Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Aug;63(8):3327–3332. doi: 10.1128/aem.63.8.3327-3332.1997

PCR primers to amplify 16S rRNA genes from cyanobacteria.

U Nübel 1, F Garcia-Pichel 1, G Muyzer 1
PMCID: PMC168636  PMID: 9251225

Abstract

We developed and tested a set of oligonucleotide primers for the specific amplification of 16S rRNA gene segments from cyanobacteria and plastids by PCR. PCR products were recovered from all cultures of cyanobacteria and diatoms that were checked but not from other bacteria and archaea. Gene segments selectively retrieved from cyanobacteria and diatoms in unialgal but nonaxenic cultures and from cyanobionts in lichens could be directly sequenced. In the context of growing sequence databases, this procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. We demonstrate the use of this specific PCR in combination with denaturing gradient gel electrophoresis to probe the diversity of oxygenic phototrophic microorganisms in cultures, lichens, and complex microbial communities.

Full Text

The Full Text of this article is available as a PDF (318.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Ben-Porath J., Zehr J. P. Detection and characterization of cyanobacterial nifH genes. Appl Environ Microbiol. 1994 Mar;60(3):880–887. doi: 10.1128/aem.60.3.880-887.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benson D. A., Boguski M. S., Lipman D. J., Ostell J. GenBank. Nucleic Acids Res. 1997 Jan 1;25(1):1–6. doi: 10.1093/nar/25.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergsland K. J., Haselkorn R. Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120. J Bacteriol. 1991 Jun;173(11):3446–3455. doi: 10.1128/jb.173.11.3446-3455.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  6. Ferris M. J., Muyzer G., Ward D. M. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol. 1996 Feb;62(2):340–346. doi: 10.1128/aem.62.2.340-346.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ferris M. J., Ruff-Roberts A. L., Kopczynski E. D., Bateson M. M., Ward D. M. Enrichment culture and microscopy conceal diverse thermophilic Synechococcus populations in a single hot spring microbial mat habitat. Appl Environ Microbiol. 1996 Mar;62(3):1045–1050. doi: 10.1128/aem.62.3.1045-1050.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fox G. E., Wisotzkey J. D., Jurtshuk P., Jr How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol. 1992 Jan;42(1):166–170. doi: 10.1099/00207713-42-1-166. [DOI] [PubMed] [Google Scholar]
  9. Garcia-Pichel F., Prufert-Bebout L., Muyzer G. Phenotypic and phylogenetic analyses show Microcoleus chthonoplastes to be a cosmopolitan cyanobacterium. Appl Environ Microbiol. 1996 Sep;62(9):3284–3291. doi: 10.1128/aem.62.9.3284-3291.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lerman L. S., Fischer S. G., Hurley I., Silverstein K., Lumelsky N. Sequence-determined DNA separations. Annu Rev Biophys Bioeng. 1984;13:399–423. doi: 10.1146/annurev.bb.13.060184.002151. [DOI] [PubMed] [Google Scholar]
  11. Maidak B. L., Olsen G. J., Larsen N., Overbeek R., McCaughey M. J., Woese C. R. The RDP (Ribosomal Database Project). Nucleic Acids Res. 1997 Jan 1;25(1):109–111. doi: 10.1093/nar/25.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Muyzer G., Teske A., Wirsen C. O., Jannasch H. W. Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments. Arch Microbiol. 1995 Sep;164(3):165–172. doi: 10.1007/BF02529967. [DOI] [PubMed] [Google Scholar]
  13. Muyzer G., de Waal E. C., Uitterlinden A. G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993 Mar;59(3):695–700. doi: 10.1128/aem.59.3.695-700.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Neilan B. A. Identification and phylogenetic analysis of toxigenic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR. Appl Environ Microbiol. 1995 Jun;61(6):2286–2291. doi: 10.1128/aem.61.6.2286-2291.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neilan B. A., Jacobs D., Goodman A. E. Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. Appl Environ Microbiol. 1995 Nov;61(11):3875–3883. doi: 10.1128/aem.61.11.3875-3883.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nelissen B., De Baere R., Wilmotte A., De Wachter R. Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis. J Mol Evol. 1996 Feb;42(2):194–200. doi: 10.1007/BF02198845. [DOI] [PubMed] [Google Scholar]
  17. Nelissen B., Van de Peer Y., Wilmotte A., De Wachter R. An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences. Mol Biol Evol. 1995 Nov;12(6):1166–1173. doi: 10.1093/oxfordjournals.molbev.a040289. [DOI] [PubMed] [Google Scholar]
  18. Nübel U., Engelen B., Felske A., Snaidr J., Wieshuber A., Amann R. I., Ludwig W., Backhaus H. Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol. 1996 Oct;178(19):5636–5643. doi: 10.1128/jb.178.19.5636-5643.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Palenik B. Cyanobacterial community structure as seen from RNA polymerase gene sequence analysis. Appl Environ Microbiol. 1994 Sep;60(9):3212–3219. doi: 10.1128/aem.60.9.3212-3219.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Reysenbach A. L., Giver L. J., Wickham G. S., Pace N. R. Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol. 1992 Oct;58(10):3417–3418. doi: 10.1128/aem.58.10.3417-3418.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Romanowski G., Lorenz M. G., Wackernagel W. Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils. Appl Environ Microbiol. 1993 Oct;59(10):3438–3446. doi: 10.1128/aem.59.10.3438-3446.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rouhiainen L., Sivonen K., Buikema W. J., Haselkorn R. Characterization of toxin-producing cyanobacteria by using an oligonucleotide probe containing a tandemly repeated heptamer. J Bacteriol. 1995 Oct;177(20):6021–6026. doi: 10.1128/jb.177.20.6021-6026.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sheffield V. C., Cox D. R., Lerman L. S., Myers R. M. Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci U S A. 1989 Jan;86(1):232–236. doi: 10.1073/pnas.86.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suzuki M. T., Giovannoni S. J. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol. 1996 Feb;62(2):625–630. doi: 10.1128/aem.62.2.625-630.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Teske A., Wawer C., Muyzer G., Ramsing N. B. Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments. Appl Environ Microbiol. 1996 Apr;62(4):1405–1415. doi: 10.1128/aem.62.4.1405-1415.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Van de Peer Y., Chapelle S., De Wachter R. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Res. 1996 Sep 1;24(17):3381–3391. doi: 10.1093/nar/24.17.3381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ward D. M., Santegoeds C. M., Nold S. C., Ramsing N. B., Ferris M. J., Bateson M. M. Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures. Antonie Van Leeuwenhoek. 1997 Feb;71(1-2):143–150. doi: 10.1023/a:1000131426164. [DOI] [PubMed] [Google Scholar]
  28. Weller R., Weller J. W., Ward D. M. 16S rRNA sequences of uncultivated hot spring cyanobacterial mat inhabitants retrieved as randomly primed cDNA. Appl Environ Microbiol. 1991 Apr;57(4):1146–1151. doi: 10.1128/aem.57.4.1146-1151.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES