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

• Published in: Microbiology (Society for General Microbiology) Vol. 154; no. Pt 8; pp. 2427 - 2436
• Main Authors: Janis, Carole, Bischof, Daniela, Gourgues, Géraldine, Frey, Joachim, Blanchard, Alain, Sirand-Pugnet, Pascal
• Format: Journal Article
• Language: English
• Published: 01-08-2008
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.2008/017640-0

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
γδ
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.