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William B Whitman - One of the best experts on this subject based on the ideXlab platform.

  • post translational methylation of arginine in methyl coenzyme m reductase has a profound impact on both methanogenesis and growth of Methanococcus maripaludis
    Journal of Bacteriology, 2019
    Co-Authors: Nana Shao, Evert C Duin, Chauwen Chou, Ricky Patel, William B Whitman
    Abstract:

    Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl-coenzyme M reductase or Mcr and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved post-translational modifications (PTM) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmp10 (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the deletion mutant Δmmp10 was complemented with the wild-type gene expressed by either a strong or weak promoter, the methylation was fully restored. Compared to the parental strain, the maximal rates of methane formation by whole cells were reduced by 40-60 % in the Δmmp10. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmp10 resulted in a differential loss of the arginine methylation among the mutants in vivo, suggesting activities of Mmp10 directly modulated the methylation. The R275 was present in a highly conserved PxRR275(A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity. Importance Mcr is the key enzyme in methanogenesis and a promising candidate for bioengineering the conversion of methane to liquid fuel. Our knowledge of Mcr is still limited. In terms of complexity, uniqueness and environmental importance, Mcr is more comparable to photosynthetic reaction centers than conventional enzymes. PTMs have long been hypothesized to play key roles in modulating Mcr activity. Here, we directly link the mmp10 gene to the arginine PTM of Mcr, demonstrate its association with methanogenesis activity, and offer insights on its substrate specificity and putative cofactor binding sites. This is also the first time that a PTM of McrA has been shown to have a substantial impact on both methanogenesis and growth in the absence of additional stressors.

  • the archaeal rna chaperone tram0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis
    PLOS Genetics, 2019
    Co-Authors: Jie Li, William B Whitman, Bo Zhang, Liguang Zhou, Lei Qi, Wenting Zhang, Huicai Cheng, Xiuzhu Dong
    Abstract:

    TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

  • assembly of methyl coenzyme m reductase in the methanogenic archaeon Methanococcus maripaludis
    Journal of Bacteriology, 2018
    Co-Authors: Chauwen Chou, Liangliang Wang, Robel Ghebreab, Dennis Phillips, Evert C Duin, William B Whitman
    Abstract:

    ABSTRACT Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile Methanothermococcus okinawensis (rMCR ok ) was expressed in the mesophile Methanococcus maripaludis. The rMCR ok was posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F 430 . Subunits of the native M. maripaludis (MCR mar ) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-tagged mcrA from M. maripaludis and the mcrBDCG from M. okinawensis in M. maripaludis. The His-tagged purified rMCR then contained the M. maripaludis McrA and the M. okinawensis McrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here. IMPORTANCE Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts for about 74% of the total global methane production, up to 25% of which is consumed by anaerobic oxidation of methane (AOM). Methyl coenzyme M reductase (MCR) is the key enzyme in both methanogenesis and AOM. MCR is assembled as a dimer of two heterotrimers, where posttranslational modifications and F 430 cofactors are embedded in the active sites. However, this complex assembly process remains unknown. Here, we established a heterologous expression system for MCR to learn how MCR is assembled.

  • engineering the autotroph Methanococcus maripaludis for geraniol production
    ACS Synthetic Biology, 2016
    Co-Authors: Rachit Jain, Peyton Smith, Travis Fetchko, William B Whitman
    Abstract:

    The rapid autotrophic growth of the methanogenic archaeon Methanococcus maripaludis on H2 and CO2 makes it an attractive microbial chassis to inexpensively produce biochemicals. To explore this potential, a synthetic gene encoding geraniol synthase (GES) derived from Ocimum basilicum was cloned into a M. maripaludis expression vector under selection for puromycin resistance. Recombinant expression of GES in M. maripaludis during autotrophic growth on H2/CO2 or formate yielded geraniol at 2.8 and 4.0 mg g–1 of dry weight, respectively. The yield of geraniol decreased 2–3-fold when organic carbon sources were added to stimulate heterotrophic growth. In the absence of puromycin, geraniol production during autotrophic growth on formate increased to 4.6 mg g–1 of dry weight. A conceptual model centered on the autotrophic acetyl coenzyme A biosynthetic pathway identified strategies to divert more autotrophic carbon flux to geraniol production.

