title: Insertional mutagenesis and illegitimate recombination in mycobacteria.
abstract: Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG.
authors.affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: G V
authors.initials: GV
authors.lastname: Kalpana
authors.forename: B R
authors.initials: BR
authors.lastname: Bloom
authors.forename: W R
authors.initials: WR
authors.lastname: Jacobs
mesh_terms: D015139:Blotting, Southern; D004270:DNA, Circular / Q000235:genetics; D005798:Genes, Bacterial; D016296:Mutagenesis; D009154:Mutation; D009161:Mycobacterium / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic
chemical_list: D004270:DNA, Circular