1887

Abstract

Nanoflagellates are ecologically important, but morphological identification requires techniques which are not practicable for use in quantitative studies of populations; alternative methods of accurate recognition of nanoflagellate species in mixed populations are therefore desirable. Fluorescent oligonucleotide probes which hybridize with unique sequences of the small subunit (SSU) rRNA have been exploited as ‘phylogenetic stains’ in the identification of bacteria. In this paper we describe the preparation and application of probes which specifically hybridize with a common nanoflagellate species, . The sequence of nucleotides in the SSU rRNA gene of this flagellate was determined and compared with those of related species to select unique sequences 18-21 nucleotides in length. Five sequences in different parts of the SSU rRNA gene were used to design 5 -fluorescently labelled oligonucleotide probes. Published sequences were used to make probes that hybridized with all eukaryotes (EUK) or any cellular organism (UNI), and probes were designed not to hybridize with rRNA (CON). Optimum conditions for hybridization were determined. In all cases, UNI probes hybridized with the cells, but CON probes were only bound to a limited extent. All five probes targeted to proved to be species-specific; they hybridized well with this species, but not with three other species of the same genus, nor with three more distantly related flagellate species, nor with a ciliate, nor with bacteria. These probes provide a means of quantitatively measuring the proportion of cells in samples of mixed protists.

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1997-05-01
2024-10-09
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References

