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Microbiology 154 (2008), 2427-2436; DOI  10.1099/mic.0.2008/017640-0
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Microbiology 154 (2008), 2427-2436; DOI  10.1099/mic.0.2008/017640-0
© 2008 Society for General Microbiology

Unmarked insertional mutagenesis in the bovine pathogen Mycoplasma mycoides subsp. mycoides SC: characterization of a lppQ mutant

Carole Janis1,2,{dagger}, Daniela Bischof3, Géraldine Gourgues1,2, Joachim Frey3, Alain Blanchard1,2 and Pascal Sirand-Pugnet1,2

1 INRA, UMR 1090, 71 avenue Edouard Bourlaux, F-33140 Villenave d'Ornon, France
2 Université Victor Segalen Bordeaux 2, UMR 1090, 71 avenue Edouard Bourlaux, F-33140 Villenave d'Ornon, France
3 Institute of Veterinary Bacteriology, Universität Bern, Laenggassstrasse 122, CH-3012 Berne, Switzerland

Correspondence
Pascal Sirand-Pugnet
sirand{at}bordeaux.inra.fr

Mycoplasma mycoides subspecies mycoides small colony (SC) is the aetiologic agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease causing important losses in cattle production. The publication of the genome sequence of M. mycoides subsp. mycoides SC should facilitate the identification of putative virulence factors. However, real progress in the study of molecular mechanisms of pathogenicity also requires efficient molecular tools for gene inactivation. In the present study, we have developed a transposon-based approach for the random mutagenesis of M. mycoides subsp. mycoides SC. A PCR-based screening assay enabled the characterization of several mutants with knockouts of genes potentially involved in pathogenicity. The initial transposon was further improved by combining it with the transposon {gamma}{delta} TnpR/res recombination system to allow the production of unmarked mutations. Using this approach, we isolated a mutant free of antibiotic-resistance genes, in which the gene encoding the main lipoprotein LppQ was disrupted. The mutant was found to express only residual amounts of the truncated N-terminal end of LppQ. This approach opens the way to study virulence factors and pathogen–host interactions of M. mycoides subsp. mycoides SC and to develop new, genetically defined vaccine strains.

Abbreviations: CBPP, contagious bovine pleuropneumonia; IR, inverted repeat

{dagger}Present address: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.






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