  • the putative trna 2 thiouridine synthetase ncs6 is an essential sulfur carrier in Methanococcus maripaludis
    FEBS Letters, 2014
    Co-Authors: Feng Long, Liangliang Wang, Dieter Soll, William B Whitman
    Abstract:

    Abstract Thiolation of carbon-2 of uridine located in the first position of the anticodons of tRNA UUG Gln , tRNA UUC Glu , and tRNA UUU Lys is a conserved RNA modification event requiring the 2-thiouridine synthetase Ncs6/Ctu1 in archaea and eukaryotes. Ncs6/Ctu1 activates uridine by adenylation, but its role in sulfur transfer is unclear. Here we show that Mmp1356, the Ncs6/Ctu1 homolog in the archaeon Methanococcus maripaludis, forms a persulfide enzyme adduct with an active site cysteine; this suggests that Mmp1356 directly participates in sulfur transfer as a persulfide carrier. Transposon mutagenesis shows that Mmp1356 is likely to be an essential protein.

John A Leigh - One of the best experts on this subject based on the ideXlab platform.

  • exploring hydrogenotrophic methanogenesis a genome scale metabolic reconstruction of Methanococcus maripaludis
    Journal of Bacteriology, 2016
    Co-Authors: Matthew A Richards, John A Leigh, Juan Zhang, Stephen W Ragsdale, Nathan D Price
    Abstract:

    ABSTRACT Hydrogenotrophic methanogenesis occurs in multiple environments, ranging from the intestinal tracts of animals to anaerobic sediments and hot springs. Energy conservation in hydrogenotrophic methanogens was long a mystery; only within the last decade was it reported that net energy conservation for growth depends on electron bifurcation. In this work, we focus on Methanococcus maripaludis, a well-studied hydrogenotrophic marine methanogen. To better understand hydrogenotrophic methanogenesis and compare it with methylotrophic methanogenesis that utilizes oxidative phosphorylation rather than electron bifurcation, we have built iMR539, a genome scale metabolic reconstruction that accounts for 539 of the 1,722 protein-coding genes of M. maripaludis strain S2. Our reconstructed metabolic network uses recent literature to not only represent the central electron bifurcation reaction but also incorporate vital biosynthesis and assimilation pathways, including unique cofactor and coenzyme syntheses. We show that our model accurately predicts experimental growth and gene knockout data, with 93% accuracy and a Matthews correlation coefficient of 0.78. Furthermore, we use our metabolic network reconstruction to probe the implications of electron bifurcation by showing its essentiality, as well as investigating the infeasibility of aceticlastic methanogenesis in the network. Additionally, we demonstrate a method of applying thermodynamic constraints to a metabolic model to quickly estimate overall free-energy changes between what comes in and out of the cell. Finally, we describe a novel reconstruction-specific computational toolbox we created to improve usability. Together, our results provide a computational network for exploring hydrogenotrophic methanogenesis and confirm the importance of electron bifurcation in this process. IMPORTANCE Understanding and applying hydrogenotrophic methanogenesis is a promising avenue for developing new bioenergy technologies around methane gas. Although a significant portion of biological methane is generated through this environmentally ubiquitous pathway, existing methanogen models portray the more traditional energy conservation mechanisms that are found in other methanogens. We have constructed a genome scale metabolic network of Methanococcus maripaludis that explicitly accounts for all major reactions involved in hydrogenotrophic methanogenesis. Our reconstruction demonstrates the importance of electron bifurcation in central metabolism, providing both a window into hydrogenotrophic methanogenesis and a hypothesis-generating platform to fuel metabolic engineering efforts.

  • hydrogenase independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis
    The ISME Journal, 2014
    Co-Authors: Svenja T Lohner, John A Leigh, Jorg S Deutzmann, Bruce E Logan, Alfred M Spormann
    Abstract:

    Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.

  • vhud facilitates electron flow from h2 or formate to heterodisulfide reductase in Methanococcus maripaludis
    Journal of Bacteriology, 2013
    Co-Authors: Kyle C Costa, John A Leigh
    Abstract:

    Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H2 via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F420-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth.

