IS1549 FROM MYCOBACTERIUM-SMEGMATIS FORMS LONG DIRECT REPEATS UPON INSERTION

A new insertion element, IS1549, was identified serendipitously from M ycobacterium smegmatis LR222 during experiments using a vector designe d to detect the excision of IS6110 from between the promoter region an d open reading frame (ORF) of an aminoglycoside phosphotransferase gen e. Six of the kanamycin-resistant isolates had a previously unidentifi ed insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C conte nt in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member o f the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually l ong and variable-length (71- to 246-bp) direct repeats of the target D NA during transposition, Southern blot analysis revealed that five cop ies of IS1549 are present in LR222, but not all M. smegmatis strains c arry this element, Only strains with a 65-kDa antigen gene with a PCR- restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549, None of 13 other species of Mycobacteria l tested by PCR with two sets of primers specific for IS1549 were posi tive for the expected amplified product.