-dependent formate formation could not be observed in the mutant indicating involvement of the respective isoform in the process. However, addition of formaldehyde, which spontaneously reacts with tetrahydrosarcinapterin (H_4SPT) to methenyl-H_4SPT, led to near-wild-type formate production rates, which argues for an alternative route of formate formation in this organism.

  • development of β lactamase as a tool for monitoring conditional gene expression by a tetracycline riboswitch in Methanosarcina Acetivorans
    Archaea, 2014
    Co-Authors: Shemsi Demolli, Michael Rother, Nicole Matschiavelli, Miriam M Geist, Julia E Weigand, Beatrix Suess
    Abstract:

    The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina Acetivorans. By using a signal peptide of a putative flagellin from M. Acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. Acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

  • a heme based redox sensor in the methanogenic archaeon Methanosarcina Acetivorans
    Journal of Biological Chemistry, 2013
    Co-Authors: Bastian Molitor, Marc Stassen, Anuja Modi, Samir F Elmashtoly, Christoph Laurich, Wolfgang Lubitz, John H Dawson, Michael Rother, Nicole Frankenbergdinkel
    Abstract:

    Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina Acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. Acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor).

  • function and regulation of isoforms of carbon monoxide dehydrogenase acetyl coenzyme a synthase in Methanosarcina Acetivorans
    Journal of Bacteriology, 2012
    Co-Authors: Michael Rother, Ellen Oelgeschläger, Nicole Matschiavelli, Berardino Cocchiararo, Johannes Finke
    Abstract:

    Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO(2) reduction and cleavage/synthesis of acetyl-CoA. Methanosarcina Acetivorans C2A contains two very similar copies of a six-gene operon (cdh genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in M. Acetivorans, mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of cdh disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are bona fide CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of cdh1, suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.

  • Genetic analysis of MA4079, an aldehyde dehydrogenase homolog, in Methanosarcina Acetivorans
    Archives of Microbiology, 2012
    Co-Authors: Michael Kliefoth, Ellen Oelgeschläger, Julian D. Langer, Nicole Matschiavelli, Michael Rother
    Abstract:

    When Methanosarcina Acetivorans grows on carbon monoxide (CO), it synthesizes high levels of a protein, MA4079, homologous to aldehyde dehydrogenases. To investigate the role of MA4079 in M. Acetivorans , mutants lacking the encoding gene were generated and phenotypically analyzed. Loss of MA4079 had no effect on methylotrophic growth but led to complete abrogation of methylotrophic growth in the presence of even small amounts of CO, which indicated the mutant’s inability to acclimate to the presence of this toxic gas. Prolonged incubation with CO allowed the isolation of a strain in which the effect of MA4079 deletion is suppressed. The strain, designated Mu3, tolerated the presence of high CO partial pressures even better than the wild type. Immunological analysis using antisera against MA4079 suggested that it is not abundant in M. Acetivorans . Comparison of proteins differentially abundant in Mu3 and the wild type revealed an elevated level of methyl-coenzyme M reductase and a decreased level of one isoform of carbon monoxide dehydrogenase/acetyl-coenzyme A synthase, which suggests that pleiotropic mutation(s) compensating for the loss of MA4079 affected catabolism. The data presented point toward a role of MA4079 to enable M. Acetivorans to properly acclimate to CO.