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Journal of Bacteriology, August 1999, p. 4780-4789, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Martin S. Pavelka Jr.1,* and William R. Jacobs Jr.1,2

Howard Hughes Medical Institute2 and Department of Microbiology and Immunology,1 Albert Einstein College of Medicine, Bronx, New York 10461

Received 13 April 1999/Accepted 15 June 1999

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.

* Corresponding author. Present address: Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Box 672, Rochester, NY 14642. Phone: (716) 275-4670. Fax: (716) 473-9573. E-mail: Martin_Pavelka{at}urmc.rochester.edu.

Journal of Bacteriology, August 1999, p. 4780-4789, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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