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Transposition of IS117 of Streptomyces coelicolor A3(2) in Mycobacterium smegmatis

Department of Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK

ABSTRACT

Derivatives of IS117, the Streptomyces coelicolor A3(2) 2·6 kb minicircle, transpose efficiently in Mycobacterium smegmatis, targeting chromosomal sites resembling translation start signals. Two IS117 derivatives, plJ4696 and plJ4697, containing a Streptomyces hygromycin-resistance gene in opposite orientations were introduced into M. smegmatis by electroporation and found to integrate into one of three specific sites. Integrations at sites A and B were frequent while integration at site C was observed only once. Only one site was occupied in each transformant. Sites A and B had either single or tandem integrations. PFGE analysis located these sites on different genomic Asel fragments. The sequences of the chromosome-IS117 junctions confirmed that integration was via the same IS117 attachment site as in Streptomyces, that there was no target site duplication, and that the orientation of IS117 at each site was fixed. In contrast to the situation in Streptomyces lividans, no deletions were created by the transposition and no circular forms could be detected. Comparison of the three M. smegmatis chromosomal IS117 target sites (att_B) with known primary and secondary S. lividans att_B sites showed that only a 2 bp ‘AG’ sequence at the crossover point was conserved. Dividing the att_B sites into two groups produced two longer consensus target sites, GtcAAGg and gCCGATAGg. Most of the IS117 target sites resemble translational start sites, and site C resembles strongly the amino-terminal sequence of a Mycobacterium tuberculosis aminopeptidase. The level of hygromycin resistance in the transformants was high and independent of the site of integration, the number of copies integrated, or the orientation of the hyg gene. plJ4696 at all three sites was stable in M. smegmatis in the absence of selection for at least 60 cell divisions. plJ4696, plJ4697 and other IS117 derivatives are promising vectors for the stable, integrative cloning of genes in M. smegmatis.

Author for correspondence: Apoorva Bhatt. Tel: + 44 1603 452571. Fax: + 44 1603 456844. e-mail: bhatta@bbsrc.ac.uk

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