Advertisement

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

  • Karl-Erik Johansson
  • Malin U. K. Heldtander
  • Bertil Pettersson
Part of the Methods in Molecular Biology™ book series (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.

Keywords

Ribosomal Database Project rRNA Operon Nucleotide Substitution Rate European Molecular Biology Laboratory Universal Region 
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.

References

  1. 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.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 3.
    Olsen, G. J. and Woese, C. R. (1993) Ribosomal RNA: a key to phylogeny. FASEB J. 7, 113–123.PubMedGoogle Scholar
  4. 4.
    Woese, C. R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221–271.PubMedGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 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.PubMedGoogle Scholar
  7. 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.PubMedGoogle Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. 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.CrossRefGoogle Scholar
  10. 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.PubMedGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.CrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 15.
    Frydenberg, J. and Christiansen, C. (1985) The sequence of 16S rRNA from Mycoplasma strain PG50. DNA 4, 127–137.PubMedCrossRefGoogle Scholar
  16. 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.PubMedCrossRefGoogle Scholar
  17. 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.PubMedGoogle Scholar
  18. 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.PubMedGoogle Scholar
  19. 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.PubMedGoogle Scholar
  20. 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. 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. 21a.
    Sanger, F., Nicklen, S., and Couldson, A. R. (1977) DNA sequencing with chainterminating inhibitiors. Proc. Natl. Acad. Sci USA 74, 5463–5467.PubMedCrossRefGoogle Scholar
  23. 22.
    Smith, S. (1992) Genetic Data Environment (Version 2.2). Millipore Imaging Systems, Ann Arbor, MI.Google Scholar
  24. 23.
    Program Manual for the Wisconsin Package (Version 8). Genetics Computer Group, Madison, WI.Google Scholar
  25. 24.
    Felsenstein, J. (1993) PHYLIP: Phylogeny inference package (Version 3.52). University of Washington, Seattle, WA.Google Scholar
  26. 25.
    Swofford, D. L. (1991) PAUP: Phylogenetic analysis using parsimony (Version 3.1.1.) Illinois Natural History Survey, Champaign, IL.Google Scholar
  27. 26.
    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. 27.
    Felsenstein, J. (1988) Phylogenies from molecular sequences: inference and reliability. Annu. Rev. Gen. 22, 521–565.CrossRefGoogle Scholar
  29. 28.
    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. 29.
    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.PubMedGoogle Scholar
  31. 30.
    Morrow, C. J. (1990) Pathogenicity, immunogenicity and strain identification of Australian isolates of M. synoviae, PhD thesis, University of Melbourne, Australia.Google Scholar
  32. 31.
    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.CrossRefGoogle Scholar
  33. 32.
    Taschke, C., Ruland, K., and Herrmann, R. (1987) Nucleotide sequence of the 16S rRNA of Mycoplasma hyopneumoniae. Nucleic Acids Res. 15, 3918.PubMedCrossRefGoogle Scholar
  34. 33.
    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.PubMedCrossRefGoogle Scholar
  35. 34.
    Gutell, R. R. (1994) Collection of small subunit (16S-and 16S-like) ribosomal RNA structures: 1994. Nucleic Acids Res. 22, 3502–3507.PubMedCrossRefGoogle Scholar
  36. 35.
    Saitou, N. and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.PubMedGoogle Scholar
  37. 36.
    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. 37.
    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. 38.
    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. 39.
    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. 40.
    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. 41.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1998

Authors and Affiliations

  • Karl-Erik Johansson
    • 1
  • Malin U. K. Heldtander
    • 1
  • Bertil Pettersson
    • 1
  1. 1.Department of BacteriologyNational Veterinary Institute (SVA)UppsalaSweden

Personalised recommendations