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Methods

Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization

Edward F. DeLong, Lance Trent Taylor, Terence L. Marsh, Christina M. Preston
Edward F. DeLong
Monterey Bay Aquarium Research Institute, Moss Landing, California, and
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Lance Trent Taylor
Monterey Bay Aquarium Research Institute, Moss Landing, California, and
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Terence L. Marsh
Center for Microbial Ecology, East Lansing, Michigan
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Christina M. Preston
Monterey Bay Aquarium Research Institute, Moss Landing, California, and
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DOI: 10.1128/AEM.65.12.5554-5563.1999
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  • Fig. 1.
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    Fig. 1.

    Phylogenetic position of the planktonic euryarchaeal 23S rRNA gene contained on clone G2lsu-1A10. The number at each bifurcation represents the percentage of 1,000 bootstrap resamplings that yielded the branching pattern appearing to the right of the value. The scale bar represents the estimated number of fixed mutations per nucleotide position.

  • Fig. 2.
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    Fig. 2.

    Surface seawater sample from Monterey Bay hybridized with a fluorescein-labeled 23S group II archaeal probe and a CY-3-labeled 16S group II archaeal probe (Table 2). Images of the same field were captured by using the fluorescein filter set (A), the CY-3 filter set (B), and the DAPI filter set (C). Bars, 5 μm.

  • Fig. 3.
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    Fig. 3.

    Similarity plots comparing rRNA sequences of templates used to generate probes to homologous regions in other bacteria or archaea. Each data point represents the unrestricted sequence similarity value along a 100-nucleotide stretch. Nonoverlapping similarity values were calculated in 100-nucleotide sequence segments along the length of the 16S and 23S genes. (A) Group I archaea (C. symbiosum) rRNA compared to homologous regions of other bacteria and archaea. (B) Group II 16S (SB95-72) and 23S (G2lsu-1A10) rRNAs compared to homologous regions of other bacteria and archaea. The respective accession numbers of the small-subunit (SSU) and large-subunit (LSU) rRNA sequences used are as follows: 4B7, U39635 andAF198456 ; 101G10, AF083071 and AF083071 ; Escherichia coli,U00006 and U00006 ; Sulfolobus solfataricus, X03235 andU32322 ; Archaeoglobus fulgidus, X05567 and M64487 ; T. acidophilum, M38637 and M32298 .

  • Fig. 4.
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    Fig. 4.

    Epifluorescence micrographs of picoplankton visualized with polynucleotide probes and FISH on polycarbonate filters. (A and B) Seawater sample, collected from a 200-m depth at a nearshore station in Monterey Bay, hybridized with the CY-3-labeled group I probe and viewed by using the CY-3 (A) or the DAPI (B) filter set. (C and D) Surface seawater sample, from a nearshore station in Monterey Bay, hybridized with the fluorescein-labeled 23S rRNA group II probe and viewed with the fluorescein (C) or the DAPI (D) filter set. (E to G) A 100-m sample, from an offshore station in Monterey Bay, dually hybridized with the fluorescein-labeled 23S group II probe and the CY-3-labeled group I probe. The sample was viewed with the fluorescein (E), DAPI (F), and CY-3 (G) filter sets. (H) Seawater sampled at an 80-m depth at 177 miles offshore of Moss Landing, Calif. The sample was dually hybridized with the fluorescein-labeled bacterial probe and the CY-3-labeled group I probe. Images were captured independently by using the fluorescein or CY-3 filter set, and the separate images were overlaid in Adobe PhotoShop. Scale bars, 5 μm.

  • Fig. 5.
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    Fig. 5.

    Group I archaea and bacterial cell concentrations at various depths, determined by polyribonucleotide probe hybridization and FISH and performed in triplicate. Error bars represent standard errors, and where not visible, they are smaller than the symbols. Methods are described in the text.

  • Fig. 6.
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    Fig. 6.

    Cell densities of group I and II archaea determined by polyribonucleotide probe hybridization (Hyb) and FISH, compared to the percentage of rRNA from each group in the same sample estimated by quantitative oligonucleotide probe hybridization. Methods are described in the text.

  • Fig. 7.
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    Fig. 7.

    Densities of group I archaea and bacterial cells at various depths, determined by polyribonucleotide probe hybridization and FISH. The sampling site was 177 miles offshore of Moss Landing, Calif. Methods are described in the text. Eubac, eubacterial.

