1887

Abstract

The phylogenically related and species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for type-strain PG45 or for type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of and field isolates. Results indicated that approximately 70 % of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one isolate, which was first classified as , reacted with 15 % of the PG2-specific probes, while several isolates reacted with 15 % of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of (ICEF) was found in most field isolates and in a few isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of - and -specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of and .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27590-0
2005-02-01
2019-10-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/2/mic1510475.html?itemId=/content/journal/micro/10.1099/mic.0.27590-0&mimeType=html&fmt=ahah

References

  1. Akopyants, N. S., Fradkov, A., Diatchenko, L., Hill, J. E., Siebert, P. D., Lukyanov, S. A., Sverdlov, E. D. & Berg, D. E. ( 1998; ). PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci U S A 95, 13108–13113.[CrossRef]
    [Google Scholar]
  2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  3. Aluotto, B. B., Wittler, R. G., Williams, C. O. & Faber, J. E. ( 1970; ). Standardized bacteriologic techniques for the characterization of mycoplasma species. Int J Syst Bacteriol 20, 35–58.[CrossRef]
    [Google Scholar]
  4. Bashiruddin, J., Frey, J., Konigsson, M. H. & 9 other authors ( 2004; ). Evaluation of PCR systems for the identification and differentiation of Mycoplasma agalactiae and Mycoplasma bovis: a collaborative trial. Vet J (in press).
    [Google Scholar]
  5. Bergonier, D., De Simone, F., Russo, P., Solsona, M., Lambert, M. & Poumarat, F. ( 1996; ). Variable expression and geographic distribution of Mycoplasma agalactiae surface epitopes demonstrated with monoclonal antibodies. FEMS Microbiol Lett 143, 159–165.[CrossRef]
    [Google Scholar]
  6. Bergonier, D., Berthelot, X. & Poumarat, F. ( 1997; ). Contagious agalactia of small ruminants: current knowledge concerning epidemiology, diagnosis and control. Rev Sci Tech 16, 848–873.
    [Google Scholar]
  7. Bogush, M. L., Velikodvorskaya, T. V., Lebedev, Y. B. & 9 other authors ( 1999; ). Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization. Mol Gen Genet 262, 721–729.[CrossRef]
    [Google Scholar]
  8. Calcutt, M. J., Lewis, M. S. & Wise, K. S. ( 2002; ). Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18. J Bacteriol 184, 6929–6941.[CrossRef]
    [Google Scholar]
  9. Chao, K. M., Pearson, W. R. & Miller, W. ( 1992; ). Aligning two sequences within a specified diagonal band. Comput Appl Biosci 8, 481–487.
    [Google Scholar]
  10. Chavez Gonzalez, Y. R., Ros Bascunana, C., Bolske, G., Mattsson, J. G., Fernandez Molina, C. & Johansson, K. E. ( 1995; ). In vitro amplification of the 16S rRNA genes from Mycoplasma bovis and Mycoplasma agalactiae by PCR. Vet Microbiol 47, 183–190.[CrossRef]
    [Google Scholar]
  11. Church, G. M. & Gilbert, W. ( 1984; ). Genomic sequencing. Proc Natl Acad Sci U S A 81, 1991–1995.[CrossRef]
    [Google Scholar]
  12. Fleury, B., Bergonier, D., Berthelot, X., Schlatter, Y., Frey, J. & Vilei, E. M. ( 2001; ). Characterization and analysis of a stable serotype-associated membrane protein (P30) of Mycoplasma agalactiae. J Clin Microbiol 39, 2814–2822.[CrossRef]
    [Google Scholar]
  13. Kuo, C. S., Hong, H. S. & Chiou, J. F. ( 2000; ). Application of representational difference analysis to cloning a Mycoplasma arthritidis specific DNA fragment. J Microbiol Immunol Infect 33, 127–130.
    [Google Scholar]
  14. Marenda, M. S., Vilei, E. M., Poumarat, F., Frey, J. & Berthelot, X. ( 2004; ). Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2. Vet Res 35, 199–212.[CrossRef]
    [Google Scholar]
  15. Middendorf, B. & Gross, R. ( 1999; ). Representational difference analysis identifies a strain-specific LPS biosynthesis locus in Bordetella spp. Mol Gen Genet 262, 189–198.[CrossRef]
    [Google Scholar]
  16. Pfutzner, H. & Sachse, K. ( 1996; ). Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle. Rev Sci Tech 15, 1477–1494.
    [Google Scholar]
  17. Pilo, P., Fleury, B., Marenda, M., Frey, J. & Vilei, E. M. ( 2003; ). Prevalence and distribution of the insertion element ISMag1 in Mycoplasma agalactiae. Vet Microbiol 92, 37–48.[CrossRef]
    [Google Scholar]
  18. Poumarat, F., Perrin, B. & Longchambon, D. ( 1991; ). Identification of ruminant mycoplasmas by dot immunobinding on membrane filtration (MF dot). Vet Microbiol 29, 329–338.[CrossRef]
    [Google Scholar]
  19. Radnedge, L., Gamez-Chin, S., McCready, P. M., Worsham, P. L. & Andersen, G. L. ( 2001; ). Identification of nucleotide sequences for the specific and rapid detection of Yersinia pestis. Appl Environ Microbiol 67, 3759–3762.[CrossRef]
    [Google Scholar]
  20. Razin, S. & Freundt, E. A. ( 1984; ). The mycoplasmas. In Bergey's Manual of Systematic Bacteriology, p. 749. Edited by J. G. Holt. Baltimore: Williams & Wilkins.
  21. Subramaniam, S., Bergonier, D., Poumarat, F., Capaul, S., Schlatter, Y., Nicolet, J. & Frey, J. ( 1998; ). Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR. Mol Cell Probes 12, 161–169.[CrossRef]
    [Google Scholar]
  22. Tinsley, C. R. & Nassif, X. ( 1996; ). Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: two closely related bacteria expressing two different pathogenicities. Proc Natl Acad Sci U S A 93, 11109–11114.[CrossRef]
    [Google Scholar]
  23. Tully, J. G. ( 1995; ). Culture medium formulation for primary isolation and maintenance of mollicutes. In Molecular and Diagnostic Procedures in Mycoplasmology: Molecular Characterization, pp. 33–39. Edited by J. G. Tully. San Diego: Academic Press.
  24. Winstanley, C. ( 2002; ). Spot the difference: applications of subtractive hybridization to the study of bacterial pathogens. J Med Microbiol 51, 459–467.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27590-0
Loading
/content/journal/micro/10.1099/mic.0.27590-0
Loading

Data & Media loading...

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