TY - JOUR
T1 - A novel transposon trap for mycobacteria
T2 - Isolation and characterization of IS1096
AU - Cirillo, J. D.
AU - Barletta, R. G.
AU - Bloom, B. R.
AU - Jacobs, W. R.
PY - 1991
Y1 - 1991
N2 - In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a Δasd::aph allele. Attempts to replace the wild-type asd gene with the Δasd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which contains β-galactosidase expressed from a mycobacteriophage promoter and Δasd::aph, was integrated into the chromosome of M. smegmatis by a homologous, single-crossover, recombination event. Visual screening for inactivation of the β-galactosidase gene in the resulting strain allowed the isolation of a novel mycobacterial insertion element from M. smegmatis. This insertion element, which is unique to M. smegmatis, was designated IS1096 and transposes at a frequency of 7.2 x 10-5 per cell in an apparently random fashion. IS1096 is 2,275 bp in length and contains two open reading frames which are predicted to encode proteins involved in transposition. This insertion element exhibits several characteristics that suggest it may be a useful tool for genetic analysis of mycobacteria, possibly including the study of mechanisms of pathogenesis.
AB - In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a Δasd::aph allele. Attempts to replace the wild-type asd gene with the Δasd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which contains β-galactosidase expressed from a mycobacteriophage promoter and Δasd::aph, was integrated into the chromosome of M. smegmatis by a homologous, single-crossover, recombination event. Visual screening for inactivation of the β-galactosidase gene in the resulting strain allowed the isolation of a novel mycobacterial insertion element from M. smegmatis. This insertion element, which is unique to M. smegmatis, was designated IS1096 and transposes at a frequency of 7.2 x 10-5 per cell in an apparently random fashion. IS1096 is 2,275 bp in length and contains two open reading frames which are predicted to encode proteins involved in transposition. This insertion element exhibits several characteristics that suggest it may be a useful tool for genetic analysis of mycobacteria, possibly including the study of mechanisms of pathogenesis.
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U2 - 10.1128/jb.173.24.7772-7780.1991
DO - 10.1128/jb.173.24.7772-7780.1991
M3 - Article
C2 - 1660454
AN - SCOPUS:0026350742
SN - 0021-9193
VL - 173
SP - 7772
EP - 7780
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 24
ER -