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 (ϳ7%) 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.The mycobacterial genus contains plethora of species that are pathogenic for either humans or animals. The most wellknown are undoubtedly Mycobacterium leprae, M. tuberculosis, and M. ulcerans, the etiologic agents of human leprosy, tuberculosis, and Buruli ulcer, respectively (47-49). M. avium subsp. paratuberculosis, responsible for Johnes disease in ruminants, is also a serious health concern since it is suspected to be a threat to human via infected milk (9, 10). M. abscessus is an emerging pathogen involved in pulmonary and disseminated infection in young cystic fibrosis patients (26,36). M. abscessus can cause nosocomial infections of skin and soft tissues in immunosuppressed patients (28,35). It is also able to cross the blood-brain barrier and to cause meningoencephalitis (42). M. abscessus is phylogenetically related to M. chelonae and, indeed, these species have long been grouped together under the designation of the "M. abscessus-chelonae complex" (6). M. abscessus is a rapid grower that forms colonies in 5 days. Like other mycobacterial species, M. abscessus is equipped with a robust waxy cell wall that, as in other species, probably contributes to virulence (12). The emerging and growing interest in M. abscessus has led to its genome being sequenced (accession no. NC010397) (F. Ripoll et al., unpublished data) and to the development of DNA microarrays (Jean-Yves Coppée, unpublished data).The availability of genomic resources and animal models (32) makes M. abscessus a very attractive system. However, there is no defined mutagenesis system for this species and, to the best of our knowledge, no defined mutants have been constructed thus far. The consequence is that the study of this organism has been restricted to spontaneous variants. Utilization of spontaneous mutants has, nevertheless, allowed the characterization of morphotypically rough isolates that are hypervirulent both in vitro and in vivo (7,8,17). These rough isolates are low glycopep...