Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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.
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
- Table 1.
Oligonucleotide PCR primers used in this study
Primer Sequencea Targetb E. coli position Reference Ar20-F TTC CGG TTG ATC CYG CCR G Archaeal SSU 6–25 34 GILSUT7-R GCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGC GCT TCC ATC AGG CAG AG Group I LSU 2610–2623 This study LSU190-F GAA YTG AAR CAT CTY AGT A Prokaryote LSU 190–207 This study LSU2445a-R CCC YGG GGT ARC TTT TCT ST Archaeal LSU 2432–2447 This study Eub27f AGA GTT TGA TCC TGG CTC AG Bacterial SSU 8–27 18 LSUT71933e-R GCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGA CCC GAC AAG GAA TTT CGC Bacterial LSU 1933–1951 This study Ar20T7-F GCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGT TCC GGT TGA TCC YGC CRG Archaeal SSU 6–25 This study Eub27T7-F GCC AGT GAA TTG TAA TAC GAC TCA CTA TAG GGA GAG TTT GAT CCT GGC TCA G Bacterial SSU 8–27 This study G1LSU-R CGC TTC CAT CAG GCA GAG Group I LSU 2610–2623 This study LSU1933e-R ACC CGA CAA GGA ATT TCG C Bacterial LSU 1933–1951 4 - Table 2.
Primers and templates used to generate amplicons for transcription reactions and optimized hybridization conditions for FISH
Primer pair DNA templates Probe targeta FISH conditionsb Ar20-F + G1-LSUT7-R Fosmids 101G10,c 4B7,d74A4e Group 1 planktonic archaeal SSU + LSU 70 (65, 45) Ar20-F + M13-F Plasmidsf SB95-74, SB95-75, SB95-76 Group 2 planktonic archaeal SSU 70 (65, 45) LSU190-F + M13-F Plasmid G2lsu-1A10g Group 2 planktonic archaeal LSU 70 (65, 45) Eub27-F + LSU1933e-R Picoplankton DNA extracts Planktonic bacteria 50 (55, 45) Ar20T7-F + G1-LSU Fosmids 101G10, 4B7, 74A4 Negative control 50 (55, 45) Eub27T7-F + 1933e-R Picoplankton DNA extracts Negative control 50 (55, 45) - Table 3.
Percentages of total epifluorescent cell counts enumerated by FISH probes
Sample Depth (m) Cell density (104/ml) % of total cells via FISH Total counts (DAPI) FISH group I FISH group II FISH bacteria 34498 M2 0 147 10.1 18.7 113 96.6 34498 M2 10 120 6.9 13.2 98.8 98.9 34498 M2 20 143 7.0 13.8 107 89.2 34498 M2 30 149 7.8 16.7 132 105 34498 M2 40 140 8.6 14.6 112 96.6 34498 M2 60 95.5 11.7 7.5 73.2 96.8 34498 M2 80 53.9 11.6 7.9 36.4 104 34498 M2 100 45.6 10.6 4.9 31.6 103 34498 M2 150 42.0 7.9 3.9 31.3 102 34498 M2 200 34.0 6.7 2.2 24.0 96.7 34498 M2 500 18.6 4.3 1.7 14.2 108 34498 M2 1,000 13.8 4.4 1.8 3.9 73.3 18198 M2 0 131 1.8 10.8 111 95.1 18198 M2 20 132 0.8 14.7 128 108 18198 M2 40 98.1 8.2 10.7 74.7 95.4 18198 M2 60 78.1 17.4 9.1 50.5 98.6 18198 M2 80 61.4 15.1 4.5 30.0 80.8 18198 M2 100 57.3 11.6 2.9 39.0 93.3 18198 M2 150 47.6 12.4 2.1 22.0 76.7 18198 M2 200 37.2 9.9 0.9 22.1 88.5 18198 M2 500 18.6 5.6 0.9 8.8 82.1
