Skip to main content

Characterization of Mycoplasmas by PCR and Sequence Analysis with Universal 16S rDNA Primers

  • Protocol
Mycoplasma Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 104))

Abstract

Ribosomes are present in all self-replicating cells and constitute their protein-synthesizing machinery. The ribosomes are composed of ribosomal proteins and ribosomal RNA (rRNA). Bacteria have three kinds of rRNA (5S, 16S, and 23S rRNA), and the genetic information of these molecules is organized in the genome in the form of rRNA operons. The nucleotide sequences of the rRNA molecules contain well-defined segments of different evolutionary variability, which in the 16S rRNA molecule are referred to as universal (U), semiconserved (S), and variable (V) regions (1). The universal regions are numbered U1–U8 from the 5′-terminus. A more refined model for the nucleotide substitution rates in bacterial rRNA was recently presented, and it was shown that the nucleotide substitution rates within one of the above regions can vary substantially (2). Ribosomal RNA has the same important function in the cell, irrespective of species, which means that the corresponding genes are under approximately the same evolutionary pressure. These properties together make sequence analysis of rRNA extremely suitable for phylogenetic (3) and evolutionary (4) studies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gray, M. W., Sankoff, D., and Cedergren, R. J. (1984) On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA. Nucleic Acids Res. 12, 5837–5852.

    Article  PubMed  CAS  Google Scholar 

  2. Van de Peer, Y., Chapelle, S., and De Wachter, R. (1996) A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Res. 24, 3381–3391.

    Article  PubMed  Google Scholar 

  3. Olsen, G. J. and Woese, C. R. (1993) Ribosomal RNA: a key to phylogeny. FASEB J. 7, 113–123.

    PubMed  CAS  Google Scholar 

  4. Woese, C. R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221–271.

    PubMed  CAS  Google Scholar 

  5. Taschke, C., Klinkert, M.-Q., Wolters, J., and Herrmann, R. (1986) Organization of ribosomal RNA genes in Mycoplasma hyopneumoniae: the 5S rRNA is separated from the 16S and 23S rRNA genes. Mol. Gen. Genet. 205, 428–433.

    Article  PubMed  CAS  Google Scholar 

  6. Chen, X. and Finch, L. R. (1989) Novel arrangement of rRNA genes in Mycoplasma gallisepticum: separation of the 16S gene of one set from the 23S and 5S genes. J. Bacteriol. 171, 2876–2878.

    PubMed  CAS  Google Scholar 

  7. Nakagawa, T., Uemori, T., Asada, K., Kato, I., and Harasawa, R. (1992) Acholeplasma laidlawii has tRNA genes in the 16S–23S spacer of the rRNA operon. J. Bacteriol. 174, 8163–8165.

    PubMed  CAS  Google Scholar 

  8. Maidak, B. L., Olsen G. J., Larsen N., Overbeek R., McCaughey M. J., and Woese C. R. (1996) The ribosomal database project (RDP). Nucleic Acids Res. 24, 82–85.

    Article  PubMed  CAS  Google Scholar 

  9. Van de Peer, Y., Jansen, J., De Rijk, P., De Wachter, R. (1997) Database on the structure of small ribosomal subunit RNA. Nucleic Acids Res. 24, 111–116.

    Article  Google Scholar 

  10. Weisburg, W. G., Tully, J. G., Rose, D. L., Petzel, J. P., Oyaizu, H., Yang, D., Mandelco, L., Sechrest, J., Lawrence, T. G., van Etten, J., Maniloff, J., and Woese, C. R. (1989) A phylogenetic analysis of the mycoplasmas: basis for their classification. J. Bacteriol. 171, 6455–6467.

    PubMed  CAS  Google Scholar 

  11. Pettersson, B., Uhlén, M., and Johansson, K.-E. (1996) Phylogeny of some mycoplasmas from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group. Int. J. Syst. Bacteriol. 46, 1093–1098.

