SEEP-SRB1: phylogeny, subclusters and probe design
In a first step to test the proposed association between SEEP-SRB1 bacteria and ANME-2 archaea, the diversity of bacterial 16S rRNA genes in two ANME-2 dominated AOM enrichment cultures was examined. One enrichment originated from the sediment above gas hydrates at Hydrate Ridge (named HR enrichment) and was previously described (Nauhaus et al., 2007; Holler et al., 2009). The other one was prepared from sediments of the Mediterranean Isis Mud Volcano (named Isis enrichment). Both enrichments were grown over years in the lab and showed similar microbial compositions based on FISH. They were dominated by ANME-2c cells, but also contained a significant population of ANME-2a. The HR enrichment contained 17% ANME-2a and 64% ANME-2c cells, while the Isis enrichment contained 20% ANME-2a and 49% ANME-2c cells. DSS associated with the two ANME-2 subgroups accounted for 18% of the cell population in the HR enrichment and for 26% in the Isis enrichment.
Bacterial 16S rRNA gene clone libraries were constructed from the AOM enrichments. Both clone libraries contained mostly 16S rRNA genes affiliating with Deltaproteobacteria (HR: 71%; Isis: 74%). In addition, the libraries contained sequences related to Firmicutes (HR: 11%; Isis: 2%), Thermomicrobia (HR: 5%; Isis: 6%), Bacteroidetes/Chlorobi (HR: 6%; Isis: 7%) and to a few other groups (for details see Supporting Information, Table S1). Within the deltaproteobacterial sequences, most sequences affiliated with the SEEP-SRB1 group (Knittel et al., 2003). They represented 56% (HR) and 65% (Isis) of the phylotypes in the two libraries, and were all closely related forming a single operational taxonomic unit at a 97% sequence-similarity cut-off.
In 2003, when Knittel and colleagues defined group SEEP-SRB1, the group comprised only 16 sequences (Knittel et al., 2003). In this study, a detailed phylogenetic analysis of currently available SEEP-SRB1 and related deltaproteobacterial sequences was performed. Altogether, more than 150 sequences could be assigned to SEEP-SRB1 (for a selection see Fig. 1). The SEEP-SRB1 group included sequences from well-investigated AOM habitats such as methane seeps and sulfate-methane transition zones, but also from, e.g. mangrove soils or hypersaline mats (Table S2). Cultured representatives did not affiliate with SEEP-SRB1. Based on sequences longer than 1200 bp, the sequence divergence within the SEEP-SRB1 group is currently up to 14%. Phylogenetic analysis with all of the used algorithms consistently yielded six well-supported subgroups within SEEP-SRB1. These subgroups are from hereon referred to as SEEP-SRB1a to SEEP-SRB1f (Fig. 1). The sequence similarities within subgroups SEEP-SRB1a, 1b, 1c, 1d and 1f ranged from ≥ 86% to ≥ 92%. Sequence similarities within SEEP-SRB1e were with ≥ 97% higher, suggesting a more coherent group at the level of a genus. The phylogenetic position of SEEP-SRB1c is still unresolved. The cluster branched only in some calculations together with the other SEEP-SRB1 groups, in other calculations SEEP-SRB1c showed a closer relationship to cultivated DSS microorganisms. The phylogenetic position is therefore shown as a multifurcation. The fact that SEEP-SRB1c sequences are not targeted by the general DSS probe DSS658 (one mismatch next to last position of the probe) is an indication that the dominant partner of ANME-2 is most likely not from the SEEP-SRB1c group.
Almost all of the SEEP-SRB1 sequences obtained from the two enrichments affiliated with subgroup SEEP-SRB1a (HR: 100%, Isis: 95%) (Table S1). Intriguingly, other SEEP-SRB1a sequences exclusively originated from AOM habitats. Moreover, in 16S rRNA gene libraries constructed for other ANME-2 dominated habitats, a fraction of the SEEP-SRB1 sequences often affiliated with the SEEP-SRB1a subgroup. The only exceptions were observed for the Tommeliten and Gullfaks oil field from which only sequences affiliated with SEEP-SRB1d and 1e were obtained (Table S2). However, CARD-FISH confirmed the presence of SEEP-SRB1a bacteria at least in sediments from the Gullfaks oil field (Fig. 2C, Table 3). Thus, SEEP-SRB1a sequences at these sites were possibly missed due to biased clone libraries or an insufficient number of clones analysed.
