A large fraction of globally produced methane is converted to CO2 by anaerobic oxidation in marine sediments. Strong geochemical evidence for net methane consumption in anoxic sediments is based on methane profiles, radiotracer experiments and stable carbon isotope data. But the elusive microorganisms mediating this reaction have not yet been isolated, and the pathway of anaerobic oxidation of methane is insufficiently understood. Recent data suggest that certain archaea reverse the process of methanogenesis by interaction with sulphate-reducing bacteria. Here we provide microscopic evidence for a structured consortium of archaea and sulphate-reducing bacteria, which we identified by fluorescence in situ hybridization using specific 16S rRNA-targeted oligonucleotide probes. In this example of a structured archaeal-bacterial symbiosis, the archaea grow in dense aggregates of about 100 cells and are surrounded by sulphate-reducing bacteria. These aggregates were abundant in gas-hydrate-rich sediments with extremely high rates of methane-based sulphate reduction, and apparently mediate anaerobic oxidation of methane.

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group-and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell ؊1 day ؊1 ), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.

A 16S ribosomal DNA (rDNA) clone library from permanently cold marine sediments was established. Screening 353 clones by dot blot hybridization with group-specific oligonucleotide probes suggested a predominance of sequences related to bacteria of the sulfur cycle (43.4% potential sulfate reducers). Within this fraction, the major cluster (19.0%) was affiliated with Desulfotalea sp. and other closely related psychrophilic sulfate reducers isolated from the same habitat. The cloned sequences showed between 93 and 100% similarity to these bacteria. Two additional groups were frequently encountered: 13% of the clones were related to Desulfuromonas palmitatis, and a second group was affiliated withMyxobacteria spp. and Bdellovibrio spp. Many clones (18.1%) belonged to the γ subclass of the classProteobacteria and were closest to symbiotic or free-living sulfur oxidizers. Probe target groups were further characterized by amplified rDNA restriction analysis to determine diversity within the groups and within the clone library. Rarefaction analysis suggested that the total diversity assessed by 16S rDNA analysis was very high in these permanently cold sediments and was only partially revealed by screening of 353 clones.

Fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization with 16S rRNA-targeted oligonucleotide probes were used to investigate the phylogenetic composition of a marine Arctic sediment (Svalbard). FISH resulted in the detection of a large fraction of microbes living in the top 5 cm of the sediment. Up to 65.4% ؎ 7.5% of total DAPI (4,6-diamidino-2-phenylindole) cell counts hybridized to the bacterial probe EUB338, and up to 4.9% ؎ 1.5% hybridized to the archaeal probe ARCH915. Besides ␦-proteobacterial sulfate-reducing bacteria (up to 16% 52) members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in this sediment, accounting for up to 12.8% of total DAPI cell counts and up to 6.1% of prokaryotic rRNA. Furthermore, members of the order Planctomycetales accounted for up to 3.9% of total cell counts. In accordance with previous studies, these findings support the hypothesis that these bacterial groups are not simply settling with organic matter from the pelagic zone but are indigenous to the anoxic zones of marine sediments. Members of the ␥-proteobacteria also constituted a significant fraction in this sediment (6.1% ؎ 2.5% of total cell counts, 14.4% ؎ 3.6% of prokaryotic rRNA). A new probe (GAM660) specific for sequences affiliated with free-living or endosymbiotic sulfur-oxidizing bacteria was developed. A significant number of cells was detected by this probe (2.1% ؎ 0.7% of total DAPI cell counts, 13.2% ؎ 4.6% of prokaryotic rRNA), showing no clear zonation along the vertical profile. Gram-positive bacteria and the ␤-proteobacteria were near the detection limit in all sediments.Knowledge of the microbial diversity of marine pelagic and benthic communities has been greatly extended recently by molecular studies based on the analysis of 16S rDNA sequences (see, for example, references 9, 10, 20, 43, 44, 48, 50, 52, and 67). Numerous new 16S rDNA sequences have been retrieved both from marine sediments and from the water column, indicating that the vast majority of species has not been cultivated yet. Several studies using the cultivation-independent approach of 16S rDNA cloning have helped to elucidate common features within the microbial communities of specific habitats such as marine benthic environments (10,35,36,52,67). Furthermore, they have provided additional sequence information for the design and evaluation of nucleic acid probes for the identification and quantification of distinct bacterial populations.While microbial diversity can be readily studied by PCRbased 16S rDNA cloning, community structure cannot be deduced from cloning studies (3) due to potential biases introduced during DNA retrieval and amplification (17,53,65). For reliable characterization of community structure, quantitative methods such as fluorescence in situ hybridization (FISH) or rRNA slot blot hybridization are more suitable (3). To date, a number of studies have been performed using either of these two methods to quantify different groups in marine sediments (15,37,38,51,(57)(5...

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