"Methanoplasmatales," Thermoplasmatales-related archaea in termite guts and other environments, are the seventh order of methanogens

Abstract

The Euryarchaeota comprise both methanogenic and nonmethanogenic orders and many lineages of uncultivated archaea with unknown properties. One of these deep-branching lineages, distantly related to the Thermoplasmatales, has been discovered in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals. By comparative phylogenetic analysis, we connected this lineage of 16S rRNA genes to a large clade of unknown mcrA gene sequences, a functional marker for methanogenesis, obtained from the same habitats. The identical topologies of 16S rRNA and mcrA gene trees and the perfect congruence of all branches, including several novel groups that we obtained from the guts of termites and cockroaches, strongly suggested that they stem from the same microorganisms. This was further corroborated by two highly enriched cultures of closely related methanogens from the guts of a higher termite (Cubitermes ugandensis) and a millipede (Anadenobolus sp.), which represented one of the arthropod-specific clusters in the respective trees. Numerous other pairs of habitat-specific sequence clusters were obtained from the guts of other termites and cockroaches but were also found in previously published data sets from the intestinal tracts of mammals (e.g., rumen cluster C) and other environments. Together with the recently described Methanomassiliicoccus luminyensis isolated from human feces, which falls into rice cluster III, the results of our study strongly support the idea that the entire clade of "uncultured Thermoplasmatales" in fact represents the seventh order of methanogenic archaea, for which the provisional name "Methanoplasmatales" is proposed.

Fig 1

Phylogenetic tree showing the relationships among uncultured archaea related to Thermoplasmatales and to representatives of all other orders of methanogenic archaea and the ANME-1 group. Clusters of clones from termite (TC) and cockroach (CC) guts are indicated. The tree is based on a maximum-likelihood analysis of an alignment of archaeal 16S rRNA genes (1,250 bp) of archaea in public databases; sequences obtained in this study are marked in boldface. Sequences of Trinervitermes sp., M. michaelseni, and A. trestus were shorter and were added to the tree using the ARB parsimony tool. The bullets indicate bootstrap support (●, >95%; ○, >70%). The scale bar indicates substitutions per site.

Fig 2

Phylogenetic tree showing the relationships among the novel mcrA genes and to representative mcrA genes of all other orders of methanogenic archaea and the ANME-1 group. Clusters of clones from termite (TC) and cockroach (CC) guts are indicated. The tree is based on a maximum-likelihood analysis of an alignment of the mcrA genes (140 amino acids) of archaea in public databases; sequences obtained in this study are marked in boldface. The bullets indicate bootstrap support (●, >95%; ○, >70%). The scale bar indicates substitutions per site.

Fig 3

Time course of methane partial pressure in the headspace of the enrichment culture MpT1 (N₂-CO₂; 80/20) inoculated from a methanol-starved preculture into basal medium supplemented with different substrates: H₂ (50 kPa in headspace), methanol (50 mM), or acetate (30 mM). The values are means of two cultures; mean standard deviations are shown only if they are larger than the symbols.

Fig 4

Phase-contrast photomicrographs of the methanogenic enrichment cultures MpT1 (A) and MpM2 (B) after several transfers in basal medium supplemented with H₂ and methanol. Both cultures consisted mostly of small roundish cells (diameter, 0.6 to 1.0 μm).

Fig 5

Composition of the enrichment culture of strain MpT1, determined by quantitative real-time PCR of bacterial and archaeal 16S rRNA genes. 454 pyrotag sequencing revealed that the bacterial contaminants belonged almost exclusively to families of the order Clostridiales.

Fig 6

Tanglegram illustrating the congruence of the phylogenies of Thermoplasmatales-related archaea (16S rRNA) and the mcrA genes of unknown origin (for details, see Fig. 1 and 2). Sequence pairs stemming from the same study are connected by dashed lines. Sequences obtained in this study are marked in boldface. The bullets indicate bootstrap support (●, >95%; ○, >70%). The scale bars indicate substitutions per site.