title: Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries.
abstract: The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants. Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative. The pCG79 system thus seems to be a useful tool for the study of M. smegmatis genetics and may be applicable to other mycobacteria, such as the M. tuberculosis complex.
authors.affiliation: Unité de Génétique Mycobactérienne, Centre National de la Recherche Scientifique URA 1300, Institut Pasteur, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Guilhot
authors.forename: I
authors.initials: I
authors.lastname: Otal
authors.forename: I
authors.initials: I
authors.lastname: Van Rompaey
authors.forename: C
authors.initials: C
authors.lastname: Martìn
authors.forename: B
authors.initials: B
authors.lastname: Gicquel
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D009161:Mycobacterium / Q000235:genetics*; D011687:Purines / Q000378:metabolism; D014815:Vitamins / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011687:Purines; D014815:Vitamins