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Efficient Allelic Exchange and Transposon Mutagenesis in Mycobacterium avium by Specialized Transduction

Joel Otero,^1, William R. Jacobs Jr.,¹ and Michael S. Glickman^2*

Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461,¹ Immunology Program, Division of Infectious Diseases, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York 10021²

Received 10 February 2003/ Accepted 4 June 2003

Mycobacterium tuberculosis and Mycobacterium avium are pathogenic slow-growing mycobacteria that cause distinct human diseases. In contrast to recent advances in M. tuberculosis genetics and pathogenesis investigation, M. avium has remained genetically intractable and, consequently, its pathogenic strategies remain poorly understood. Here we report the successful development of efficient allelic exchange and transposon mutagenesis in an opaque clinical strain of M. avium by specialized transduction. Efforts to disrupt the leuD gene of M. avium by specialized transduction were successful but were complicated by inefficient isolation of recombinants secondary to high spontaneous antibiotic resistance. However, by using this leucine auxotroph as a genetic host and the Streptomyces coelicolor leuD gene as a selectable marker, we achieved efficient allelic exchange at the M. avium pcaA locus. A leuD-marked transposon delivered by specialized transduction mutagenized M. avium with efficiencies similar to M. tuberculosis. These results establish a system for random and directed mutagenesis of M. avium. In combination with the forthcoming M. avium genome sequence, these tools will allow the distinct physiologic and pathogenic properties of M. avium to be dissected in molecular detail.

Present address: Cell and Molecular Biology Program, Molecular and Human Genetics Department, Baylor College of Medicine, Houston, TX 77030.

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