Figure 2. Cell aggregates of ANME-2 and ANME-3 in AOM enrichments, a Black Sea mat, and sediments from Hydrate Ridge, the Gulf of Mexico, the Gullfaks oil field and the Haakon Mosby Mud Volcano, visualized by CARD-FISH. A. ANME-2/SEEP-SRB1a aggregate as detected by using probe ANME2-538 (red) and probe SEEP1a-1441 (green) in the Isis enrichment. B. ANME-2-aggregate (ANME2-538, red) surrounded by SEEP-SRB1a bacteria (SEEP1a-1441, green) as detected in Hydrate Ridge sediment (station 19-2). C. ANME-2/SEEP-SRB1a-aggregate (ANME2-538, red; SEEP1a-473, green) detected in Gullfaks oil field sediment. D. Association of ANME-2 with a bacterial partner not belonging to the SEEP-SRB1a group. The aggregate was observed in a Gulf of Mexico sediment sample after hybridization with probes ANME2-538 (red), SEEP1a-1441 (green, not present in micrograph) and a simultaneous DAPI-staining (blue). E. ANME-2a-aggregate (ANME2a-647, red) with associated SEEP-SRB1a bacteria (SEEP1a-473, green) as detected in Hydrate Ridge sediment (station 19-2). F. ANME-2c-aggregate (ANME2c-760, red) associated with SEEP-SRB1a bacteria (SEEP1a-473, green) as detected in Hydrate Ridge sediment (station 19-2). G. ANME-3/SEEP-SRB1a aggregates, labelled with probe SEEP1a-1441 (green) and probe ANME3-1249 (Niemann et al., 2006; Lösekann et al., 2007; red) as observed in sediments from the Haakon Mosby Mud Volcano. H. ANME-2/SEEP-SRB1a aggregate (ANME2-538, red; SEEP1a-473, green) as observed in a Black Sea microbial mat sample. All scale bars = 5 µm.
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Table 3. Abundances of single SEEP-SRB1a and ANME-2 cells in sediments at different AOM sites.
| ||Hydrate ridge, St. 19-2a||Hydrate ridge, St. 38a||Isis mud volcano||Gulf of Mexico||Gullfaks oil fieldb|
|Total number of single cells (cm−3)||2.9 × 109||3.5 × 109||1.5 × 109||3.6 × 109||6.7 × 109|
|Single cells showing a signal with probe DSS658||3%||3%||5%||6%||19%|
|8.7 × 107 cm−3||1.1 × 108 cm−3||7.5 × 107 cm−3||2.2 × 108 cm−3||1.3 × 109 cm−3|
|SEEP1a-1441 labelled cells relative to DSS658 labelled cells||17%||9%||13%||8%||n.d. |
|Calculated single SEEP-SRB1a cells||0.5%||0.3%||0.7%||0.5%||9%c|
|1.5 × 107 cm−3||1.1 × 107 cm−3||1.1 × 107 cm−3||1.8 × 107 cm−3||6.0 × 108 cm−3|
|Single cells showing a signal with probe ANME2-538||1%||1%||1%||1%||10%|
|2.9 × 107 cm−3||3.5 × 107 cm−3||1.5 × 107 cm−3||3.6 × 107 cm−3||6.7 × 108 cm−3|
Oligonucleotide probes were designed and tested for each of the six SEEP-SRB1 subgroups (Table S3). Two probes were designed for SEEP-SRB1a, probe SEEP1a-473 and probe SEEP1a-1441 (Table 1). Probe SEEP1a-1441 targeted 98% of all SEEP-SRB1a sequences. Besides SEEP-SRB1a sequences, the probe also targeted a few sequences from Acidobacteria and Desulfobacterales not affiliated with the SEEP-SRB1 group (Table 1). The second probe designed for subgroup SEEP-SRB1a, probe SEEP1a-473, targeted 77% of the sequences within group SEEP-SRB1a. SEEP-SRB1a sequences not targeted by SEEP1a-473 exhibited 1–4 mismatches to the probe sequence. SEEP1a-473 is currently not targeting any non-SEEP-SRB1a sequence. The probe was used in combination with two helper probes (Fuchs et al., 2000), HSEEP1a-453 and HSEEP1a-491 (Table 1), to increase signal intensity. Probe SEEP1c-1309 was designed for the SEEP-SRB1c subcluster. It targeted 92% of all SEEP-SRB1c affiliated sequences. Non-SEEP-SRB1c sequences targeted by the probe included sequences from Fibrobacteres, Bacteroidetes/Chlorobi and non-SEEP-SRB1 Deltaproteobacteria (Table 1). Furthermore, probe SEEP1f-153 was designed for group SEEP-SRB1f. The probe targeted 90% of all SEEP-SRB1f affiliated sequences. Besides that, the probe also targeted a few sequences from Acidobacteria, Chloroflexi and Deltaproteobacteria not affiliated with the SEEP-SRB1 group (Table 1). Probes designed for subgroups SEEP-SRB1b, 1d and 1e (Table S3) did not show sufficient signal intensity or specificity during probe testing, even when used in combination with unlabelled helper or competitor oligonucleotides respectively. However, they may be used for other molecular techniques in future studies, e.g. as primers for polymerase chain reactions.
