Loss of the
            <i>mtr</i>
            operon in
            <i>Methanosarcina</i>
            blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway

Welander, Paula V.; Metcalf, William W.

doi:10.1073/pnas.0502623102

Cited by 85 publications

(71 citation statements)

References 31 publications

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“…Whereas most methanogenic species grow by obligate CO 2 reduction with H 2 , methyl reduction with H 2 , aceticlastic fermentation of acetate, or methylotrophic catabolism of methanol, methylated amines, and dimethylsulfide, most Methanosarcina spp. can grow by all four catabolic pathways (49). Methanosarcina acetivorans was recently reported to grow also nonmethanogenically with CO (35).…”

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The Methanosarcina barkeri Genome: Comparative Analysis with Methanosarcina acetivorans and Methanosarcina mazei Reveals Extensive Rearrangement within Methanosarcinal Genomes

et al. 2006

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We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. The genome of M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcina spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in M. barkeri, 746 had homologs with better than 80% identity to both M. acetivorans and M. mazei, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for N-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in M. barkeri. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large M. acetivorans genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the M. barkeri genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short M. mazei genome most closely approximates the putative ancestral organizational state of these species.Biological methanogenesis by the methane-producing archaea has a significant role in the global carbon cycle. This process is one of several anaerobic degradative processes that complement aerobic degradation by utilizing alternative electron acceptors in habitats where O 2 is not available (39). The efficiency of this microbial process is directly dependent upon the interaction of three metabolically distinct groups of microorganisms: the fermentative and acetogenic bacteria and the methanogenic archaea. The methanogenic archaea have two pivotal roles in methanogenic consortia (28). By consuming hydrogen for methanogenesis and effectively lowering its partial pressure by the process of interspecies hydrogen exchange, the methanogens provide a thermodynamically favorable environment for the fermentative and acetogenic species to utilize protons as electron acceptors. This interaction enables fermenters to conserve more energy by producing a more oxidized product, acetate, which is also a substrate for methanogenesis. The second role of the methanogens is the fermentation of acetate, which accounts for 70% of the global methane produced by biological methane production (28). The net effect of these microbial interactions is the diversion of protons to hydrogen and carbon to acetate, which ultimately yields methane and carbon dioxide via methanogenesis.The genus M...

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The Methanosarcina barkeri Genome: Comparative Analysis with Methanosarcina acetivorans and Methanosarcina mazei Reveals Extensive Rearrangement within Methanosarcinal Genomes

et al. 2006

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“…This reaction is catalyzed by the N 5 -methyl-H 4 MPT:CoM methyltransferase (Mtr), an eight-subunit membrane-bound enzyme encoded by the mtrECDBAFGH operon (10). We have demonstrated that Mtr is dispensable in M. barkeri under a variety of conditions but is required for growth via the hydrogenotrophic, aceticlastic, and methylotrophic pathways (26). Interestingly, although a ⌬mtr deletion mutant is unable to utilize methanol as a growth substrate, ⌬mtr cells are still able to oxidize methanol to CO 2 , clearly demonstrating that the Mtr-catalyzed reaction must be bypassed in M. barkeri.…”

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“…Labeling studies showed that growth on this combination of substrates occurred via a new methanogenic pathway in which acetate oxidation was used to drive methanogenic reduction of methanol. Because Mtr activity is sodium dependent, it seems possible that this pathway may be physiologically relevant in low-sodium environments (26).…”

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Mutagenesis of the C1 Oxidation Pathway in Methanosarcina barkeri : New Insights into the Mtr/Mer Bypass Pathway

Welander

Metcalf

2008

J Bacteriol

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(⌬ftr) all failed to grow using either methanol or H 2 /CO 2 as a growth substrate, indicating that there is an absolute requirement for the C1 oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the ⌬mtr and ⌬mer mutants were capable of producing methane from this substrate, whereas the ⌬mtd and ⌬ftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-H 4 MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in ⌬mtr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and H 4 MPT in vitro. Both the ⌬mer and ⌬mtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.Biologically mediated methanogenesis is carried out by a diverse group of anaerobic organisms, known as methanoarchaea, that obtain all of their energy for growth by converting a limited number of substrates to methane (CH 4 ) (5). These organisms are found in a variety of environments throughout the world, including both man-made and natural anaerobic habitats in which there are limited amounts of light, sulfate, and nitrate but which contain complex organic compounds (4). Despite the enormous phylogenetic diversity among the methanogens, the metabolism of these organisms is extremely specialized, and all known species are obligate methanogens. Four discrete methanogenic pathways are known: (i) the CO 2 reduction or hydrogenotrophic pathway involves the reduction of CO 2 to CH 4 with hydrogen gas (H 2 ) as the reductant; (ii) the methylotrophic pathway involves the disproportionation of methylated compounds, such as methanol and methylamines, to CO 2 and CH 4 ; (iii) the methyl reduction pathway results in the direct reduction of methanol to CH 4 with H 2 as the reductant; and (iv) the acetoclastic pathway involves the dismutation of acetate to CO 2 and CH 4 (7). A key series of reactions in both the methylotrophic and hydrogenotrophic pathways involves the reversible oxidation/reduction of coenzyme-bound one-carbon units (the C1 oxidation/reduction pathway). Extensive in vitro biochemical studies over the years have characterized the unique proteins involved in this pathway, leading to elucidation of the pathway shown in Fig. 1 (4, 7, 23). However, in vivo studies of the pathway were not possible until the relatively recent development of tools for facile genetic manipulation of Methanosarcina species (21).Genetic studies of the C1 oxidation/reduction pathway in Methanosarcina barkeri have shown ...

