1887

Abstract

We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from A 70°C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus , which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with α-P-labeled primers, again with extension at 70°C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and as the outgroup. Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously. Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed.

Loading

Article metrics loading...

/content/journal/ijsem/10.1099/00207713-45-4-811
1995-10-01
2023-01-31
Loading full text...

Full text loading...

/deliver/fulltext/ijsem/45/4/ijs-45-4-811.html?itemId=/content/journal/ijsem/10.1099/00207713-45-4-811&mimeType=html&fmt=ahah

References

  1. An Q., Buxton D., Hendricks A., Robinson L., Shah J., Lu L., Vera-Garcia M., King W., Olive D. M. 1995; Comparison of amplified Qβ replicase and PCR assays for detection of Mycobacterium tuberculosis. . J. Clin. Microbiol. 33:860–867
    [Google Scholar]
  2. Barnard G. F., Puder M., Begum N. A., Chen L. B. 1994; PCR product sequencing with [α-33P] and [α-32P]dATP. BioTechniques 16:572–573
    [Google Scholar]
  3. Barns S. M., Lane D. J., Sogin M. L., Bibeau C., Weisburg W. G. 1991; Evolutionary relationships among pathogenic Candida species and relatives. J. Bacteriol. 173:2250–2255
    [Google Scholar]
  4. Beck K.-F., Stathopulos I., Berninger M. G., Scheweizer M. 1993; Overcoming GC compression in nucleotide sequencing. BioTechniques 14:375
    [Google Scholar]
  5. Boddinghaus B., Rogall T., Flohr T., Blocker H., Bottger E. C. 1990; Detection and identification of mycobacteria by amplification of rRNA. J. Clin. Microbiol. 28:1751–1759
    [Google Scholar]
  6. Bottger E. C. 1989; Rapid determination of bacterial ribosomal RNA sequences by direct sequencing of enzymatically amplified DNA. FEMS Microbiol. Lett. 65:171–176
    [Google Scholar]
  7. Butler W. R., O’Conner S. P., Yakrus M. A., Gross W. M. 1994; Cross-reactivity of genetic probe for detection of Mycobacterium tuberculosis with newly described species Mycobacterium celatum. . J. Clin. Microbiol. 32:536–538
    [Google Scholar]
  8. Casanova J.-L., Pannetier C., Jaulin C., Kourilsky P. 1990; Optimal conditions for directly sequencing double-stranded PCR products with Sequenase. Nucleic Acids Res. 18:4028
    [Google Scholar]
  9. Coyle M. B., Carlson L. C., Wallis C. K., Leonard R. B., Raisys V. A., Kilburn J. O., Samadpour M., Bottger E. C. 1992; Laboratory aspects of Mycobacterium genavense, a proposed species isolated from AIDS patients. J. Clin. Microbiol. 30:3206–3212
    [Google Scholar]
  10. De Soete G. 1983; A least squares algorithm for fitting additive trees to proximity data. Psychometrika 48:621–626
    [Google Scholar]
  11. Fichot O., Girard M. 1990; An improved method for sequencing of RNA templates. Nucleic Acids Res. 18:6162
    [Google Scholar]
  12. Ford E. G., Snead S. J., Todd J., Warren N. G. 1993; Strains of Mycobacterium terrae complex which react with DNA probes for M. tuberculosis complex.. J. Clin. Microbiol. 31:2805–2806
    [Google Scholar]
  13. Frothingham R., Hills H. G., Wilson K. H. 1994; Extensive DNA sequence conservation throughout the Mycobacterium tuberculosis complex. J. Clin. Microbiol. 32:1639–1643
    [Google Scholar]
  14. Gaynor C. D., Clark R. A., Koontz F. P., Emler S., Hirschel B., Schlesinger L. S. 1994; Disseminated Mycobacterium genavense infection in two patients with AIDS. Clin. Infect. Dis. 18:455–457
    [Google Scholar]
  15. Good R. C. 1991; The genus Mycobacterium—medical,. 1238–1270 Balows A., Trüper H. G., Dworkin M., Harder W., Schleifer K.-H. The prokaryotes, 2. II Springer-Verlag; New York:
    [Google Scholar]
  16. Gutell R. R., Gray M. W., Schnare M. N. 1993; A compilation of large subunit (23S- and 23S-like) ribosomal RNA structures. Nucleic Acids Res. 21:3055–3074
    [Google Scholar]
  17. Gyllensten U. B., Erlich H. A. 1988; Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQalpha locus.. Proc. Natl. Acad. Sci.USA 85:7652–7656
    [Google Scholar]
  18. Hartmans S., De Bont J. A. M. 1991; The genus Mycobacteriumnonmedical,. 1214–1237 Balows A., Trüper H. G., Dworkin M., Harder W., Schleifer K.-H. The prokaryotes, 2. II Springer-Verlag; New York:
    [Google Scholar]
