Construction of Mycobacterium abscessus defined glycopeptidolipid mutants: comparison of genetic tools

Mycobacterium abscessus is a rapidly growing mycobacterial species that can be involved in pulmonary and disseminated infections in immunosuppressed or young cystic fibrosis patients. It is an emerging pathogen and has attracted recent attention due to the numerous cases of infection; furthermore, genomic tools have been developed for this species. Nevertheless, the study of this species has until now been limited to spontaneous variants. We report here a comparison of three different mutagenesis systems--the ts-sacB, the phage, and the recombineering systems--and show that there are important differences in their efficiency for the construction of allelic-exchange mutants. We show, using the mmpL4b gene of the glycopeptidolipid pathway as a target, that allelic-exchange mutants can be constructed with a reasonable efficiency (approximately 7%) using the recombineering system. These observations will facilitate genetic and cellular microbiology experiments involving the construction and use of well-defined mutants to study the virulence determinant of this emerging pathogen.