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“…Methanosarcina is the only known genus of methanogens with members that can utilize all of the known methanogenic pathways (acetoclastic, methylotrophic, hydrogenotrophic, and methyl reducing) (71). This metabolic diversity makes these species more permissive to metabolic and genetic manipulations than other methanogens.…”

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Genome-Scale Metabolic Reconstruction and Hypothesis Testing in the Methanogenic Archaeon Methanosarcina acetivorans C2A

et al. 2012

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Methanosarcina acetivorans strain C2A is a marine methanogenic archaeon notable for its substrate utilization, genetic tractability, and novel energy conservation mechanisms. To help probe the phenotypic implications of this organism's unique metabolism, we have constructed and manually curated a genome-scale metabolic model of M. acetivorans, iMB745, which accounts for 745 of the 4,540 predicted protein-coding genes (16%) in the M. acetivorans genome. The reconstruction effort has identified key knowledge gaps and differences in peripheral and central metabolism between methanogenic species. Using flux balance analysis, the model quantitatively predicts wild-type phenotypes and is 96% accurate in knockout lethality predictions compared to currently available experimental data. The model was used to probe the mechanisms and energetics of by-product formation and growth on carbon monoxide, as well as the nature of the reaction catalyzed by the soluble heterodisulfide reductase HdrABC in M. acetivorans. The genome-scale model provides quantitative and qualitative hypotheses that can be used to help iteratively guide additional experiments to further the state of knowledge about methanogenesis. Methanogenic archaea are unique in their ability to grow on low-energy substrates, such as acetic acid, by converting them into methane and other by-products. Methanogens are a critical part of the global carbon cycle, consuming by-products of other natural bioprocesses that would otherwise be recalcitrant in sulfate-poor, anaerobic environments (12). They also play an important role in global warming, since methane is a greenhouse gas 20 times as potent as carbon dioxide (42) and methanogenesis is the primary mechanism for the emission of methane into the atmosphere (2).Methanosarcina is the only known genus of methanogens with members that can utilize all of the known methanogenic pathways (acetoclastic, methylotrophic, hydrogenotrophic, and methyl reducing) (71). This metabolic diversity makes these species more permissive to metabolic and genetic manipulations than other methanogens. To capitalize on this characteristic, the genomes of three Methanosarcina species have been sequenced (15,22,38). In addition, genetic tools have been developed for several of these species, including methods for directed mutagenesis and regulated expression of specific genes (3,34,73,74).The constraint-based reconstruction and analysis (COBRA) strategy is a powerful paradigm for consolidating large amounts of metabolic knowledge and synthesizing that knowledge into quantitative phenotypic predictions (45, 51). For the performance of constraint-based analysis on an individual organism, its metabolic network is reconstructed from the bottom up, beginning with a sequenced and annotated genome and ending with a network of reactions and reaction-gene associations that directly link genotype and phenotype (68). Many metabolic reconstructions have been curated by hand and have been used to make useful predictions, such as the identification of put...

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Loss of the mtr operon in Methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway

Cited by 85 publications

References 31 publications

The Methanosarcina barkeri Genome: Comparative Analysis with Methanosarcina acetivorans and Methanosarcina mazei Reveals Extensive Rearrangement within Methanosarcinal Genomes

The Methanosarcina barkeri Genome: Comparative Analysis with Methanosarcina acetivorans and Methanosarcina mazei Reveals Extensive Rearrangement within Methanosarcinal Genomes

Mutagenesis of the C1 Oxidation Pathway in Methanosarcina barkeri : New Insights into the Mtr/Mer Bypass Pathway

Genome-Scale Metabolic Reconstruction and Hypothesis Testing in the Methanogenic Archaeon Methanosarcina acetivorans C2A

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