Advertisement

Microbial Ecology

, Volume 21, Issue 1, pp 191–198 | Cite as

Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria

  • W. Liesack
  • H. Weyland
  • E. Stackebrandt
Article

Abstract

The 16S rDNA genes of an apparently pure culture of a psychrophilic and strict barophilic bacterium (WHB 46) were studied by PCR-mediated amplification and cloning into phage M13 mp18. Sequence analysis of five individual clones revealed the presence of two different 16S rDNA types. The homology value of 90% indicates that culture WHB 46 is actually composed of two closely related species (WHB 46-1 and 46-2). Both strains are members of the γ-subdivision of proteobacteria. Analysis of a sixth clone (WHB 46-1/2) leads to the conclusion that it represents a 16S rDNA hybrid molecule assembled during the PCR reaction. This hypothesis was confirmed by secondary structure analysis of the chimeric rDNA. The appearance of such hybrid molecules point to a potential risk in studies on the diversity of bacterial populations by analysis of rDNA pattern via PCR-mediated amplification because they suggest the existence of organisms that do not actually exist in the sample investigated.

Keywords

Filamentous Bacterium Diatom Frustule Peritrophic Membrane Gastric Mill Fine Organic Matter 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Casanova J-L., Pannetier C, Kourilsky P (1990) Optimal conditions for directly sequencing double-stranded PCR products with Sequenase, Nucl Acids Res 18:4028PubMedGoogle Scholar
  2. 2.
    Deming JW, Somers LK, Straube WL, Swartz DG, MacDonell MT (1988) Isolation of an obligately barophilic bacterium and description of a new genus,Colwellia gen. nov. System Appl Microbiol 10:152–160Google Scholar
  3. 3.
    Fitch WM (1981) A non-sequential method for constructing trees and hierarchical classifications, J Mol Evol 18:30–37CrossRefPubMedGoogle Scholar
  4. 4.
    Giovannoni SJ, Britschgi TB, Moyer CL, Field KG (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345:60–62CrossRefPubMedGoogle Scholar
  5. 5.
    Hagenbuechle O, Santer M, Steitz JA, Mans RJ (1978) Conservation of primary structure at 3′ end of 18S rRNA from eucaryotic cells. Cell 13:551–563CrossRefGoogle Scholar
  6. 6.
    Helmke E, Weyland H (1986) Effect of hydrostatic pressure and temperature on the activity and synthesis of chitinases of Antarctic Ocean bacteria. Mar Biol 91:1–7CrossRefGoogle Scholar
  7. 7.
    Higuchi RG, Ochman H (1989) Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction, Nucl Acids Res 17:5865PubMedGoogle Scholar
  8. 8.
    Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR protocols: A guide to methods and applications. Academic Press, San DiegoGoogle Scholar
  9. 9.
    Kemmerling C, Witt D, Liesack W, Weyland H, Stackebrandt E (1989) Approaches for the molecular identification of streptomycetes in marine environment. In: Miyachi S, Karube I, Ishida Y (eds) Current topics in marine biotechnology. Jpn Soc Marine Biotech, Tokyo, pp 423–426Google Scholar
  10. 10.
    Liesack W, Menke M, Stackebrandt E (1990) Rapid generation of vector-free Digoxygenin-dUTP labeled probes for nonradioactive hybridization using polymerase chain reaction (PCR) method. System Appl Microbiol 13:255–256Google Scholar
  11. 11.
    MacDonell MT, Colwell RR (1985) Phylogeny of the Vibrionaceae, and recommendation for two new genera,Listonella andShewanella. System Appl Microbiol 6:171–182Google Scholar
  12. 12.
    Meyerhans A, Vartanian JP, Wain-Hobson S (1990) DNA recombination during PCR. Nucl Acids Res 18:1687PubMedGoogle Scholar
  13. 13.
    Neefs JM, Van de Peer Y, Hendriks Y, De Wachter R (1990) Compilation of small ribosomal subunit RNA sequences. Nucl Acids Res 18:2237–2317PubMedGoogle Scholar
  14. 14.
    Ogram A, Sayler GS, Barkay T (1987) The extraction and purification of microbial DNA from sediments. J Microbiol Meth 7:57–66CrossRefGoogle Scholar
  15. 15.
    Puetz J, Meinert F, Wyss U, Ehlers R-U, Stackebrandt E (1990) Development and application of oligonucleotide probes for molecular identification ofXenorhabdus species. Appl Environ Microbiol 56:181–186Google Scholar
  16. 16.
    Saiki RK, Gelfand DH, Stoffel SJ, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491CrossRefPubMedGoogle Scholar
  17. 17.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  18. 18.
    Sattath S, Tversky A (1977) Additive similarity trees. Psychomet 42:319–345CrossRefGoogle Scholar
  19. 19.
    Steffan RJ, Goksoyr J, Bey AK, Atlas RM (1988) Recovery of DNA from soils and sediments. Appl Environ Microbiol 54:2908–2915PubMedGoogle Scholar
  20. 20.
    Valle O, Dorsch M, Wiik R, Stackebrandt E (1990) Nucleotide sequence of the 16S rRNA fromVibrio anguillarum. Syst Appl Microbiol 13:257Google Scholar
  21. 21.
    Ward DM, Weller R, Bateson MM (1990) 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345:63–65CrossRefPubMedGoogle Scholar
  22. 22.
    Weller R, Ward DM (1989) Selective recovery of 16S rRNA sequences from natural microbial communities in the form of cDNA. Appl Environ Microbiol 55:1818–1822PubMedGoogle Scholar
  23. 23.
    Weyland H, Helmke E (1989) Barophilic and psychrophilic bacteria in the Antarctic Ocean. In: Hottori T, Ishida Y, Maruyama Y, Morita RY, Uchida A (eds) Recent advances in microbial ecology. Jpn Sci Soc Press, Tokyo, pp 43–47Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1991

Authors and Affiliations

  • W. Liesack
    • 1
  • H. Weyland
    • 2
  • E. Stackebrandt
    • 1
  1. 1.Department of MicrobiologyUniversity of QueenslandSt. LuciaAustralia
  2. 2.Alfred-Wegener-Institut für Polar- und MeeresforschungBremerhavenFederal Republic of Germany

Personalised recommendations