  1. Amann R. I., Krumholz L., Stahl D. A. 1990; Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. . J Bacteriol 172:762–770
    [Google Scholar]
  2. Amann R. I., Ludwig W., Schleifer K.-H. 1992; Identification and in situ detection of individual bacterial cells. FEMS Microbiol Lett 100:45–50
    [Google Scholar]
  3. Amann R. I., Ludwig W., Schleifer K.-H. 1995; Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169
    [Google Scholar]
  4. Azam F., Fenchel T., Field J. G., Meyer-Reil L. A., Thingstad F. 1983; The ecological role of water column microbes in the sea. Mar Ecol Prog Ser 10:257–263
    [Google Scholar]
  5. Bertin B., Broux O., Van Hoegaerden M. 1990; Flow cytometric detection of yeast by in situ hybridisation with a fluorescent ribosomal RNA probe. J Microbiol Methods 12:1–12
    [Google Scholar]
  6. Bohlool B. B., Schmidt E. L. 1980; The immunofluorescence approach in microbial ecology. Adv Microb Ecol 4:203–241
    [Google Scholar]
  7. Breslauer K. J., Frank R., Blocker H., Markey L A. 1986; Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA 83:3746–3750
    [Google Scholar]
  8. Bresser J., Evinger-Hodges M. 1987; Comparison and optimisation of in situ hybridisation procedures yielding rapid, sensitive mRNA detections. Gene Anal Tech 4:89–104
    [Google Scholar]
  9. Burkill P. H., Edwards E. S., Landry M., Paranjape M., Reckermann M., Sieracki M., Sleigh M. A., Stoecker D. K., Verity P. 1994; Microzooplankton biomass : JGOFS protocols. Edited by A. H. Knap. Paris : UNESCO. . In Protocols for the Joint Global Ocean Flux Study (JGOFS) Core Measurements, lntergouernmental Oceanographic Commission Manual and Guide. 29:147–151
    [Google Scholar]
  10. Campbell L., Shapiro L. P., Haugen E. 1994; Immunochemical characterization of eukaryotic ultraplankton from the Atlantic and Pacific oceans. J Plankton Res 16:35–51
    [Google Scholar]
  11. Clarholm M. 1984; Microbes as predators or prey. Heterotrophic, free-living protozoa : neglected microorganisms with an important task in regulating bacterial populations. Edited by M. J. Klug & C. A. Reddy. Washington, DC: . In Current Perspectives in Microbial Ecology American Society for Microbiology;321–326
    [Google Scholar]
  12. Dale R. M. K., Ward D. C. 1975; Mercurated polynucleotides : new probes for hybridisation and selective polymer fractionation.. Biochemistry 14:2458–2469
    [Google Scholar]
  13. DeLong E. F., Wickam G. S., Pace N. R. 1989; Phylogenetic stains : ribosomal RNA-based probes for the identification of single cells. Science 243:1360–1363
    [Google Scholar]
  14. Delong E. F., Wu K. Y., Prezelin B. B., Jovine R. V. M. 1994; High abundance of Archaea in Antarctic marine picoplankton. Nature 371:695–697
    [Google Scholar]
  15. Devereux R., Kane M. D., Winfrey J., Stahl D. A. 1992; Genus- and group-specific hybridisation probes for determination and environmental studies of sulphate reducing bacteria. Syst Appl Microbiol 15:601–609
    [Google Scholar]
  16. Felsenstein J. 1995; Phylip Manual (version 3.5c).Seattle : University of Washington..
    [Google Scholar]
  17. Fenchel T. 1982; Ecology of heterotrophic microflagellates. IV. Quantitative occurrence and importance as bacterial consumers. Mar Ecol Prog Ser 9:35–42
    [Google Scholar]
  18. Fenchel T. 1988; Marine plankton food chains. Annu Rev Ecol Syst 19:19–38
    [Google Scholar]
  19. Giovannoni S. J., De Long E. F., Olsen G. J., Pace N. R. 1988; Phylogenetic group-specific oligodeoxynucleotide probes for the identification of single microbial cells. J Bacteriol 170:720–726
    [Google Scholar]
  20. Giovannoni S. J., Britschgi T. B., Moyer C. L, Field K. G. 1990; Genetic diversity in Sargasso Sea bacterioplankton. Nature 345:60–63
    [Google Scholar]
  21. Göbel U. B., Geiser A., Stanbridge E. J. 1987; Oligonucleotide probes complementary to variable regions of ribosomal RNA discriminate between Mycoplasma species. J Gen Microbiol 133:1969–1974
    [Google Scholar]
  22. Hicks R. E., Amann R. I., Stahl D. A. 1992; Dual staining of natural bacterioplankton with 4’,6-diamidino-2-phenylindolea and fluorescent oligonucleotide probes targeting Kingdom-level 16s rRNA sequences. Appl Environ Microbiol 58:2158–2163
    [Google Scholar]
  23. Katz S. 1963; The reversible reaction of Hg(I1) and doublestranded polynucleotides. A step function theory and its significance. Biochim Biophys Acta 68:240–253
    [Google Scholar]
  24. Kemp P. F. 1994; Single-cell RNA content of natural marine planktonic bacteria measured by hybridization with multiple 16s rRNA-targeted fluorescent probes. Limnol Oceanogr 39:869–879
    [Google Scholar]
  25. Lee S., Malone C., Kemp P. F. 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
    [Google Scholar]
  26. Lim E. L, Amaral L. A., Caron D.A., DeLong E. F. 1993; Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl Environ Microbiol 59:1647–1655
    [Google Scholar]
  27. Lim E. L., Caron D. A., Delong E. F. 1996; Development and field application of a quantitative method for examining natural assemblages of protists with oligonucleotide probes. Appl Environ Microbiol 62:1416–1423
    [Google Scholar]
  28. Manz W., Szewzyk U., Ericsson P., Amann R., Schleifer K.-H., Stenstrbm T.-A. 1993; In situ identification of bacteria in drinking water and adjoining biofilms by hybridisation with 16s and 23s rRNA-directed fluorescent oligonucleotide probes. Appl Environ Microbiol 59:2293–2298
    [Google Scholar]
  29. Neefs J.-M., Van de Peer Y., Hendricks L., De Wachter R. 1990; Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 18:suppl.2237–2317
    [Google Scholar]
  30. Neefs J.-M., Van de Peer Y., De Rijk P., Goris A., De Wachter R. 1991; Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 19:suppl.1987–2018
    [Google Scholar]
  31. Nickrent D. L., Sargent M. L. 1991; An overview of the secondary structure of the V4 region of Eukaryotic small-subunit ribosomal RNA. Nucfeic Acids Res 19:227–235
    [Google Scholar]
  32. Olsen G. J. 1994; Archaea, Archaea, everywhere. Nature 371:657–658
    [Google Scholar]
  33. Schildkraut C., Lifson S. 1965; Dependence of the melting temperature of DNA on salt concentration. Biopolymers 3:195–208
    [Google Scholar]
  34. Sherr E. B., Sherr B. F. 1994; Bacterivory and herbivory: key roles of phagotrophic protists in pelagic food webs. . Microb Ecol 28:223–235
    [Google Scholar]
  35. Stahl D. A., Amann R. 1991; Development and application of nucleic acid probes. Edited by E. Stackebrandt & M. Goodfellow. Chichester : Wiley. In Nucleic Acid Techniques in Bacterial Systematics205–244
    [Google Scholar]
  36. Stahl D. A., Flesher B., Mansfield H. R., Montgomery L. 1988; Use of phylogenetically based hybridisation probes for studies of ruminal microbial ecology. Appl Environ Microbiol 54:1079–1089
    [Google Scholar]
  37. Thompson J. D., Higgins D. G., Gibson T. J. 1994; Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
    [Google Scholar]
  38. Trebesius K., Amann R., Ludwig W., MUhlegger K., Schleifer K.-H. 1994; Identification of whole fixed bacterial cells with nonradioactive 23S rRNA-targeted polynucleotide probes. Appl Environ Microbiol 60:3228–3235
    [Google Scholar]
  39. Ward D. M., Weller R., Bateson M. M. 1990; 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345:63–65
    [Google Scholar]
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