  • phenotypic evidence that the function of the fe hydrogenase hmd in Methanococcus maripaludis requires seven hcg hmd co occurring genes but not hmdii
    Fems Microbiology Letters, 2013
    Co-Authors: Kyle C Costa, Varun Sakesan, John A Leigh
    Abstract:

    The H2-dependent methylene-tetrahydromethanopterin dehydrogenase (Hmd), also known as the [Fe]-hydrogenase, is found only in methanogens without cytochromes. In contrast to the binuclear metal centers of the [NiFe]- and [FeFe]-hydrogenases, the [Fe]-hydrogenase contains only a single Fe atom, which is coordinated by a novel guanylylpyridinol cofactor in the active site. The biosynthesis of the cofactor is not well understood and the responsible genes are unknown. However, seven genes ( hmd co-occurring genes, hcg ) encoding proteins of unknown function are always associated with the hmd gene. In the model methanogen Methanococcus maripaludis , we used a genetic background in which a deletion of hmd had a distinct growth phenotype, and made null-mutations in each hcg gene as well as in a gene encoding the Hmd paralog HmdII, which is hypothesized to function as a scaffold for cofactor synthesis. Deletions in all seven hcg genes resulted in the same growth phenotype as a deletion in hmd , suggesting they are required for Hmd function. In all cases, genetic complementation of the mutation restored the wild-type phenotype. A deletion in hmdII had no effect.

  • hydrogen regulation and global responses to electron carbon and nitrogen sources of Methanococcus maripaludis
    2013
    Co-Authors: John A Leigh
    Abstract:

    Methanogens catalyze the critical, methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter. This project has generated the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and non-coding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 58 different steady-state and time course experiments that were performed in chemostats or batch cultures, under a spectrum of environmental perturbations that modulated methanogenesis. Analyses of the EGRIN model have revealed novel components of methanogenesis that included at least three additional protein-coding genes of previously unknown function as well as one non-coding RNA. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to inter-coordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel transcription factors in the regulation of phosphate-dependent repression of formate dehydrogenase a key enzyme inmore » the methanogenesis pathway. The EGRIN model demonstrates regulatory affiliations within methanogenesis as well as between methanogenesis and other cellular functions. In addition, we have published an analysis of transcriptome architecture in M. maripaludis and an analysis of the effects of H2 and formate on growth yield and regulation of methanogenesis in M. maripaludis.« less

Ken F. Jarrell - One of the best experts on this subject based on the ideXlab platform.

  • Identification and characterization of the 4-epimerase AglW from the archaeon Methanococcus maripaludis
    Glycoconjugate Journal, 2018
    Co-Authors: Sulav Sharma, Ken F. Jarrell, Yan Ding, Inka Brockhausen
    Abstract:

    Archaea are ubiquitous single-cell microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis , a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with N-linked tetrasaccharides that are essential for the proper assembly and function of archaella. The aglW gene, encoding the putative 4-epimerase AglW, plays a key role in the synthesis of the tetrasaccharide. The goal of our work was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop assays to determine its substrate specificity and properties. We carried out assays using UDP-Galactose, UDP-Glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and N-acetylglucosamine/N-acetylgalactosamine-diphosphate – lipid as substrates, coupled with specific glycosyltransferases. We showed that AglW has a broad specificity towards UDP-sugars and that Tyr151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The glycosyltransferase-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases.

  • bypassing the need for the transcriptional activator eara through a spontaneous deletion in the bre portion of the fla operon promoter in Methanococcus maripaludis
    Frontiers in Microbiology, 2017
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, Ken F. Jarrell
    Abstract:

    In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (∆earA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ∆earA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ∆earA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA helps recruit transcription factor B to a weak BRE in the fla promoter of wildtype cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.

  • phylogenetic distribution of the euryarchaeal archaellum regulator eara and complementation of a Methanococcus maripaludis eara mutant with heterologous eara homologues
    Microbiology, 2017
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, Ken F. Jarrell
    Abstract:

    Archaella are the swimming organelles in the Archaea. Recently, the first archaellum regulator in the Euryarchaeota, EarA Mma , was identified in Methanococcus maripaludis, one of the model organisms used for archaellum studies. EarA Mma binds to 6 bp consensus sequences upstream of the fla promoter to activate the transcription of the fla operon, which encodes most of the proteins required for archaella synthesis. In this study, synteny analysis showed that earA homologues are widely distributed in the phylum of Euryarchaeota, with the notable exception of extreme halophiles. We classified Euryarchaeota species containing earA homologues into five classes based on the genomic location of the earA genes relative to fla and chemotaxis operons. EarA homologues from Methanococcus vannielii, Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii successfully complemented the function of EarA Mma in a ΔearAMma mutant, demonstrated by the restoration of FlaB2 expression in Western blot analysis and the appearance of archaella on the cell surface in complemented cells. Furthermore, the 6 bp consensus sequence was also found in the fla promoter region in these methanogens, indicating that the EarA homologues ly use a similar mechanism to activate transcription of the fla operons in their own hosts. Attempts to demonstrate complementation of the function of EarAMma in a ΔearAMma mutant by the EarA homologue of Pyrococcus furiosus were unsuccessful, despite the presence of a copy of the 6 bp consensus EarA-binding sequence upstream of the fla promoter in the P. furiosus genome.

