Methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A (mcrA) genes in hydrothermal sediments of the Guaymas Basin

Abstract

The methanogenic community in hydrothermally active sediments of Guaymas Basin (Gulf of California, Mexico) was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Members of the Methanomicrobiales and Methanosarcinales dominated the mcrA and 16S rRNA clone libraries from the upper 15 cm of the sediments. Within the H2/CO2- and formate-utilizing family Methanomicrobiales, two mcrA and 16S rRNA lineages were closely affiliated with cultured species of the genera Methanoculleus and Methanocorpusculum. The most frequently recovered mcrA PCR amplicons within the Methanomicrobiales did not branch with any cultured genera. Within the nutritionally versatile family Methanosarcinales, one 16S rRNA amplicon and most of the mcrA PCR amplicons were affiliated with the obligately acetate utilizing species Methanosaeta concilii. The mcrA clone libraries also included phylotypes related to the methyl-disproportionating genus Methanococcoides. However, two mcrA and two 16S rRNA lineages within the Methanosarcinales were unrelated to any cultured genus. Overall, the clone libraries indicate a diversified methanogen community that uses H2/CO2, formate, acetate, and methylated substrates. Phylogenetic affiliations of mcrA and 16S rRNA clones with thermophilic and nonthermophilic cultured isolates indicate a mixed mesophilic and thermophilic methanogen community in the surficial Guaymas sediments.

FIG. 1.

Phylogenetic tree based on translated, partial amino acid sequences of mcrA genes from methanogenic archaea. Sequences were retrieved from Guaymas core B sediment layers (1 to 15 cm). The tree topology and posterior probability were constructed using Bayesian analyses. For sequences representing multiple, near-identical clones, the number of additional clones is given in parentheses.

FIG. 2.

Phylogenetic tree based on 16S rRNA sequences of archaeal clones from Guaymas core B sediment layers (1 to 15 cm). The tree was constructed with PAUP*. Bootstrap values based on 500 replicates for each distance and parsimony are shown for branches with more than 50% bootstrap support. The labeled brackets define order- or phylum-level lineages of cultured and uncultured archaea and specific lineages of methane-oxidizing archaea (ANME-1, ANME-2).

FIG. 3.

Concentration profiles of sulfate, acetate, propionate, and formate (A) and of n-butyrate, isobutyrate, and isovalerate (B) and in situ temperature profiles (B) in Guaymas sediments. Acetate concentrations in panel A are scaled down by a factor of 10 to match the scale of the other low-molecular-weight compounds. See Materials and Methods for details on temperature profiles.