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Microbial Ecology

Diversity and Distribution of Methanotrophic Archaea at Cold Seeps

Katrin Knittel, Tina Lösekann, Antje Boetius, Renate Kort, Rudolf Amann
Katrin Knittel
1Max Planck Institute for Marine Microbiology
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  • For correspondence: kknittel@mpi-bremen.de
Tina Lösekann
1Max Planck Institute for Marine Microbiology
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Antje Boetius
1Max Planck Institute for Marine Microbiology
2International University of Bremen, Bremen
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Renate Kort
3ICBM-Geomicrobiology Group, University of Oldenburg, Oldenburg, Germany
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Rudolf Amann
1Max Planck Institute for Marine Microbiology
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DOI: 10.1128/AEM.71.1.467-479.2005
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    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.
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    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.
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    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.
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    FIG. 4.

    Detection and quantification of ANME-1 cells in Hydrate Ridge sediments by FISH.

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  • TABLE 1.

    Geochemical characterization of Hydrate Ridge and Black Sea stations and abundance of ANME-2a/DSS and ANME-2c/DSS aggregatesa

    Embedded Image
    • ↵ 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

    ProbeSpecificitySequence (5′ to 3′)PositionaFormamide concn (%, vol/vol)bReference
    GroupTaxon
    DSS658 Desulfosarcina-Desulfococcus SRB of Delta-proteobacteriaTCC ACT TCC CTC TCC CAT658-68560 32
    ARCH915Most archaea Archaea GTG CTC CCC CGC CAA TTC CT915-93435 2
    ANME-1-350ANME-1 Euryarchaeota AGT TTT CGC GCC TGA TGC350-36740 7
    EeIMS932ANME-2 Euryarchaeota AGC TCC ACC CGT TGT AGT932-94965 7
    ANME-2a-647ANME-2a Euryarchaeota TCT TCC GGT CCC AAG CCT647-66450This study
    ANME-2c-622ANME-2c Euryarchaeota CCC TTG GCA GTC TGA TTG622-63950This study
    ANME-2c-760ANME-2c Euryarchaeota CGC CCC CAG CTT TCG TCC760-77760This study
    MBGB-280Marine benthic group B Crenarchaeota TCA CGG CCC CTA TCG ATT280-29750This study
    MBGB-335Marine benthic group B Crenarchaeota TGC GCC TCG TAA GGC CTG335-35250-60This study
    MBGB-380Marine benthic group B Crenarchaeota GTA ACC CCG TCA CAC TTT380-39750This study
    MBGB-525cMarine benthic group B Crenarchaeota AGA GCT GGT TTT ACC GCG525-54240-50This study
    • ↵ a Position in the 16S rRNA of Escherichia coli.

    • ↵ b Formamide concentration in the hybridization buffer.

    • ↵ c Probe MBGB-525 also targets several sequences from korarchaeota and other archaea due to a single terminal mismatch.

  • TABLE 3.

    Phylogenetic affiliations of 16S rRNA gene clones retrieved from Hydrate Ridge sediments and Black Sea microbial mats

    Phylogenetic groupNo. of clones
    Hydrate RidgeBlack Sea microbial mat
    Beggiatoa mat (station 19-2)Beggiatoa mat (station 19-2)Calyptogena field (station 38-1)PCR from extracted DNAPCR from fixed cells
    Station 55-4Station 55-4 (black/pink)Station 14 (nodule)
    2-4 cm6-7 cm2-6 cmBlackBlack/pinkPink
    Euryarchaeota
        ANME-1a1131
        ANME-1b431429253282
        ANME-1 unaffiliated1
        ANME-2a7522
        ANME-2c20461
        ANME-2 unaffiliated1
        ANME-33
        Methanomicrobiales1
        Marine benthic group D1
    Crenarchaeota
        Marine benthic group B309
        Unaffiliated3
    Total no. of clones per library315720292533216
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Diversity and Distribution of Methanotrophic Archaea at Cold Seeps
Katrin Knittel, Tina Lösekann, Antje Boetius, Renate Kort, Rudolf Amann
Applied and Environmental Microbiology Jan 2005, 71 (1) 467-479; DOI: 10.1128/AEM.71.1.467-479.2005

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Diversity and Distribution of Methanotrophic Archaea at Cold Seeps
Katrin Knittel, Tina Lösekann, Antje Boetius, Renate Kort, Rudolf Amann
Applied and Environmental Microbiology Jan 2005, 71 (1) 467-479; DOI: 10.1128/AEM.71.1.467-479.2005
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KEYWORDS

Archaea
Cold Temperature
Geologic Sediments
methane
seawater

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