Abstract
New molecular approaches relying on 16S rRNA sequences allow qualitative and quantitative analysis of marine microbial diversity. Here we report on (1) continued development of ‘lists’ of taxa present in marine environments, in temperate coastal waters, and (2) new fluorescent in situ hybridization (FISH) approaches to quantify taxonomic compositions, with an initial focus on archaea. Our cloning results come from Long Island Sound on the Atlantic coast (February and August), and Malibu (April) and offshore Monterey Bay (September), California. The clones were dominated (39 of 45 total clones) by proteobacteria, with the α subdivision (33 clones), and the SAR11 cluster (17) in particular, being quite abundant. There were also clones from the β (2) and γ (4) subdivisions, the cyanobacteria (4, from Monterey Bay only) and the Cytophaga group (2). Some clones were very similar to those previously reported from open ocean or deep sea environments, but others were not close relatives of any of those previously reported. The FISH results used doubly-labeled probes that were ‘universal’, bacterial, and archaeal (single and multiple), in combination with chloramphenicol treatment and probe detection by intensified video microscopy. Universal probes detected ca. 75–95% of total DAPI counts. Of 2 depth profiles from mesotrophic-oligotrophic California waters, a September one, to 300 m, indicated a low but detectable presence of archaea (about 10% above control values) as measured with single probes. A second profile in May with 4 archaeal probes showed <5% at 100 m depth, but the percentage relative to total DAPI counts increased to about 40% at 600 m depth. Samples from the French Mediterranean coast showed few detectable archaea (analyzed with single probes) in surface waters of Villefranche-sur-Mer Bay, but about 60% archaea at 200 m depth outside the bay. These results point in general to the suitability of this single cell FISH method to quantify taxonomic composition of marine samples, and the specific results indicate the high abundance of archaea in at least some midwater locations.
Similar content being viewed by others
References
Alm EW, Oerther DB, Larsen M, Stahl DA and Raskin L (1996) The Oligonucleotide Probe Database. Appl Environ Microbiol 62(10): 3557-3559
Amann RI, Krumholz L and Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and evironmental studies in microbiology. J Bacteriol 172: 762-770
Amann R, Ludwig L and Schleifer K (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59: 143-169
Azam F, Fenchel T, Gray JG, Meyer-Reil LA and Thingstad T (1983) The ecological role of water-column microbes in the sea. Mar Ecol Prog Ser 10: 257-263
Braun-Howland EB, Danielson SA and Nierzwicki-Bauer SA (1992) Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes. BioTechniques 13: 928-933
Britschgi T and Giovannoni SJ (1991) Phylogenetic analysis of a natural marine bacterioplankton population by rRNA gene cloning and sequencing. Appl Environ Microbiol 57: 1707-1713
Brunk CF, Avaniss-Aghajani E, Brunk CA (1996) A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences. Appl Environ Microbiol 62(3): 872-879
DeLong EF (1992) Archaea in coastal marine enviroments. Proc Natl Acad Sci 89: 5685-5689
DeLong EF, Wickham GS and Pace NR (1989) Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243: 1360-1363
DeLong EF, Franks DG and Alldredge AL (1993) Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Limnol Oceanogr 38(5): 924-934
Farrelly V, Rainey FA and Stackebrandt E (1995) Effect of genome size and rrn gene copy number on PCR amplification of 16s rRNA genes from a mixture of bacterial species. Appl Environ Microbiol 61(7): 2798-2801
Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17: 368-376
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791
Felsenstein J (1993) Phylip 3.5. Phylogeny inference package, distributed by author, University of Wahington, Seattle, Washington
Ferguson RL, Buckley EN and Palumbo AV (1984) Response ofmarine bacterioplankton to differential filtration and confinement. Appl Environ Microbiol 47: 49-55
Field KG, Gordon D, Wright T, Rappe M, Urbach E, Vergin K and Giovannoni SJ (1997) Diversity and depth specific distribution of SAR 11 cluster rRNA genes from marine planktonic bacteria. Appl Environ Microbiol 63: 63-70
Fuhrman JA (1992) Bacterioplankton roles in cycling of organic matter: the microbial food web. In Falkowski PG and Woodhead AD (eds), Primary Productivity and Biogeochemical Cycles in the Sea. Plenum Press, New York, pp. 361-383
Fuhrman JA and Azam F (1982) Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: evaluation and field results. Mar Biol 66: 109-120
Fuhrman JA and Davis AA (1997) Widespread Archaeaand novel Bacteriafrom the deep sea as shown by 16S rRNA gene sequences. Mar Ecol Prog Ser 150: 275-285
Fuhrman JA, Comeau DE, Hagström Å and Chan AM (1988) Extraction of DNA suitable for molecular biological studies from natural planktonic microorganisms. Appl Environ Microbiol 54: 1426-1429
Fuhrman JA, Lee SH, Masuchi Y, Davis AA and Wilcox RM (1994) Characterization of marine prokaryotic communities via DNA and RNA. Microb Ecol 28: 133-145
Fuhrman JA, McCallum K and Davis AA (1992) Novel major archaebacterial group from marine plankton. Nature (London) 356: 148-149
Fuhrman JA, McCallum K and Davis AA (1993) Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans. Appl Environ Microbiol 59: 1294- 1302
Fuhrman JA and Noble RT (1995) Viruses and protists cause similar bacterial mortality in coastal seawater. Limnol Oceanogr 40: 1236-1242
Fuhrman JA and Suttle CA (1993) Viruses in marine plankton. Oceanography 6: 51-63.