  • Bypassing the need for the transcriptional activator eara through a spontaneous deletion in the BRE portion of the fla operon promoter in Methanococcus maripaludis
    Frontiers in Microbiology, 2017
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, Ken F. Jarrell
    Abstract:

    © 2017 Ding, Berezuk, Khursigara and Jarrell. In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (ΔearA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ΔearA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ΔearA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.

  • complementation of an aglb mutant of Methanococcus maripaludis with heterologous oligosaccharyltransferases
    PLOS ONE, 2016
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, James Schneider, Helen A Vrionis, Ken F. Jarrell
    Abstract:

    : The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.

Michael Rother - One of the best experts on this subject based on the ideXlab platform.

  • selenium dependent gene expression in Methanococcus maripaludis involvement of the transcriptional regulator hrsm
    Biochimica et Biophysica Acta, 2018
    Co-Authors: Vivien Quitzke, Julia Fersch, Deniz Seyhan, Michael Rother
    Abstract:

    Abstract Background The archaeon Methanococcus maripaludis strain JJ employs several selenocysteine (Sec)-containing proteins in its primary energy metabolism, methanogenesis. Upon selenium deprivation, or when the pathway for selenoprotein synthesis is disrupted, they are replaced by cysteine (Cys)-containing isoforms, thus allowing for selenium-independent growth. Methods Expression of a fusion of the promoter region of frcA (encoding a subunit of the selenium-independent hydrogenase Frc) and bla [encoding β-lactamase (Bla)] in M. maripaludis JJ was assessed in response to the selenium supply, growth substrate, and growth phase. Random transposon mutants of the reporter strain were screened for deregulated bla expression, which identified HrsM, a LysR-type transcriptional regulator (LTTR). Its involvement in selenium-dependent gene regulation was further assessed by analyzing in vivo transcription, synthesis of selenoproteins and of HrsM, and by analyzing in vitro binding of HrsM to DNA. Results HrsM, which is not required for selenoprotein synthesis, acts as a positive effector of selenoprotein gene expression and as a negative effector of Cys-encoding isogene expression, but its own expression is independent of the selenium availability. Specific binding in vitro of HrsM to a promoter region under in vivo HrsM control verified its role in selenium-dependent gene regulation. Conclusions HrsM exerts a key role in regulating expression of selenoprotein genes and their Cys-encoding isogenes in M. maripaludis in a selenium-dependent fashion. However, this activity is not achieved via autoregulation but probably by a mechanism, which modulates the DNA-binding of HrsM. General significance Although LTTRs are abundant in Bacteria, HrsM represents only the second characterized member of this group in Archaea.

  • complete genome sequence of the Methanococcus maripaludis type strain jj dsm 2067 a model for selenoprotein synthesis in archaea
    Genome Announcements, 2018
    Co-Authors: Anja Poehlein, Daniel Heym, Vivien Quitzke, Julia Fersch, Rolf Daniel, Michael Rother
    Abstract:

    ABSTRACT Methanococcus maripaludis type strain JJ (DSM 2067) is an important organism because it serves as a model for primary energy metabolism and hydrogenotrophic methanogenesis and is amenable to genetic manipulation. The complete genome (1.7 Mb) harbors 1,815 predicted protein-encoding genes, including 9 encoding selenoproteins.

  • selenocysteine independent suppression of uga codons in the archaeon Methanococcus maripaludis
    Biochimica et Biophysica Acta, 2015
    Co-Authors: Deniz Seyhan, Julia Fersch, Michael Rother, Nico Jehmlich, Martin Von Bergen
    Abstract:

