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 report er transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions, A plasmid that is tempera ture-sensitive for replication in mycobacteria was used to deliver promoter less lacZ or aph reporter genes to Mycobacterium smegmatis as transcription al (lacZ), or translational ('aph) fusions. Mutants containing lacZ produce d varying intensities of blue colour on indicator media. This reporter acti vity could be used as a quantitative measure of promoter strength. Mutants displaying varying Levels of resistance to kanamycin were obtained by trans positional insertion of the aph reporter lacking a promoter, ribosome bindi ng site and start codon to form functionally active translational fusions, Finally, inclusion of the R6K gamma 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 transcription al and translational reporter derivatives of Tn5367 are functional, and the y supplement the growing range of molecular tools available for the study o f mycobacteria. (C) 2000 Elsevier Science B.V. All rights reserved.