Tables

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  • Table 1.

    Oligonucleotide PCR primers used in this study

    PrimerSequenceaTargetbE. coli positionReference
    Ar20-FTTC CGG TTG ATC CYG CCR GArchaeal SSU6–2534
    GILSUT7-RGCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGC GCT TCC ATC AGG CAG AGGroup I LSU2610–2623This study
    LSU190-FGAA YTG AAR CAT CTY AGT AProkaryote LSU190–207This study
    LSU2445a-RCCC YGG GGT ARC TTT TCT STArchaeal LSU2432–2447This study
    Eub27fAGA GTT TGA TCC TGG CTC AGBacterial SSU8–2718
    LSUT71933e-RGCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGA CCC GAC AAG GAA TTT CGCBacterial LSU1933–1951This study
    Ar20T7-FGCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGT TCC GGT TGA TCC YGC CRGArchaeal SSU6–25This study
    Eub27T7-FGCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGA GAG TTT GAT CCT GGC TCA GBacterial SSU8–27This study
    G1LSU-RCGC TTC CAT CAG GCA GAGGroup I LSU2610–2623This study
    LSU1933e-RACC CGA CAA GGA ATT TCG CBacterial LSU1933–19514
    • ↵a The T7 RNA polymerase promoter sequence is in boldface type.

    • ↵b SSU, small subunit; LSU, large subunit.

  • Table 2.

    Primers and templates used to generate amplicons for transcription reactions and optimized hybridization conditions for FISH

    Primer pairDNA templatesProbe targetaFISH conditionsb
    Ar20-F + G1-LSUT7-RFosmids 101G10,c 4B7,d74A4eGroup 1 planktonic archaeal SSU + LSU70 (65, 45)
    Ar20-F + M13-FPlasmidsf SB95-74, SB95-75, SB95-76Group 2 planktonic archaeal SSU70 (65, 45)
    LSU190-F + M13-FPlasmid G2lsu-1A10gGroup 2 planktonic archaeal LSU70 (65, 45)
    Eub27-F + LSU1933e-RPicoplankton DNA extractsPlanktonic bacteria50 (55, 45)
    Ar20T7-F + G1-LSUFosmids 101G10, 4B7, 74A4Negative control50 (55, 45)
    Eub27T7-F + 1933e-RPicoplankton DNA extractsNegative control50 (55, 45)
    • ↵a SSU, small subunit; LSU, large subunit.

    • ↵b Percent formamide in hybridization buffer (hybridization and wash temperatures [°C]).

    • ↵c Reference 43.

    • ↵d Reference 50.

    • ↵e This study.

    • ↵f Reference 34.

    • ↵g This study.

  • Table 3.

    Percentages of total epifluorescent cell counts enumerated by FISH probes

    SampleDepth (m)Cell density (104/ml)% of total cells via FISH
    Total counts (DAPI)FISH group IFISH group IIFISH bacteria
    34498 M2014710.118.711396.6
    34498 M2101206.913.298.898.9
    34498 M2201437.013.810789.2
    34498 M2301497.816.7132105
    34498 M2401408.614.611296.6
    34498 M26095.511.77.573.296.8
    34498 M28053.911.67.936.4104
    34498 M210045.610.64.931.6103
    34498 M215042.07.93.931.3102
    34498 M220034.06.72.224.096.7
    34498 M250018.64.31.714.2108
    34498 M21,00013.84.41.83.973.3
    18198 M201311.810.811195.1
    18198 M2201320.814.7128108
    18198 M24098.18.210.774.795.4
    18198 M26078.117.49.150.598.6
    18198 M28061.415.14.530.080.8
    18198 M210057.311.62.939.093.3
    18198 M215047.612.42.122.076.7
    18198 M220037.29.90.922.188.5
    18198 M250018.65.60.98.882.1
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Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization
Edward F. DeLong, Lance Trent Taylor, Terence L. Marsh, Christina M. Preston
Applied and Environmental Microbiology Dec 1999, 65 (12) 5554-5563; DOI: 10.1128/AEM.65.12.5554-5563.1999

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Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization
Edward F. DeLong, Lance Trent Taylor, Terence L. Marsh, Christina M. Preston
Applied and Environmental Microbiology Dec 1999, 65 (12) 5554-5563; DOI: 10.1128/AEM.65.12.5554-5563.1999
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KEYWORDS

Archaea
bacteria
phylogeny
Plankton
seawater

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