  19. Higuchi R. G., Ochman H. 1989; Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Res. 17:5865
    [Google Scholar]
  20. Hughes M. S., Skuce R. A., Veck L.-A., Neill S. D. 1993; Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences. J. Clin. Microbiol. 31:3216–3222
    [Google Scholar]
  21. Kirschner P., Meier A., Bottger E. C. 1993; Genotypic identification and detection of mycobacteria—facing novel and uncultured pathogens,. 173–190 Persing D. H., Smith T. F., Tenover F. C., White T. J. Diagnostic molecular microbiology: principles and applications American Society for Microbiology; Washington, D.C:
    [Google Scholar]
  22. Lane D. J. 1991; 16S/23S rRNA sequencing,. 115–176 Stackebrandt E., Goodfellow M. Nucleic acid techniques in bacterial systematics J. Wiley & Sons; New York:
    [Google Scholar]
  23. Lane D. J., Pace B., Olsen G. J., Stahl D. A., Sogin M. L., Pace N. R. 1985; Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses.. Proc. Natl. Acad. Sci.USA 82:6955–6959
    [Google Scholar]
  24. Maidak B. Personal communication.
  25. Maidak B. L., Larsen N., McCaughey M. J., Overbeek R., Olsen G. J., Fogel K., Blandy J., Woese C. R. 1994; The Ribosomal Database Project. Nucleic Acids Res. 22:3485–3487
    [Google Scholar]
  26. Mitchell L. G., Merill C. R. 1989; Affinity generation of single-stranded DNA for dideoxy sequencing following the polymerase chain reaction. Anal. Biochem. 178:239–242
    [Google Scholar]
  27. Myers T. W., Gelfand D. H. 1991; Reverse transcriptions and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30:7661–7666
    [Google Scholar]
  28. O’Brien D. P., Billadeau D., Van Ness B. 1994; RT-PCR assay for detection of transcripts from very few cells using whole cell lysates. BioTechniques 16:586–590
    [Google Scholar]
  29. Olsen G. J. 1988; Phylogenetic analysis using ribosomal RNA. Methods Enzymol. 164:793–812
    [Google Scholar]
  30. Pitulle C., Dorsch M., Kazda J., Wolters J., Stackebrandt E. 1992; Phylogeny of rapidly growing members of the genus Mycobacterium. . Int. J. Syst. Bacteriol. 42:337–343
    [Google Scholar]
  31. Reynolds T. R., Uliana S. R. B., Floeter-Winter L. M., Buck G. A. 1993; Optimization of coupled PCR amplification and cycle sequencing of cloned and genomic DNA. BioTechniques 15:462–467
    [Google Scholar]
  32. Rogall T., Wolters J., Flohr T., Bottger E. 1990; Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. . Int. J. Syst. Bacteriol. 40:323–330
    [Google Scholar]
  33. Ruan C. C., Fuller C. W. 1991; Using T7 gene 6 exonuclease to prepare single-stranded templates for sequencing. Comments U. S. Biochemical Corp. 18:1–8
    [Google Scholar]
  34. Ruano G., Kidd K. K. 1991; Coupled amplification and sequencing of genomic DNA.. Proc. Natl. Acad. Sci.USA 88:2815–2819
    [Google Scholar]
  35. Shinnick T. M., Good R. C. 1994; Mycobacterial taxonomy. Eur. J. Clin. Microbiol. Infect. Dis. 13:884–901
    [Google Scholar]
  36. Stackebrandt E., Goebel B. M. 1994; Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44:846–849
    [Google Scholar]
  37. Stahl D. A., Urbance J. W. 1990; The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J. Bacteriol. 172:116–124
    [Google Scholar]
  38. Telenti A., Marchesi F., Balz M., Bally F., Bottger E. C., Bodmer T. 1993; Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J. Clin. Microbiol. 31:175–178
    [Google Scholar]
  39. Teske A., Boddinghaus B., Rogall T., Ohrdorf A., Bottger E. C. 1991; A general method for generation of taxon-specific DNA probes: application for the detection and identification of mycobacteria by amplification of rRNA followed by hybridization with specific oligonucleotides. Adv. Mol. Gen. 3:265–276
    [Google Scholar]
  40. Walther W., Stein U., Eder C. 1994; RNA analysis using miniprep RNA in reverse transcription PCR. BioTechniques 17:674–675
    [Google Scholar]
  41. Wang R.-F., Cao W. W., Slavik M. F. 1991; Comparison and modification of rRNA sequencing methods. BioTechniques 11:438–439
    [Google Scholar]
  42. Weisburg W. G., Barns S. M., Pelletier D. A., Lane D. J. 1991; 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697–703
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/ijsem/10.1099/00207713-45-4-811
Loading
/content/journal/ijsem/10.1099/00207713-45-4-811
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error