Production of avirulent Mycobacterium bovis strains by illegitimate recombination with deoxyribonucleic acid fragments containing an interrupted ahpC gene

Abstract

Setting: Molecular genetic techniques have been used to elucidate virulence determinants and to produce vaccines for many bacterial pathogens but reliable techniques for slow-growing mycobacteria have not been available. Objective: Oxidative defence genes including ahpC are involved in isoniazid resistance of strains of the Mycobacterium tuberculosis complex. The aim of this study was to inactivate ahpC by allelic exchange and to screen the expected background of illegitimate recombinants for their inability to grow in minimal medium (auxotrophy). Design: M. bovis ATCC35723 was electroporated with linear fragments of deoxyribonucleic acid (DNA) containing an ahpC gene interrupted by a kanamycin resistance gene. Kanamycin-resistant colonies were screened for allelic exchange and auxotrophy. Results: Southern blotting of DNA from kanamycin-resistant colonies revealed that no allelic exchange had occurred. Four of these recombinants were auxotrophs and subsequently were found to be avirulent in guinea pigs. The fragment insertion sites in the chromosome of each auxotroph were determined by DNA sequencing. In three cases, large chromosomal deletions had occurred. Conclusion: The M. bovis ahpC gene was not inactivated by this linear fragment approach but illegitimate insertion of such a fragment can be successfully used to produce avirulent auxotrophs which have potential for vaccine development.