Giovannoni SJ, Britschgi TB, Moyer CL and Field KG (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345: 60-63
Giovannoni SJ, Rappé MS, Vergin KL and Adair NI (1996) 16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Nonsulfur bacteria. Proc Nat Acad Sci USA 93: 7979-7984
Giovannoni SJ, DeLong EF, Olsen GJ and Pace NR (1988) Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol 170: 720-726
Glöckner FO, Amann R, Alfreider A, Pernthaler J, Psenner R, Trebesius K and Schleifer K (1996) An in situ hybridization protocol for detection and identification of planktonic bacteria. System Appl Microbiol 19: 403-406
Hicks RE, Amann RI and Stahl DA (1992) Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences. Appl Environ Microbiol 58: 2158-2163
Jannasch HW and Jones GE (1959) Bacterial populations in sea water as determined by different methods of enumeration. Limnol Oceanogr 4: 128-139
Karner M and Fuhrman JA (1997) Determination of active marine bacterioplankton: a comparison of universal 16S rRNA probes, autoradiography, and nucleiod staining. Appl Environ Microbiol 63: 1208-1213
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML and Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci 82: 6955-6959
Lee SH and Fuhrman JA (1991) Confinement effect on species composition of bacterioplankton studied at the level of community DNA. Mar Ecol Prog Ser 79: 195-201
Lee SH, Malone C and Kemp PF (1993) Use of multiple 16S rRNA-targeted fluorescent probes to increase signal strength and measure cellular RNA from natural planktonic bacteria. Mar Ecol Prog Ser 101: 193-201
Maidak BL, Olsen GJ, Larsen N, Overbeek R, McCaughey MJ and Woese CR (1997) The Ribosomal Database Project. Nucleic Acids Res 25: 109-111
Maniatis T, Fritsch EF and Sambrook J (1982) Molecular Cloning, A Laboratory Manual 545 p. Cold Springs Harbor Laboratory, Cold Springs Harbor
Massana R, Murray A, Preston C and DeLong E (1997) Vertical distribution and phylogenetic characterization of marine planktonic archaea in the Santa Barbara Channel. Appl Environ Microbiol 63: 50-56
Mullins TD, Britschgi TB, Krest RL and Giovannoni SJ (1995) Genetic comparisons reveal the same unknown bacterial lineages in Atlantic and Pacific bacterioplankton communities. Limnol Oceanogr 41: 148-158
Olsen GJ, Lane DL, Giovannoni SJ and Pace NR (1986) Microbial ecology and evolution: a ribosomal RNA approach. Ann Rev Microbiol 40: 337-365
Olsen GJ, Matsuda H, Hagstrom R and Overbeek R (1994) fastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput Appl Biosci 10: 41-48
Ouverney CC and Fuhrman JA (1997) Increase in fluorescence intensity of 16S rRNA in situhybridization in natural samples treated with chloramphenicol. Appl Environ Microbiol 63(7): 2735-2740
Pace NR (1997) A Molecular view of microbial diversity and the biosphere. Science 276: 734-740
Pace NR, Stahl DA, Lane DL and Olsen GJ (1986) The analysis of natural microbial populations by rRNA sequences. Adv Microbiol Ecol 9: 1-55
Rappé MS, Kemp PF and Giovannoni SJ (1997) Phylogenetic diversity of marine coastal picoplankton 16S rRNA genes cloned from the continental shelf off Cape Hatteras, North Carolina. Limnol Oceanogr 42(5): 811-826
Schmidt TM, Long EFD and Pace NR (1991) Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol 173: 4371-4378
Smith SW, Overbeek R, Woese CR, Gilbert W and Gillevet PM (1994) The genetic data environment, an expandable GUI for multiple sequence analysis. CABIOS 10: 671-675
Suzuki MT and Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16s rRNA genes by PCR. Appl Environ Microbiol 62: 625-630
Suzuki MT, Rappé MS, Haimberger ZW, Winfield H, Adair N, Strobel J and Giovannoni SJ (1997) Bacterial diversity among small subunit rRNA gene clones and cellular isolates from the same seawater sample. Appl Environ Microbiol 63: 983-989
Tabor PS and RA Neihof (1982) Improved method for determination of respiring individual organisms in natural waters. Appl Environ Microbiol 43: 1249-1255
Woese CR (1987) Bacterial evolution. Microbiol Rev 51: 221-271
Woese CR, Kandler O and Wheelis ML (1990) Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eukarya. Proc Natl Acad Sci USA 87: 4576-4579
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Fuhrman, J.A., Ouverney, C.C. Marine microbial diversity studied via 16S rRNA sequences: cloning results from coastal waters and counting of native archaea with fluorescent single cell probes. Aquatic Ecology 32, 3–15 (1998). https://doi.org/10.1023/A:1009974817127
Issue Date:
DOI: https://doi.org/10.1023/A:1009974817127