Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries.
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.
Guilhot
C
C
Unité de Génétique Mycobactérienne, Centre National de la Recherche Scientifique URA 1300, Institut Pasteur, Paris, France.
Otal
I
I
Van Rompaey
I
I
Martìn
C
C
Gicquel
B
B
D000596:Amino Acids / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000379:methods*; D009161:Mycobacterium / Q000235:genetics*; D011687:Purines / Q000378:metabolism; D014815:Vitamins / Q000378:metabolism
D000596:Amino Acids; D011687:Purines; D014815:Vitamins