    Abstract Background Proteins containing selenocysteine (sec) are found in Bacteria, Eukarya, and Archaea. While selenium-dependence of methanogenesis from H 2  + CO 2 in the archaeon Methanococcus maripaludis JJ is compensated by induction of a set of cysteine-containing homologs, growth on formate is abrogated in the absence of sec due to the dependence of formate dehydrogenase (Fdh) on selenium. Despite this dependence, formate-dependent growth occurs after prolonged incubation of M. maripaludis mutants lacking sec. Methods To study this phenomenon, a M. maripaludis strain with only one Fdh isoform and an FdhA selenoprotein C-terminally tagged for affinity enrichment was constructed. Factors required for sec synthesis were deleted in this strain and translation of UGA in fdhA was analyzed physiologically, enzymatically, immunologically, and via mass spectrometry. Results M. maripaludis JJ mutants lacking sec synthesis grew at least five times more slowly than the wild type on formate due to a 20–35-fold reduction of Fdh activity. The enzyme in the mutant strains lacked sec but was still produced as a full-length protein. Peptide mass spectrometry revealed that both cysteine (cys) and tryptophan (trp) were inserted at the UGA encoding sec without apparent mutations in tRNA cys or tRNA trp , respectively. Conclusions We demonstrate that M. maripaludis has the inherent capacity to translate UGA with cys and trp; other mechanisms to replace sec with cys in the absence of selenium could thereby be ruled out. General significance This study exemplifies how an organism uses the inherent flexibility in its canonical protein synthesis machinery to recover some activity of an essential selenium-dependent enzyme in the absence of sec.

  • Random mutagenesis identifies factors involved in formate-dependent growth of the methanogenic archaeon Methanococcus maripaludis
    Molecular Genetics and Genomics, 2013
    Co-Authors: Christian Sattler, Julia Fersch, Sandro Wolf, Stefan Goetz, Michael Rother
    Abstract:

    Methane is a key intermediate in the carbon cycle and biologically produced by methanogenic archaea. Most methanogens are able to conserve energy by reducing CO_2 to methane using molecular hydrogen as electron donor (hydrogenotrophic methanogenesis), but several hydrogenotrophic methanogens can also use formate as electron donor for methanogenesis. Formate dehydrogenase (Fdh) oxidizes formate to CO_2 and is involved in funneling reducing equivalents into the methanogenic pathway, but details on other factors relevant for formate-dependent physiology of methanogens are not available. To learn more about the factors involved in formate-dependent growth of Methanococcus maripaludis strain JJ, we used a recently developed system for random in vitro mutagenesis, which is based on a modified insect transposable element to create 2,865 chromosomal transposon mutants and screened them for impaired growth on formate. Of 12 M. maripaludis transposon-induced mutants exhibiting this phenotype, the transposon insertion sites in the chromosome were mapped. Among the genes, apparently affecting formate-dependent growth were those encoding archaeal transcription factor S, a regulator of ion transport, and carbon monoxide dehydrogenase/acetyl-CoA synthase. Interestingly, in seven of the mutants, transposons were localized in a 10.2 kb region where Fdh1, one of two Fdh isoforms in the organism, is encoded. Two transcription start sites within the 10.2 kb region could be mapped, and quantification of transcripts revealed that transposon insertion in this region diminished fdhA1 expression due to polar effects.

  • disruption and complementation of the selenocysteine biosynthesis pathway reveals a hierarchy of selenoprotein gene expression in the archaeon Methanococcus maripaludis
    Molecular Microbiology, 2011
    Co-Authors: Tilmann Stock, Deniz Seyhan, Mirjam Selzer, Sarah Connery, Armin Resch, Michael Rother
    Abstract:

    : Proteins containing selenocysteine are found in members of all three domains of life, Bacteria, Eukarya and Archaea. A dedicated tRNA (tRNA(sec)) serves as a scaffold for selenocysteine synthesis. However, sequence and secondary structures differ in tRNA(sec) from the different domains. An Escherichia coli strain lacking the gene for tRNA(sec) could only be complemented with the homologue from Methanococcus maripaludis when a single base in the anticodon loop was exchanged demonstrating that this base is a crucial determinant for archaeal tRNA(sec) to function in E. coli. Complementation in trans of M. maripaludis JJ mutants lacking tRNA(sec) , O-phosphoseryl-tRNA(sec) kinase or O-phosphoseryl-tRNA(sec) :selenocysteine synthase with the corresponding genes from M. maripaludis S2 restored the mutant's ability to synthesize selenoproteins. However, only partial restoration of the wild-type selenoproteome was observed as only selenocysteine-containing formate dehydrogenase was synthesized. Quantification of transcripts showed that disrupting the pathway of selenocysteine synthesis leads to downregulation of selenoprotein gene expression, concomitant with upregulation of a selenium-independent backup system, which is not re-adjusted upon complementation. This transcriptional arrest was independent of selenophosphate but depended on the 'history' of the mutants and was inheritable, which suggests that a stable genetic switch may cause the resulting hierarchy of selenoproteins synthesized.

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  • Identification and characterization of the 4-epimerase AglW from the archaeon Methanococcus maripaludis
    Glycoconjugate Journal, 2018
    Co-Authors: Sulav Sharma, Ken F. Jarrell, Yan Ding, Inka Brockhausen
    Abstract:

    Archaea are ubiquitous single-cell microorganisms that have often adapted to harsh conditions and play important roles in biogeochemical cycles with potential applications in biotechnology. Methanococcus maripaludis , a methane-producing archaeon, is motile through multiple archaella on its cell surface. The major structural proteins (archaellins) of the archaellum are glycoproteins, modified with N-linked tetrasaccharides that are essential for the proper assembly and function of archaella. The aglW gene, encoding the putative 4-epimerase AglW, plays a key role in the synthesis of the tetrasaccharide. The goal of our work was to biochemically demonstrate the 4-epimerase activity of AglW, and to develop assays to determine its substrate specificity and properties. We carried out assays using UDP-Galactose, UDP-Glucose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and N-acetylglucosamine/N-acetylgalactosamine-diphosphate – lipid as substrates, coupled with specific glycosyltransferases. We showed that AglW has a broad specificity towards UDP-sugars and that Tyr151 within a conserved YxxxK sequon is essential for the 4-epimerase function of AglW. The glycosyltransferase-coupled assays are generally useful for the identification and specificity studies of novel 4-epimerases.

  • bypassing the need for the transcriptional activator eara through a spontaneous deletion in the bre portion of the fla operon promoter in Methanococcus maripaludis
    Frontiers in Microbiology, 2017
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, Ken F. Jarrell
    Abstract:

    In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (∆earA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ∆earA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ∆earA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA helps recruit transcription factor B to a weak BRE in the fla promoter of wildtype cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.

  • phylogenetic distribution of the euryarchaeal archaellum regulator eara and complementation of a Methanococcus maripaludis eara mutant with heterologous eara homologues
    Microbiology, 2017
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, Ken F. Jarrell
    Abstract:

    Archaella are the swimming organelles in the Archaea. Recently, the first archaellum regulator in the Euryarchaeota, EarA Mma , was identified in Methanococcus maripaludis, one of the model organisms used for archaellum studies. EarA Mma binds to 6 bp consensus sequences upstream of the fla promoter to activate the transcription of the fla operon, which encodes most of the proteins required for archaella synthesis. In this study, synteny analysis showed that earA homologues are widely distributed in the phylum of Euryarchaeota, with the notable exception of extreme halophiles. We classified Euryarchaeota species containing earA homologues into five classes based on the genomic location of the earA genes relative to fla and chemotaxis operons. EarA homologues from Methanococcus vannielii, Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii successfully complemented the function of EarA Mma in a ΔearAMma mutant, demonstrated by the restoration of FlaB2 expression in Western blot analysis and the appearance of archaella on the cell surface in complemented cells. Furthermore, the 6 bp consensus sequence was also found in the fla promoter region in these methanogens, indicating that the EarA homologues ly use a similar mechanism to activate transcription of the fla operons in their own hosts. Attempts to demonstrate complementation of the function of EarAMma in a ΔearAMma mutant by the EarA homologue of Pyrococcus furiosus were unsuccessful, despite the presence of a copy of the 6 bp consensus EarA-binding sequence upstream of the fla promoter in the P. furiosus genome.

  • Bypassing the need for the transcriptional activator eara through a spontaneous deletion in the BRE portion of the fla operon promoter in Methanococcus maripaludis
    Frontiers in Microbiology, 2017
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, Ken F. Jarrell
    Abstract:

    © 2017 Ding, Berezuk, Khursigara and Jarrell. In Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (ΔearA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ΔearA mutant in which the restoration of the transcription and translation of the fla operon (using flaB2, the second gene of the operon, as a reporter), archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ΔearA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.

  • complementation of an aglb mutant of Methanococcus maripaludis with heterologous oligosaccharyltransferases
    PLOS ONE, 2016
    Co-Authors: Yan Ding, Alison Berezuk, Cezar M Khursigara, James Schneider, Helen A Vrionis, Ken F. Jarrell
    Abstract:

    : The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.