Construction and application of mycobacterial reporter transposons.
The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria.
Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa.
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EE
Machowski
R A
RA
McAdam
K M
KM
Derbyshire
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Mizrahi
D000595:Amino Acid Sequence; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D004274:DNA, Recombinant; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005819:Genetic Markers; D019868:Kanamycin Kinase / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics*; D010641:Phenotype; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D005819:Genetic Markers; D019868:Kanamycin Kinase