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Abstract

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic growth. Although motility has not been observed in any Methanosarcineae, a flagellin gene cluster and two complete chemotaxis gene clusters were identified. The availability of genetic methods, coupled with its physiological and metabolic diversity, makes M. acetivorans a powerful model organism for the study of archaeal biology. [Sequence, data, annotations and analyses are available at http://www-genome.wi.mit.edu/.]

Figures

Figure 1
Figure 1
Three pathways for methanogenesis. Methanogenesis is a form of anaerobic respiration using a variety of one-carbon (C-1) compounds or acetic acid as a terminal electron acceptor. All three pathways converge on the reduction of methyl-CoM to methane (CH4). Many methanogens can reduce CO2 to methane using electrons derived by oxidizing H2 (the hydrogenotrophic pathway, red arrows). Others can utilize C-1 compounds such as methanol or methylamines with one molecule of C-1 compound being oxidized to provide electrons for reducing three additional molecules to methane (the methylotrophic pathway, green arrows). Still other methanogens split acetate into a methyl group and an enzyme-bound CO, with the CO subsequently oxidized to provide electrons for the reduction of the methyl group to methane (the acetoclastic pathway, blue arrows). In all cases, an electrochemical gradient is generated for use in ATP synthesis. Most methanogens possess only one of the three methanogenic pathways. Methanosarcina species possess all three. CoM, coenzyme M; H4SPT, tetrahydrosarcinapterin; MF, methanofuran.
Figure 2
Figure 2
Different morphological forms of Methanosarcina acetivorans. Thin-section electron micrographs showing M. acetivorans growing as both single cells (center of micrograph) and within multicellular aggregates (top left, bottom right). Cells were harvested during late-exponential growth in medium containing sodium acetate and prepared for electron microscopy as described previously (Sowers and Ferry 1983). Electron micrographs were taken with a JOEL JEM 100B transmission electron microscope.
Figure 3
Figure 3
Universal tree of life based on small-subunit ribosomal (SSU) RNA sequences. Selected sequenced Archaea are highlighted and have their genome sizes indicated. All known methane-producing organisms are members of the domain Archaea (shown in green). The Methanosarcineae are the most metabolically and environmentally diverse methanogens. They are also members of the lineage giving rise to the anaerobic sulfate-reducing Archaeoglobus fulgidus (shown in yellow) and the aerobic halophilic Halobacterium species (shown in blue).
Figure 4
Figure 4
Redundant and novel genes involved in methylotrophic and acetoclastic methanogenesis. (A) The pathway for conversion of one-carbon (C-1) compounds to the central-pathway intermediate methyl-CoM (see Fig. 1). Multiple copies of substrate-specific methyltransferases (MT1 and MT2) and methylotrophic corrinoid proteins were identified for each substrate (CH3-X). The gene IDs (MA numbers) for proteins involved in the metabolism of methanol (green) and methylamine (pink); dimethylamine (red) and trimethylamine (dark red) are indicated. Five corrinoid and 12 MT2 homologs without known substrates were identified. Two shown in yellow form a putative operon; three shown in light blue comprise a paralogous family with fused corrinoid and MT2 domains. Additional MT1 homologs were not identified as these proteins do not constitute a homologous family. (B) The pathway for conversion of acetate to the central-pathway intermediate methyl-H4MPT. Two nearly identical copies of genes encoding the acetyl-CoA decarbonylase/synthase (ACDS) complex were identified (homologous genes indicated by identical color, a third lone copy of cdhA [MA4399] was also identified). Two genes (cyan) encoding single-subunit, bacterial-type CO2 dehydrogenase (CODH) proteins were also identified suggesting the possibility of exogenous CO metabolism (see text for details).
Figure 5
Figure 5
Diversity of surface proteins. Consistent with the unique ability of M. acetivorans among the archaea to form complex multicellular structures, a large number and diverse array of surface proteins were identified. Illustrated are examples of domain architectures that contain β-propeller, polycystic kidney disease (PKD), and β-helix domains. The PKD and YVTN β-domains share sequence similarity to metazoan cell-adhesion molecules, suggesting a role for these proteins in the formation of multicellular structures. The domains were identified by similarity to structurally defined domains in M. mazei surface antigens (H. Jing, in prep.). The helical transmembrane regions (HTM) were predicted using PHDhtm (Rost et al. 1996). The Ca2+-binding β-hairpin motifs were predicted based on sequence similarity with a previously identified Ca2+-binding motif (Springer et al. 2000). One β-propeller domain appears intermediate between YVTN and WD40 β-propellers.

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