Table 1. SEEP-SRB1 specific oligonucleotide probes.
|Probe||Specificity||Sequence (5′ to 3′)||Position||FA conc. (%, vol/vol)||Hits in target groupa||Outgroup hitsb|
|SEEP1a-473 (S-*-SP1a-0473-a-A-23)||SEEP-SRB1a group||TTC AGT GAT ACC GTC AGT ATC CC||473–495||30||79/102 (77%)||–|
|HSEEP1a-453 (S-*-SP1a-0453-a-A-21)||Helper 1 for SEEP1a-473||RCG RTA TTR RCG CGG RAT AGG||453–472||n/a||n/a||n/a|
|HSEEP1a-496 (S-*-SP1a-0496-a-A-21)||Helper 2 for SEEP1a-473||ACG GAG TTA GCC GGT GCT TCC||496–516||n/a||n/a||n/a|
|SEEP1a-1441 (S-*-SP1a-1441-a-A-18)||SEEP-SRB1a group||CCC CTT GCG GGT TGG TCC||1441–1470||45||85/87 (98%)||Desulfobacterales||19|
|SEEP1c-1309 (S-*-SP1c-1309-a-A-21)||SEEP-SRB1c group||ATG GAG TCG AAT TGC AGA CTC||1309–1329||30||22/24 (92%)||Fibrobacteria||592|
|SEEP1f-153 (S-*-SP1f-0153-a-A-18)||SEEP-SRB1f group||AGC ATC GCT TTC GCG GTG||153–170||35||9/10 (90%)||Acidobacteria||25|
SEEP-SRB1a is the dominant partner of ANME-2 in AOM enrichment cultures
The SEEP-SRB1a probes were first applied to enrichment cultures in double FISH experiments combining either of the two newly designed SEEP-SRB1a-specific probes with an ANME-2 specific probe, probe ANME2-538 (Treude et al., 2005; Table S4). In the HR enrichment 97% (SEEP1a-1441) and 93% (SEEP1a-473) of the ANME-2-aggregates were targeted by the SEEP-SRB1a specific probes. In the Isis enrichment, probe SEEP1a-1441 hybridized to all ANME-2-aggregates (Fig. 2A), while probe SEEP1a-473 labelled the partners of 76% of the ANME-2-aggregates. This was consistent with the fact that probe SEEP1a-473 was only covering 77% of all known SEEP-SRB1a 16S rRNA sequences leading to an underestimation of the percentage of SEEP-SRB1a/ANME-2 consortia. It also proved that in the Isis enrichment there were at least two SEEP-SRB1a partners of ANME-2 consortia, one hybridizing with SEEP1a-1441 and SEEP1a-473, and one only hybridizing to probe SEEP1a-1441. The results indicated that the dominant partners of ANME-2 in both enrichments were from the SEEP-SRB1a group. Knowing about the ratio of ANME-2a to ANME-2c cells in the enrichments (HR: ANME-2a 17%, ANME-2c 64%; Isis: ANME-2a 20%, ANME-2c 49%) it was also evident that both, ANME-2a and ANME-2c, associate with bacteria of the SEEP-SRB1a group.
