Deconstructing Methanosarcina acetivorans into an acetogenic archaeon.

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide-dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

Pyruvate-dependent growth of Methanosarcina acetivorans.

Methanogenesis is a key step during anaerobic biomass degradation. Methanogenic archaea (methanogens) are the only organisms coupling methanogenic substrate conversion to energy conservation. The range of substrates utilized by methanogens is limited, with acetate and H2+CO2 being the ecologically most relevant. The only single methanogenic energy substrate containing more carbon-carbon bonds than acetate is pyruvate. Only the aggregate-forming, freshwater methanogen Methanosarcina barkeri Fusaro was shown to grow on this compound. Here, the pyruvate-utilizing capabilities of the single-celled, marine Methanosarcina acetivorans were addressed. Robust pyruvate-dependent, methanogenic, growth could be established by omitting CO2 from the growth medium. Growth rates which were independent of the pyruvate concentration indicated that M. acetivorans actively translocates pyruvate across the cytoplasmic membrane. When 2-bromoethanesulfonate (BES) inhibited methanogenesis to more than 99%, pyruvate-dependent growth was acetogenic and sustained. However, when methanogenesis was completely inhibited M. acetivorans did not grow on pyruvate. Analysis of metabolites showed that acetogenesis is used by BES-inhibited M. acetivorans as a sink for electrons derived from pyruvate oxidation and that other, thus far unidentified, metabolites are produced.IMPORTANCEThe known range of methanogenic growth substrates is very limited and M. acetivorans is only the second methanogenic species for which growth on pyruvate is demonstrated. Besides some commonalities, analysis of M. acetivorans highlights differences in pyruvate metabolism among Methanosarcina species. The observation that M. acetivorans probably imports pyruvate actively indicates that the capabilities for heterotrophic catabolism in methanogens may be underestimated. The mostly acetogenic growth of M. acetivorans on pyruvate with concomitant inhibition of methanogenesis confirms that energy conservation of methanogenic archaea can be independent of methane formation.

Genetic and Physiological Probing of Cytoplasmic Bypasses for the Energy-Converting Methyltransferase Mtr in Methanosarcina acetivorans.

Methanogenesis is a unique energy metabolism carried out by members of the domain Archaea. Unlike most other methanogens, which reduce CO2 to methane with hydrogen as the electron donor, Methanosarcina acetivorans is able to grow on methylated compounds, on acetate, and on carbon monoxide (CO). These substrates are metabolized via distinct yet overlapping pathways. For the use of any single methanogenic substrate, the membrane-integral, energy-converting N5-methyl-tetrahydrosarcinapterin (H4SPT):coenzyme M (HS-CoM) methyltransferase (Mtr) is required. It was proposed that M. acetivorans can bypass the methyl transfer catalyzed by Mtr via cytoplasmic activities. To address this issue, conversion of different energy substrates by an mtr deletion mutant was analyzed. No significant methyl transfer from H4SPT to HS-CoM could be detected with CO as the electron donor. In contrast, formation of methane and CO2 in the presence of methanol or trimethylamine was indicative of an Mtr bypass in the oxidative direction. As methane thiol and dimethyl sulfide were transiently produced during methylotrophic methanogenesis in the mtr mutant, involvement in this process of methyl sulfide-dependent methyltransferases (Mts) was analyzed in a strain lacking both the Mts system and Mtr. It could be unequivocally demonstrated that the Mts system is not involved in bypassing Mtr, thereby ruling out previous proposals. Conversion of [13C]methanol indicated that in the absence of Mtr M. acetivorans provides the reducing equivalents for methyl-S-CoM reduction to methane by oxidizing (an) intracellular compound(s) to CO2 rather than disproportioning the source of methyl groups. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans. IMPORTANCE Methanogenic archaea possess only a limited number of chemiosmotic coupling sites in their respiratory chains. Among them, N5-methyl-H4SPT:HS-CoM methyltransferase (Mtr) is the most widely distributed. Previous observations led to the conclusion that Methanosarcina acetivorans is able to bypass this reaction via methyl sulfide-dependent methyltransferases (Mts). However, strains lacking Mtr are not able to produce methane from CO. Also, these strains are unable to oxidize methylated substrates to CO2, in contrast to observations in the close relative Methanosarcina barkeri. The results also highlight the sole function of the Mts system in methyl sulfide metabolism. Thus, no in vivo Mtr bypass appears to exist in M. acetivorans.

Identification of a unique Radical SAM methyltransferase required for the sp3-C-methylation of an arginine residue of methyl-coenzyme M reductase.

The biological formation of methane (methanogenesis) is a globally important process, which is exploited in biogas technology, but also contributes to global warming through the release of a potent greenhouse gas into the atmosphere. The last and methane-releasing step of methanogenesis is catalysed by the enzyme methyl-coenzyme M reductase (MCR), which carries several exceptional posttranslational amino acid modifications. Among these, a 5-C-(S)-methylarginine is located close to the active site of the enzyme. Here, we show that a unique Radical S-adenosyl-L-methionine (SAM) methyltransferase is required for the methylation of the arginine residue. The gene encoding the methyltransferase is currently annotated as "methanogenesis marker 10" whose function was unknown until now. The deletion of the methyltransferase gene ma4551 in Methanosarcina acetivorans WWM1 leads to the production of an active MCR lacking the C-5-methylation of the respective arginine residue. The growth behaviour of the corresponding M. acetivorans mutant strain and the biophysical characterization of the isolated MCR indicate that the methylated arginine is important for MCR stability under stress conditions.