Vaamonde C, Bardarov S, Kriakov J, Jacobs WR; American Society for Microbiology. General Meeting.
Abstr Gen Meet Am Soc Microbiol. 1997 May 4-8; 97: 566 (abstract no. U128).
Howard Hughes Medical Institute, Bronx, NY.
The generation of mutants is a fundamental strategy in the genetic analysis of micro-organisms. Efficient delivery of a transposon, Tn5367, with the goal of generating insertional mutants, has recently been accomplished in Mycobacterium tuberculosis and BCG using a conditionally-replicating shuttle phasmid. While the generation of mutants in pathogens such as M. tuberculosis is the ultimate goal of these efforts, working with these organisms exclusively is cumbersome (biosafety concerns, slow growth, and suboptimal transformation efficiencies). A transposon delivery system in a rapidly growing, non-pathogenic, genetically manipulable mycobacteria would be ideal; (1) for study and refinements in the transposon delivery system, and (2) to generate a library of insertional mutants that could be used to define phenotypes for mycobacterial genes, including those of M. tuberculosis. Transposition in the best characterized of the fast growing mycobacterial lab strains, M. smegmatis, appears to be inefficient, most likely due to the presence of multiple copies of IS1096 (the backbone into which a kanamycin resistance gene was cloned to create Tn5367). We have screened a variety of fast-growing mycobacteria including M. aurum, M. chelonae, M. flavescens, M. fortuitum, and M. vaccae, and found that transposon mutants could be generated in M. phlei with efficiencies of 10(-5) to 10(-6) per 10(8) cells. The presence of Tn5367 and the randomness of it's insertion was assessed by Southern analysis of 20 kanamycin resistant clones. Current efforts are directed towards optimizing the transposon mutagenesis efficiencies and screening the libraries of mutants for phenotypes of diverse characteristics.
Publication Types:
Keywords:
- Gene Library
- Kanamycin Resistance
- Mutagenesis
- Mycobacterium
- Mycobacterium bovis
- Mycobacterium tuberculosis
- Tuberculosis
- genetics
Other ID:
UI: 102235520
From Meeting Abstracts