Distribution and ecology of SEEP-SRB1a bacteria in various methane seep systems
To test if the association between ANME-2 and SEEP-SRB1a is of general nature and not only induced by the enrichment procedure, six ANME-2 dominated environmental samples were examined by multi-colour catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) (Pernthaler et al., 2004). The samples originated from a methanotrophic microbial mat growing in the Black Sea and sediments at gas seeps at the Hydrate Ridge (NE Pacific), in the Gulf of Mexico (W Atlantic), at the Isis Mud Volcano (Eastern Mediterranean Sea) and the Gullfaks oil field (N Atlantic) (for a detailed description of the sampling sites see Table 2). In double hybridizations with the ANME-2 specific probe ANME2-538, altogether between 92% (Gulf of Mexico) and 99% (HR) of all ANME-2 consortia were targeted by DSS658 (Manz et al., 1998; Fig. 3). Subsequent application of the newly designed probes showed the presence of SEEP-SRB1a and their association with ANME-2 in all examined samples. In sediment samples from Hydrate Ridge, the Isis Mud Volcano, and the Gulf of Mexico at least 75% and up to 95% of the ANME-2 aggregates exhibited a partner from the SEEP-SRB1a group (Figs 2B and 3). In addition, SEEP-SRB1a was also frequently observed as the partner of ANME-2 in mat samples from the Black Sea (Fig. 2H) and a sediment sample from the Gullfaks oil field (Fig. 2C). Due to the complex structure of the microbial mat and very low aggregate abundances in the Gullfaks sediment, it was, however, not possible to quantify these associations. Altogether, our data from geographically distant AOM habitats indicate that SEEP-SRB1a is the dominant partner of ANME-2 at methane seeps.
Table 2. Environmental samples used in this study.
|Sample||Cruise||Site description||Position||Depth (m)||Date||Sediment layer depth||References|
|Black Sea microbial mat, P822 top||P317/3 (RV POSEIDON)||Microbial mat sample 822 from top of microbial reef, Dnieper area, northwestern Black Sea||44° 46.542′ N, 31° 58.978′ E||190||Oct 2004||n/a||Rossel et al. (2008)|
|Hydrate Ridge St. 19-2||SO148-1 (RV SONNE)||Crest of southern Hydrate Ridge, coast of Oregon; gas hydrate bearing sediment covered by Beggiatoa mat||44° 34.104′ N, 125° 08.807′ W||777||July 2000||0–1 cm||Linke and Suess (2001), Knittel et al. (2003)|
|Hydrate Ridge St. 38||SO148-1 (RV SONNE)||Crest of southern Hydrate Ridge, coast of Oregon; gas hydrate bearing sediment covered by clam fields of Calyptogena spp.||44° 34.186′ N, 125° 08.847′ W||787||July 2000||2–3 cm||Linke and Suess (2001), Knittel et al. (2003)|
|Isis Mud Volcano, St. 812||M70-2 (RV METEOR)||Isis mud volcano, Eastern Mediterranean; mud volcano sediment covered by Arcobacter mat||32° 21.669′ N, 031° 23.387′ E||992||November 2006||0–1 cm||This study, Felden and Boetius (2009)|
|Gulf of Mexico St. 156||SO174/OTEGA II (RV SONNE)||White sulfide-oxidizing bacteria occurring as a ‘mat’ on the surface of the sediment, oily hydrate||27° 46.95′ N, 91° 30.47′ W||546||October/November 2003||0–2 cm||Bohrmann and Schenck (2004), Orcutt et al. (2008)|
|Haakon Mosby Mud Volcano, ATL19-27||AWI (RV L'ATALANTE)||Mud Volcano at Norwegian-Barents-Svalbard continental margin; Beggiatoa mat site||72° 00.19′ N, 14° 43.67′ E||1250||August 2001||1–2 cm||Lösekann et al. (2007)|
|Gullfaks oil field, St. 771; Heincke seep||HE 208 (RV HEINCKE)||Coarse sand sediment densely covered with bacterial mats, gas ebullition observed||61° 10.40′°N, 02° 14.50′°E||150||May 2004||0–10 cm||Wegener et al. (2008b)|
Figure 3. ANME-2-partners in the environment. The graph shows the percentages of ANME-2/Deltaproteobacteria (probes: ANME2-538, DELTA495a/b/c), and ANME-2/DSS (probes: ANME2-538, DSS658) aggregates (Table S4) as well as of ANME-2/SEEP-SRB1a detected with probe SEEP1a-473, or probe SEEP1a-1441 (Table 1). For each sample and probe at least 110 ANME-2-aggregates were counted (for details see SI Table S5). Only ANME-2 aggregates featuring a partner were considered.
