Transposition of Tn5367 in Mycobacterium marinum, using a conditionally recombinant mycobacteriophage

Abstract

Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis. As with M. tuberculosis, M. marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. Conditions permissive for phage replication in M. tuberculosis facilitated highly efficient transposon delivery in M. marinum. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M. marinum.

FIG. 1.

Southern blot analysis of M. marinum Tn5367 mutants. Lanes 1 to 10 show BamHI digests of total chromosomal DNA hybridized to a labeled IS1096 probe. Lane L, DNA markers (sizes in kilobase pairs are indicated on the left); lane 1, wild type; lanes 2 to 9, Kan^r insertion mutants; lane 10, M. smegmatis mc²155.