1887

Abstract

The genome of strain R has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for and using the region upstream from the gene. The plasmids of (pGTLori) and (pMIori) replicated in both species, but could not support replication of pGTLori. A 180 bp section of the region of was found to be the minimal region required for plasmid replication in strain S6, the shortest region defined for mycoplasmas. Targeted gene disruption of of S6 was attempted using these plasmids. Constructs made in pPLoriC7 integrated into the genomic region, not into the targeted gene, whereas those made in pMIori disrupted the gene, which has 97 % DNA sequence identity with the gene. During passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in and , and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.

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2008-09-01
2019-10-22
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References

  1. Bearson, S. M., Collier, S. D., Bearson, B. L. & Branton, S. L. ( 2003; ). Induction of a Mycoplasma gallisepticum pMGA gene in the chicken tracheal ring organ culture model. Avian Dis 47, 745–749.[CrossRef]
    [Google Scholar]
  2. Bradbury, J. M., Abdul-Wahab, O. M., Yavari, C. A., Dupiellet, J. P. & Bove, J. M. ( 1993; ). Mycoplasma imitans sp. nov. is related to Mycoplasma gallisepticum and found in birds. Int J Syst Bacteriol 43, 721–728.[CrossRef]
    [Google Scholar]
  3. Chopra-Dewasthaly, R., Marenda, M., Rosengarten, R., Jechlinger, W. & Citti, C. ( 2005; ). Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae. FEMS Microbiol Lett 253, 89–94.[CrossRef]
    [Google Scholar]
  4. Church, G. M. & Gilbert, W. ( 1984; ). Genomic sequencing. Proc Natl Acad Sci U S A 81, 1991–1995.[CrossRef]
    [Google Scholar]
  5. Cordova, C. M., Lartigue, C., Sirand-Pugnet, P., Renaudin, J., Cunha, R. A. & Blanchard, A. ( 2002; ). Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids. J Bacteriol 184, 5426–5435.[CrossRef]
    [Google Scholar]
  6. Duret, S., Danet, J. L., Garnier, M. & Renaudin, J. ( 1999; ). Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic. J Bacteriol 181, 7449–7456.
    [Google Scholar]
  7. Dybvig, K. & Alderete, J. ( 1988; ). Transformation of Mycoplasma pulmonis and Mycoplasma hyorhinis: transposition of Tn916 and formation of cointegrate structures. Plasmid 20, 33–41.[CrossRef]
    [Google Scholar]
  8. Dybvig, K. & Cassell, G. H. ( 1987; ). Transposition of Gram-positive transposon Tn916 in Acholeplasma laidlawii and Mycoplasma pulmonis. Science 235, 1392–1394.[CrossRef]
    [Google Scholar]
  9. Glew, M. D., Browning, G. F., Markham, P. F. & Walker, I. D. ( 2000; ). pMGA phenotypic variation in Mycoplasma gallisepticum occurs in vivo and is mediated by trinucleotide repeat length variation. Infect Immun 68, 6027–6033.[CrossRef]
    [Google Scholar]
  10. Hedreyda, C. T., Lee, K. K. & Krause, D. C. ( 1993; ). Transformation of Mycoplasma pneumoniae with Tn4001 by electroporation. Plasmid 30, 170–175.[CrossRef]
    [Google Scholar]
  11. Hudson, P., Gorton, T. S., Papazisi, L., Cecchini, K., Frasca, S., Jr & Geary, S. J. ( 2006; ). Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants. Infect Immun 74, 931–939.[CrossRef]
    [Google Scholar]
  12. Janis, C., Lartigue, C., Frey, J., Wroblewski, H., Thiaucourt, F., Blanchard, A. & Sirand-Pugnet, P. ( 2005; ). Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum. Appl Environ Microbiol 71, 2888–2893.[CrossRef]