    Article  PubMed  CAS  Google Scholar 

  12. Heldtander, M. U. K., Pettersson, B., Tully, J. G., and Johansson, K.-E. (1998) Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas; Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii, and Mycoplasma yeatsii. Int. J. Syst. Bacteriol. 48, 263–268.

    Article  PubMed  CAS  Google Scholar 

  13. Tully, J. G., Bové, J. M., Laigret, F., and Whitcomb, R. F. (1993) Revised taxonomy of the class Mollicutes: proposed elevation of a monophyletic cluster of arthropod-associated mollicutes to ordinal rank (Entomoplasmatales ord. nov.), with provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceae fam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae. Int. J. Syst. Bacteriol. 43, 378–385.

    Article  Google Scholar 

  14. Iwami, M., Muto, A., Yamao, F., and Osawa, S. (1984) Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum. Mol. Gen. Genet. 196, 317–322.

    Article  PubMed  CAS  Google Scholar 

  15. Frydenberg, J. and Christiansen, C. (1985) The sequence of 16S rRNA from Mycoplasma strain PG50. DNA 4, 127–137.

    Article  PubMed  CAS  Google Scholar 

  16. Hultman, T., Ståhl, S., Hornes, E., and Uhlén, M. (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucleic Acids Res. 17, 4937–4946.

    Article  PubMed  CAS  Google Scholar 

  17. Hultman, T., Bergh, S., Moks, T., and Uhlén, M. (1991) Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA. BioTechniques 10, 84–93.

    PubMed  CAS  Google Scholar 

  18. Pettersson, B., Johansson, K.-E., and Uhlén, M. (1994) Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing. Appl. Environ. Microbiol. 60, 2456–2461.

    PubMed  CAS  Google Scholar 

  19. Pettersson, B., Leitner, T., Ronaghi, M., Bölske, G., Uhlén, M., and Johansson, K.-E. (1996) Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons. J. Bacteriol. 178, 4131–4142.

    PubMed  CAS  Google Scholar 

  20. Pettersson, B. (1997) Direct solid-phase 16S rDNA sequencing: a tool in bacterial phylogeny. PhD thesis. Royal Institute of Technology, Stockholm, Sweden.

    Google Scholar 

  21. Pettersson, B., Bölske, G., Thiaucourt, F., Uhlén, M., and Johansson, K.-E. (1998) Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes, submitted for publication.

    Google Scholar 

  22. Sanger, F., Nicklen, S., and Couldson, A. R. (1977) DNA sequencing with chainterminating inhibitiors. Proc. Natl. Acad. Sci USA 74, 5463–5467.

    Article  PubMed  CAS  Google Scholar 

  23. Smith, S. (1992) Genetic Data Environment (Version 2.2). Millipore Imaging Systems, Ann Arbor, MI.

    Google Scholar 

  24. Program Manual for the Wisconsin Package (Version 8). Genetics Computer Group, Madison, WI.

    Google Scholar 

  25. Felsenstein, J. (1993) PHYLIP: Phylogeny inference package (Version 3.52). University of Washington, Seattle, WA.

    Google Scholar 

  26. Swofford, D. L. (1991) PAUP: Phylogenetic analysis using parsimony (Version 3.1.1.) Illinois Natural History Survey, Champaign, IL.

    Google Scholar 

  27. Kumar, S., Tamura, K., and Nei, M. (1993) MEGA: molecular evolutionary genetic analysis (Version 1.01). The Pennsylvania State University, University Park, PA.

    Google Scholar 

  28. Felsenstein, J. (1988) Phylogenies from molecular sequences: inference and reliability. Annu. Rev. Gen. 22, 521–565.

    Article  CAS  Google Scholar 

  29. Swofford, D. L., Olsen, G. J., Waddell, P. J., and Hillis, D. M. (1996). Phylogenetic inference, in Molecular Systematics, 2nd ed. (Hillis, D. M., Moritz, C., and Mable, B. K., eds.), Sinauer Associates, Sunderland, MA, pp. 407–514.

