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Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase.

ABSTRACT: The sequence and transcript of the genes encoding a recently discovered coenzyme M methylase in Methanosarcina barkeri were analyzed. This 480-kDa protein is composed of two subunits in equimolar concentrations which bind one corrinoid cofactor per alphabeta dimer. The gene for the alphabeta polypeptide, mtsA, is upstream of that encoding the beta polypeptide, mtsB. The two genes are contiguous and overlap by several nucleotides. A 1.9-kb mRNA species which reacted with probes specific for either mtsA or mtsB was detected. Three possible methanogen consensus BoxA sequences as well as two sets of direct repeats were found upstream of mtsA. The 5' end of the mts transcript was 19 nucleotides upstream of the translational start site of mtsA and was positioned 25 bp from the center of the proximal BoxA sequence. The transcript was most abundant in cells grown to the late log phase on acetate but barely detectable in cells grown on methanol or trimethylamine. The amino acid sequence of MtsB was homologous to the cobalamin-binding fragment of methionine synthase from Escherichia coli and possessed the signature residues involved in binding the corrinoid, including a histidyl residue which ligates cobalt. The sequence of MtsA is homologous to the "A" and "M" isozymes of methylcobamide:coenzyme M methyltransferases (methyltransferase II), indicating that the alpha polypeptide is a new member of the methyltransferase II family of coenzyme M methylases. All three methyltransferase II homolog sequences could be aligned with the sequences of uroporphyrinogen decarboxylase from various sources. The implications of these homologies for the mechanism of corrinoid binding by proteins involved in methylotrophic methanogenesis are discussed.

SUBMITTER: Paul L

PROVIDER: S-EPMC178547 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase.

Paul L L Krzycki J A JA

Journal of bacteriology 19961101 22

The sequence and transcript of the genes encoding a recently discovered coenzyme M methylase in Methanosarcina barkeri were analyzed. This 480-kDa protein is composed of two subunits in equimolar concentrations which bind one corrinoid cofactor per alphabeta dimer. The gene for the alphabeta polypeptide, mtsA, is upstream of that encoding the beta polypeptide, mtsB. The two genes are contiguous and overlap by several nucleotides. A 1.9-kb mRNA species which reacted with probes specific for eithe ...[more]

PMID: 8932317

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Project description:A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, Methanosarcina barkeri and Methanosarcina soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV.IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina-infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

Project description: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 k cat of 0.315?s-1 and apparent Km for MPA of 12??M. 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.

Project description:The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanol-grown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C(1) compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization.

Dataset Information

Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase.

Publications

Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase.

Similar Datasets

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