Article Figures & Data
Figures
- FIG. 1.
Phylogenetic tree showing the affiliations of 16S rRNA gene sequences retrieved from Hydrate Ridge sediments and Black Sea microbial mats (boldface type) with selected reference sequences of the domain Archaea. Besides cultivated organisms all previously published clone sequences from methane-rich sites are included as references (at least one representative per phylogenetic group). The tree was constructed by using maximum-likelihood analysis in combination with filters excluding highly variable positions in a subset of 200 nearly full-length sequences (>1,300 bp). Partial sequences were inserted into the reconstructed tree by using parsimony criteria with global-local optimization, without allowing changes in the overall tree topology. Probe specificity is indicated by brackets. Bar = 10% estimated sequence divergence.
- FIG. 2.
Single cells and cell aggregates of ANME-1 and ANME-2 archaea in sediments from Hydrate Ridge sediments and thin sections of Black Sea microbial mats, visualized by FISH. Scale bars = 10 μm. Panels A to H, J, and K are confocal laser scanning micrographs. Panel I is a regular epifluorescence micrograph. (A) DAPI staining showing the embedded aggregates in the bulk biomass in Black Sea microbial mats. (B) Corresponding FISH image of ANME-2/DSS aggregates (probe ANME-2c-760 labeled with Cy5 [red] and probe DSS658 labeled with Cy3 [green]). DSS cells surrounded the archaea partially or completely. In many aggregates the ANME cells had only a few or no DSS partners. (C to F) Different types of ANME-2c/DSS aggregates (probe ANME-2c-622 labeled with Cy3 [red] and probe DSS658 labeled with Cy5 [green]), including aggregates with an inner core of archaea surrounded by a shell of SRB (shell type). (G and H) ANME-2a/DSS aggregates (probe ANME-2a-647 labeled with Cy3 [red] and probe DSS658 labeled with Cy5) from Beggiatoa site station 38 (depth, 1 to 2 cm), showing cell-to-cell association of the partners (mixed type). (I) ANME-1 cells stained with Cy3-labeled probe ANME-1-350 in Hydrate Ridge sediments (Beggiatoa site station 19-2 [depth, 5 to 6 cm]) (J) Color overlay of ANME-1 cells targeted with probe ANME-1-350 labeled with Cy3 (red) and SRB of the Desulfosarcina-Desulfococcus branch labeled with fluorescein (probe DSS658; green) in a Black Sea mat section. (K) Marine benthic group B Crenarchaeota targeted with probe MBGB-380 labeled with Cy3 in a Black Sea mat section.
- FIG. 3.
ANME-1 archaea and ANME-2/DSS aggregates in the Black Sea microbial mat: SEM images of a gold-sputtered formaldehyde-fixed mat section. (A) ANME-2/DSS aggregate, showing the typical spherical structure. (B) Cylindrical ANME-1 cells.
- FIG. 4.
Detection and quantification of ANME-1 cells in Hydrate Ridge sediments by FISH.
Tables
- TABLE 1.
Geochemical characterization of Hydrate Ridge and Black Sea stations and abundance of ANME-2a/DSS and ANME-2c/DSS aggregatesa
↵a The diagram of the chemosynthetic communities at Hydrate Ridge is from reference 65. The numbers in parentheses are references.
b Shipboard measurement.
c ND, not determined.
d The units are micromoles per gram (dry weight) per day.
e SRR, sulfate reduction rate.
- TABLE 2.
Oligonucleotide probes used in this study
Probe Specificity Sequence (5′ to 3′) Positiona Formamide concn (%, vol/vol)b Reference Group Taxon DSS658 Desulfosarcina-Desulfococcus SRB of Delta-proteobacteria TCC ACT TCC CTC TCC CAT 658-685 60 32 ARCH915 Most archaea Archaea GTG CTC CCC CGC CAA TTC CT 915-934 35 2 ANME-1-350 ANME-1 Euryarchaeota AGT TTT CGC GCC TGA TGC 350-367 40 7 EeIMS932 ANME-2 Euryarchaeota AGC TCC ACC CGT TGT AGT 932-949 65 7 ANME-2a-647 ANME-2a Euryarchaeota TCT TCC GGT CCC AAG CCT 647-664 50 This study ANME-2c-622 ANME-2c Euryarchaeota CCC TTG GCA GTC TGA TTG 622-639 50 This study ANME-2c-760 ANME-2c Euryarchaeota CGC CCC CAG CTT TCG TCC 760-777 60 This study MBGB-280 Marine benthic group B Crenarchaeota TCA CGG CCC CTA TCG ATT 280-297 50 This study MBGB-335 Marine benthic group B Crenarchaeota TGC GCC TCG TAA GGC CTG 335-352 50-60 This study MBGB-380 Marine benthic group B Crenarchaeota GTA ACC CCG TCA CAC TTT 380-397 50 This study MBGB-525c Marine benthic group B Crenarchaeota AGA GCT GGT TTT ACC GCG 525-542 40-50 This study - TABLE 3.
Phylogenetic affiliations of 16S rRNA gene clones retrieved from Hydrate Ridge sediments and Black Sea microbial mats
Phylogenetic group No. of clones Hydrate Ridge Black Sea microbial mat Beggiatoa mat (station 19-2) Beggiatoa mat (station 19-2) Calyptogena field (station 38-1) PCR from extracted DNA PCR from fixed cells Station 55-4 Station 55-4 (black/pink) Station 14 (nodule) 2-4 cm 6-7 cm 2-6 cm Black Black/pink Pink Euryarchaeota ANME-1a 1 13 1 ANME-1b 4 3 14 29 25 32 8 2 ANME-1 unaffiliated 1 ANME-2a 7 5 2 2 ANME-2c 20 46 1 ANME-2 unaffiliated 1 ANME-3 3 Methanomicrobiales 1 Marine benthic group D 1 Crenarchaeota Marine benthic group B 30 9 Unaffiliated 3 Total no. of clones per library 31 57 20 29 25 33 21 6
