This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Otero, J. Articles by Glickman, M. S. Search for Related Content PubMed PubMed Citation Articles by Otero, J. Articles by Glickman, M. S. Agricola Articles by Otero, J. Articles by Glickman, M. S.

Next Article 

Applied and Environmental Microbiology, September 2003, p. 5039-5044, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5039-5044.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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.

---

* Corresponding author. Mailing address: Division of Infectious Diseases, Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, Box 9, 1275 York Ave., New York, NY 10021. Phone: (212) 639-3191. Fax: (646) 422-2124. E-mail: glickmam{at}mskcc.org.

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

---

Applied and Environmental Microbiology, September 2003, p. 5039-5044, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5039-5044.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Park, K. T., Dahl, J. L., Bannantine, J. P., Barletta, R. G., Ahn, J., Allen, A. J., Hamilton, M. J., Davis, W. C. (2008). Demonstration of Allelic Exchange in the Slow-Growing Bacterium Mycobacterium avium subsp. paratuberculosis, and Generation of Mutants with Deletions at the pknG, relA, and lsr2 Loci. Appl. Environ. Microbiol. 74: 1687-1695 [Abstract] [Full Text]  
• Krzywinska, E., Krzywinski, J., Schorey, J. S. (2004). Naturally occurring horizontal gene transfer and homologous recombination in Mycobacterium. Microbiology 150: 1707-1712 [Abstract] [Full Text]