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The association of ANME-2 with SEEP-SRB1a seemed to be independent of the ANME-2 subgroup. A previous report (Knittel et al., 2005) showed that the two Hydrate Ridge samples examined in this study (Table 2) were dominated by different ANME-2 subgroups (station 19-2, 80% ANME-2a vs. 16% ANME-2c aggregates; station 38: 20% ANME-2a vs. 75% ANME-2c aggregates; Knittel et al., 2005). In spite of this difference, at least 87% (station 19-2) and 85% (station 38) of the ANME-2 aggregates in both samples exhibited a SEEP-SRB1a partner (Fig. 3) suggesting that both, ANME-2a and ANME-2c, were predominantly associated with bacteria of the SEEP-SRB1a group. This was confirmed by CARD-FISH hybridizations with probes specific for ANME-2a (ANME2a-647; Knittel et al., 2005) and ANME-2c (ANME2c-760; Knittel et al., 2005) (Fig. 2E and F). Quantification of the association of SEEP-SRB1a with the ANME-2 subgroups, yielded numbers in the same range as those obtained with the general ANME-2 probe (for details see Table S5).
ANME-2 associated SEEP-SRB1a were observed as coccoid cells (Fig. 2B) but also as rod/vibrio-shaped morphotypes (Fig. 2C, E and F). This morphological variability likely reflects the genomic variations within the SEEP-SRB1a group (92% 16S rRNA sequence similarity) which might be at the level of genera. Different ANME-clades, species within a particular clade, or environmental parameters seem to select for different strains within the SEEP-SRB1a group. FISH studies involving probes of a higher resolution, e.g. by targeting ITS sequences, might be useful to gain further insight into the diversity within SEEP-SRB1a.
In addition to ANME-2 dominated AOM samples, one ANME-3 dominated sediment sample from the Haakon Mosby Mud Volcano (HMMV; Table 2) was screened for the presence of SEEP-SRB1a bacteria. Sediments from this site were previously described by Lösekann and colleagues (2007) who showed that the majority of ANME-3 aggregates was associated with bacteria related to the genus Desulfobulbus. In addition, however, a small number of ANME-3 aggregates was also detected which possessed an unknown bacterial partner. In the present study, 8% and 23% of the examined ANME-3-aggregates were found to be associated with partners detected by probe SEEP1a-1441 or SEEP1a-473 respectively (Fig. 2G). This suggests that at least a fraction of the unknown bacterial partner belongs to the SEEP-SRB1a group. Most of the SEEP-SRB1a-positive aggregates (43 of 48 aggregates) consisted of only 1–3 SEEP-SRB1a and 1–3 ANME-3 cells (Fig. 2G). However, some bigger mixed-type aggregates (150–300 total cells) were also detected (Fig. 2G).
Diversity of the bacterial partners of ANME-2
The majority of the bacterial partners of ANME-2 belonged to the SEEP-SRB1a cluster within the DSS branch. However, the abundance of ANME-2/SEEP-SRB1a consortia was significantly lower than those of ANME-2/DSS consortia (Figs 2D and 3). This discrepancy might have been caused by an insufficient coverage of the developed SEEP-SRB1a-probes or microdiversity of SEEP-SRB1a microorganisms within a sample, as shown for the analysed AOM enrichments (Fig. 1). Another possible explanation is an affiliation of these DSS cells with another SEEP-SRB1 subgroup. The SEEP-SRB1f probe was used to test for the discrepancy between DSS658 and SEEP-SRB1a targeted cells. None of the examined ANME-2 aggregates featured a partner targeted by the SEEP-SRB1f probe. An association of the remaining SEEP-SRB1 subgroups (SEEP-SRB1b, 1d and 1e) with ANME-2 could not be tested as probes designed for these groups showed either no signals or insufficient specificity when evaluated (see above). However, the presence of sequences from ANME-2 dominated habitats within these subgroups suggests that the other SEEP-SRB1 subgroups either interact with ANME-2 or use short-chain alkanes (Kniemeyer et al., 2007) or other hydrocarbons occurring in the habitat.
