Genomic characterization of methanomicrobiales reveals three classes of methanogens

Abstract

Background: Methanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens.

Methodology/principal findings: In order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales.

Conclusions/significance: Our results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

Figure 1. Proposed pathway for methanogenesis in Methanomicrobiales.

Methanomicrobiales are predicted to couple formylmethanofuran formation and CoM-CoB heterodisulfide reduction to ion gradients. Fd: ferredoxin; MF: methanofuran; H₄MPT: tetrahydromethanopterin.

Figure 2. Alternate pathways for synthesis of 2-oxoglutarate from oxaloacetate.

Class I methanogens and Methanomicrobiales use a partial reductive citric acid cycle while Methanosarcinales use a partial oxidative citric acid cycle.

Figure 3. Phylogenetic tree of methanogens based on seven core enzymes of methanogenesis and cofactor biosynthesis.

See Materials and Methods for a list of the proteins and organisms included. Protein sequences were concatenated and aligned with Clustal W. The tree was generated with MrBayes 3.1.2 and viewed with TreeView.

Figure 4. Venn diagram of signature clusters.

The clusters were generated using a spectral clustering procedure (see Materials and Methods section for details). Signature protein clusters were identified as clusters for which a member protein was present in every analyzed species from one or more classes of methanogens. The number of exclusive, shared, and common signature clusters associated with each methanogen class are shown. The functions of characterized proteins belonging to exclusive signature clusters and to clusters shared between the Methanomicrobiales and the Methanosarcinales are also noted.