Molecular phylogeny of methanogens associated with flagellated protists in the gut and with the gut epithelium of termites.

The molecular phylogeny of methanogenic archaea associated with the flagellated protist species Dinenympha and Microjoenia in the gut of termites, Reticulitermes speratus and Hodotermopsis sjoestedti, and those attached to the gut epithelium was examined based on PCR-amplified small-subunit ribosomal RNA genes. The sequences identified were classified into six groups within the genus Methanobrevibacter, including groups of yet uncharacterized novel species. Closely related methanogens were shared between Microjoenia and some Dinenympha cells in each termite. The methanogens harbored by the flagellates were phylogenetically different from the methanogens associated with the gut epithelium, suggesting that distinct methanogen species showed distinct spatial distributions in the termite gut.

Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotoides, and parabasalian symbionts in the hindgut of termites.

The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent-labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole-cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology-based taxonomic groups. The analysis of the tree-root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep-branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.

Phylogenetic study of methanogens associated with rumen ciliates.

The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated. Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted. Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture. The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109. Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer. Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences. RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation. The association of Methanobrevibacter spp. with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M. smithii.

Sulphate reduction and methanogenesis in the ovine rumen and porcine caecum: a comparison of two microbial ecosystems.

A series of experiments was conducted to study the relationship between methanogenesis and sulphate reduction in ovine rumen and porcine caecum. Effect of 2-bromoethane sulphonate on hydrogen production by digesta suggested that the most important H2-disposal system in the rumen is methanogenesis and that methanogenesis is not predominant H2-disposal system in the porcine caecum. This inference was supported by the difference in predominant H2-utilizers in these two microbial ecosystems; Methanogenic bacteria (MB) were predominant in the rumen and sulphate reducing bacteria (SRB) were predominant in porcine caecum. Free sulphate levels in digesta appear to affect the relationship between MB and SRB. Sulphate levels in the rumen were likely to be insufficient for SRB to outcompete MB.