In all examined sediments, the proportion of Bacteria/ANME-2-aggregates – as quantified using the EUB338I-III probe mix (Amann et al., 1990; Daims et al., 1999; Table S4) targeting most of the Bacteria– ranged from 96% for the Isis Mud Volcano, over 98% for the Gulf of Mexico and station 38 at Hydrate Ridge, to 100% for station 19-2 at Hydrate Ridge (Table S5). Results with a general probe mix targeting Deltaproteobacteria, Delta495a/b/c (Loy et al., 2002; Macalady et al., 2006; Lücker et al., 2007; Table S4), were in the same range (Table S5). The percentage of ANME-2/DSS positive aggregates was, in contrast, lower for the Isis Mud Volcano (97% versus 94%) and especially for the Gulf of Mexico (100% versus 92%) sample (Fig. 3). The SEEP-SRB1c probe was used to test for the discrepancy between Delta495a/b/c and DSS658 targeted cells (as SEEP-SRB1c sequences are likely not targeted by DSS658). None of the examined ANME-2 aggregates featured a partner of that group. This indicates that ANME-2 may have partners distinctly different from the DSS group. Similar observations were previously described for other ANME-2 habitats such as the Eel River Basin (Pernthaler et al., 2008) or Mud Volcanoes in the Eastern Mediterranean (Omoregie et al., 2009). In sediments from the Eel River Basin, Pernthaler and colleagues (2008) identified Alphaproteobacteria and Betaproteobacteria associated with ANME-2. This finding could not be confirmed by the present study, because even though a small number of single Alphaproteobacteria and Betaproteobacteria were detected in the examined sediments, none of these bacteria showed an association with ANME-2 (data not shown). In addition, Pernthaler and colleagues (2008) also showed that ANME-2 can, similar to ANME-3 (Lösekann et al., 2007), have a bacterial partner related to Desulfobulbus spp. This observation could also not be confirmed, suggesting that the association between ANME-2 and Alphaproteobacteria, Betaproteobacteria, or Desulfobulbus-related bacteria might be restricted to certain AOM habitats.
Presence of single SEEP-SRB1a cells
Cells of SEEP-SRB1a were not only found to be associated with ANME-2, but also as single cells. In sediment samples from Hydrate Ridge, the Isis Mud Volcano and the Gulf of Mexico, DSS cells accounted for 3–6% of all DAPI-stained single cells. Out of these, 8–17% were labelled with probe SEEP1a-1441. This translated into relative abundances of single SEEP-SRB1a cells of 0.3% to 0.7% (Table 3). Contrastingly, in a sediment sample from the Gullfaks oil field, DSS cells accounted for 18% and SEEP-SRB1a for 9% of all single cells. This sediment sample also featured an unusually high abundance of single ANME-2 cells and only very few ANME-2/DSS aggregates in comparison with other AOM habitats (Table 3, Knittel et al., 2005; Wegener et al., 2008b; Omoregie et al., 2009). Considering also the nature of the sample (Wegener et al., 2008b), it is likely that the high number of single ANME-2 and SEEP-SRB1a cells were an artifact of sample preparation. Here, harsher sonication was required to remove the microorganisms from coarse sand prior to CARD-FISH analysis. This procedure most likely disrupted part of the aggregates, releasing single cells. This conclusion was supported by the analysis of a Gullfaks oil field enrichment culture from the same sample in which SEEP-SRB1a was almost exclusively observed in aggregates together with ANME-2 (data not shown).
Apart from this exception, the generally low number of single SEEP-SRB1a cells in the environment is in line with results for the Desulfobulbus-related partner of ANME-3 in sediments of the Haakon Mosby Mud Volcano. An overall low percentage of less than 0.5% of single Desulfobulbus-related cells indicated that the partner, if at all, accounts for a very low fraction of the single cells, whereas single ANME-3 cells accounted for about 25% of DAPI-stained single cells (Lösekann et al., 2007). Due to the specificity of the probes and the diversity within the SEEP-SRB1a group, it cannot conclusively be answered whether single and ANME-2 associated SEEP-SRB1a bacteria are identical. Thus, further phylogenetic analyses targeting genomic regions with a higher variability (e.g. ITS) are necessary to address this question. Finally, it may also be possible that the detected single cells are inactive without ANME partner. Altogether the results indicate that SEEP-SRB1a is highly adapted to or even depending on life in ANME-2-consortia. This is also supported by failed 13C-labelled bicarbonate uptake in lipids of the SRB from AOM sediments in the absence of methane (Wegener et al., 2008a), and the failure to stimulate sulfate reduction in AOM samples with common substrates for SRB (Nauhaus et al., 2002).