The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G؉C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G؉C content of the 16S rRNA genes (P GC ) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P GC and optimal growth temperatures (T opt ) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.
Over the past decade, many studies on the anaerobic oxidation of methane (AOM) have been published (for a review, see reference 1), emphasizing the global distribution of this process in marine sediments and its importance as a factor that reduces the level of methane emissions to the atmosphere. The oxidation of methane by archaea is assumed to be a reverse methanogenesis coupled to the reduction of sulfate by sulfate-reducing bacteria (2). This process occurs in marine habitats where sulfate from the seawater and methane of biological and geochemical origin from deeper layers meet (1). Based on the 16S rRNA phylogeny, all anaerobic methanotrophic archaea (ANME) are grouped into three distinct clusters of Euryarchaeota, namely, ANME-1, ANME-2, and ANME-3 (3, 4). Microorganisms of the ANME-2 and ANME-3 clusters belong to Methanosarcinales, whereas the ANME-1 cluster is a deep phylogenetic branch of Euryarchaeota only distantly related to the orders Methanomicrobiales and Methanosarcinales (5). Despite the great interest in these microorganisms, all three clusters remain uncultured. It is assumed that one of the major obstacles to the isolation of AOM-mediating microorganisms is their slow growth, the main reason for which is presumably bioenergetic limitations caused by the very low energy yield of AOM (6). According to theoretical calculations, the freeenergy yield (⌬G) of sulfate-dependent oxidation of methane increases with increasing temperature (7), s...