    [Google Scholar]
  13. Lartigue, C., Duret, S., Garnier, M. & Renaudin, J. ( 2002; ). New plasmid vectors for specific gene targeting in Spiroplasma citri. Plasmid 48, 149–159.[CrossRef]
    [Google Scholar]
  14. Lartigue, C., Blanchard, A., Renaudin, J., Thiaucourt, F. & Sirand-Pugnet, P. ( 2003; ). Host specificity of mollicutes oriC plasmids: functional analysis of replication origin. Nucleic Acids Res 31, 6610–6618.[CrossRef]
    [Google Scholar]
  15. Ley, D. H. & Yoder, H. W. J. ( 1997; ). Mycoplasma gallisepticum infection. In Diseases of Poultry, pp. 194–207. Edited by B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald & Y. M. Saif. London: Mosby-Wolfe.
  16. Mahairas, G. G. & Minion, F. C. ( 1989; ). Random insertion of the gentamicin resistance transposon Tn4001 in Mycoplasma pulmonis. Plasmid 21, 43–47.[CrossRef]
    [Google Scholar]
  17. Markham, P. F., Glew, M. D., Brandon, M. R., Walker, I. D. & Whithear, K. G. ( 1992; ). Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum. Infect Immun 60, 3885–3891.
    [Google Scholar]
  18. Markham, P. F., Kanci, A., Czifra, G., Sundquist, B., Hains, P. & Browning, G. F. ( 2003; ). Homologue of macrophage-activating lipoprotein in Mycoplasma gallisepticum is not essential for growth and pathogenicity in tracheal organ cultures. J Bacteriol 185, 2538–2547.[CrossRef]
    [Google Scholar]
  19. Marois, C., Dufour-Gesbert, F. & Kempf, I. ( 2001; ). Molecular differentiation of Mycoplasma gallisepticum and Mycoplasma imitans strains by pulsed-field gel electrophoresis and random amplified polymorphic DNA. J Vet Med B Infect Dis Vet Public Health 48, 695–703.[CrossRef]
    [Google Scholar]
  20. Mudahi-Orenstein, S., Levisohn, S., Geary, S. J. & Yogev, D. ( 2003; ). Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis. Infect Immun 71, 3812–3820.[CrossRef]
    [Google Scholar]
  21. Papazisi, L., Frasca, S., Jr, Gladd, M., Liao, X., Yogev, D. & Geary, S. J. ( 2002; ). GapA and CrmA coexpression is essential for Mycoplasma gallisepticum cytadherence and virulence. Infect Immun 70, 6839–6845.[CrossRef]
    [Google Scholar]
  22. Papazisi, L., Gorton, T. S., Kutish, G., Markham, P. F., Browning, G. F., Nguyen, D. K., Swartzell, S., Madan, A., Mahairas, G. & Geary, S. J. ( 2003; ). The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow. Microbiology 149, 2307–2316.[CrossRef]
    [Google Scholar]
  23. Renaudin, J., Marais, A., Verdin, E., Duret, S., Foissac, X., Laigret, F. & Bove, J. M. ( 1995; ). Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri. J Bacteriol 177, 2870–2877.
    [Google Scholar]
  24. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  25. Whetzel, P. L., Hnatow, L. L., Keeler, C. L., Jr & Dohms, J. E. ( 2003; ). Transposon mutagenesis of Mycoplasma gallisepticum. Plasmid 49, 34–43.[CrossRef]
    [Google Scholar]
  26. Whithear, K. G. ( 1993; ). Avian mycoplasmosis. In Australian Standard Diagnostic Techniques for Animal Diseases, pp. 1–12. Edited by L. A. Corner & T. J. Bagust. East Melbourne: Australian Agricultural Council (Standing Committee on Agriculture and Resource Management).
  27. Winner, F., Markova, I., Much, P., Lugmair, A., Siebert-Gulle, K., Vogl, G., Rosengarten, R. & Citti, C. ( 2003; ). Phenotypic switching in Mycoplasma gallisepticum hemadsorption is governed by a high-frequency, reversible point mutation. Infect Immun 71, 1265–1273.[CrossRef]
    [Google Scholar]
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