    Google Scholar 

  30. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.

    PubMed  CAS  Google Scholar 

  31. Morrow, C. J. (1990) Pathogenicity, immunogenicity and strain identification of Australian isolates of M. synoviae, PhD thesis, University of Melbourne, Australia.

    Google Scholar 

  32. Brown, D. R., Crenshaw, B. C., McLaughlin, G. S., Schumacher, I. M., McKenna, C. E., Klein, P. A., Jacobsen, E. R., and Brown, M. B. (1994) Taxonomic analysis of the tortoise mycoplasmas Mycoplasma agassizii and Mycoplasma testudinis by 16S rRNA gene sequence comparison. Int. J. Syst. Bacteriol. 45, 348–350.

    Article  Google Scholar 

  33. Taschke, C., Ruland, K., and Herrmann, R. (1987) Nucleotide sequence of the 16S rRNA of Mycoplasma hyopneumoniae. Nucleic Acids Res. 15, 3918.

    Article  PubMed  CAS  Google Scholar 

  34. Seemüller, E., Schneider, B., Mäurer, R., Ahrens, U., Daire, X., Kison, H., Lorenz, K.-H., Firrao, G., Avinent, L., Sears, B., and Stackebrandt, E. (1994) Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int. J. Syst. Bacteriol. 44, 440–446.

    Article  PubMed  Google Scholar 

  35. Gutell, R. R. (1994) Collection of small subunit (16S-and 16S-like) ribosomal RNA structures: 1994. Nucleic Acids Res. 22, 3502–3507.

    Article  PubMed  CAS  Google Scholar 

  36. Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.

    PubMed  CAS  Google Scholar 

  37. Jukes, T. H. and Cantor, C. R. (1969) Evolution of protein molecules, in Mammalian Protein Metabolism, vol 3. (Munro, H. N., ed.), Academic Press, New York, pp. 21–132.

    Google Scholar 

  38. Ros Bascuñana, C., Mattsson, J. G., Bölske, G., and Johansson, K.-E. (1994) Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR. J. Bacteriol. 176, 2577–2586.

    Google Scholar 

  39. Johansson, K.-E., Berg, L.-O., Bölske, G., Deniz, S., Mattsson, J., Persson, M., and Pettersson, B. (1996) Specific PCR systems based on the 16S rRNA genes of Mycoplasma agalactiae and Mycoplasma bovis, in COST 826. Agriculture and Biotechnology. Mycoplasmas of Ruminants: Pathogenicity, Diagnostics, Epidemiology and Molecular Genetics (Frey, J. and Sarris, K., eds.), European Commission, Brussels, Belgium, pp. 88–90.

    Google Scholar 

  40. Persson, A., Pettersson, B., Johansson, K.-E. (1996) Identification of Mycoplasma mycoides subsp. mycoides SC type by PCR and restriction enzyme analysis with AluI. IOM Lett. 4, 79–80.

    Google Scholar 

  41. Boyle, J. S., Bradbury, J. M., and Morrow, C. J. (1993) Further evidence that M. imitans is closely related to M. gallisepticum, unpublished.

    Google Scholar 

  42. Bradbury, J. M., Abdul-Wahab, O. M. S., Yavari, C. A., Dupiellet, J.-P., and Bové, J. M. (1993) Mycoplasma imitans sp. nov. is related to Mycoplasma gallisepticum and found in birds. Int. J. Syst. Bacteriol. 43, 721–728.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1998 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Johansson, KE., Heldtander, M.U.K., Pettersson, B. (1998). Characterization of Mycoplasmas by PCR and Sequence Analysis with Universal 16S rDNA Primers. In: Miles, R., Nicholas, R. (eds) Mycoplasma Protocols. Methods in Molecular Biology™, vol 104. Humana Press. https://doi.org/10.1385/0-89603-525-5:145

Download citation

  • DOI: https://doi.org/10.1385/0-89603-525-5:145

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-525-6

  • Online ISBN: 978-1-59259-269-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics