Transposition of IS117 of Streptomyces coelicolor A3(2) in Mycobacterium smegmatis.
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, pIJ4696 and pIJ4697, 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 15117 target sites (attB) with known primary and secondary S. lividans attB sites showed that only a 2 bp 'AG' sequence at the crossover point was conserved. Dividing the attB 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. pIJ4696 at all three sites was stable in M. smegmatis in the absence of selection for at least 60 cell divisions. pIJ4696, pIJ4697 and other IS117 derivatives are promising vectors for the stable, integrative cloning of genes in M. smegmatis.
Bhatt
Apoorva
A
Kieser
Tobias
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D015139:Blotting, Southern; D002934:Cinnamates; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D018274:Electroporation / Q000379:methods; D005798:Genes, Bacterial; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics*; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D006921:Hygromycin B; C026273:hygromycin A