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The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.

ABSTRACT: Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1. However, two other nearly identical copies of mtbB1, designated mtbB2 and mtbB3, were also found in the genome. A 6.8-kb transcript was detected with probes to mttB and mtbB1, as well as to mtbC and mttC, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP, encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order mtbC, mttB, mttC, mttP, and mtbB1, form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC. The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1, mtbB2, or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M. barkeri mttB gene. The UAG codon is also present in mttB transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.

SUBMITTER: Paul L

PROVIDER: S-EPMC111316 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.

Paul L L Ferguson D J DJ Krzycki J A JA

Journal of bacteriology 20000501 9

Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1. However, two other nearly identical co ...[more]

PMID: 10762254

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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.

Project description:BackgroundAnaerobic digestion of easily degradable biowaste can lead to the accumulation of volatile fatty acids, which will cause environmental stress to the sensitive methanogens consequently. The metabolic characteristics of methanogens under acetate stress can affect the overall performance of mixed consortia. Nevertheless, there exist huge gaps in understanding the responses of the dominant methanogens to the stress, e.g., Methanosarcinaceae. Such methanogens are resistant to environmental deterioration and able to utilize multiple carbon sources. In this study, transcriptomic and proteomic analyses were conducted to explore the responses of Methanosarcina barkeri strain MS at different acetate concentrations of 10, 25, and 50 mM.ResultsThe trend of OD600 and the regulation of the specific genes in 50 mM acetate, indicated that high concentration of acetate promoted the acclimation of M. barkeri to acetate stress. Acetate stress hindered the regulation of quorum sensing and thereby eliminated the advantages of cell aggregation, which was beneficial to resist stress. Under acetate stress, M. barkeri allocated more resources to enhance the uptake of iron to maintain the integrities of electron-transport chains and other essential biological processes. Comparing with the initial stages of different acetate concentrations, most of the genes participating in acetoclastic methanogenesis did not show significantly different expressions except hdrB1C1, an electron-bifurcating heterodisulfide reductase participating in energy conversion and improving thermodynamic efficiency. Meanwhile, vnfDGHK and nifDHK participating in nitrogen fixation pathway were upregulated.ConclusionIn this work, transcriptomic and proteomic analyses are combined to reveal the responses of M. barkeri to acetate stress in terms of central metabolic pathways, which provides basic clues for exploring the responses of other specific methanogens under high organics load. Moreover, the results can also be used to gain insights into the complex interactions and geochemical cycles among natural or engineered populations. Furthermore, these findings also provide the potential for designing effective and robust anaerobic digesters with high organic loads.

Dataset Information

The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.

Publications

The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.

Similar Datasets

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