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Isolation of a novel insertion sequence from Mycobacterium fortuitum using a trap vector based on inactivation of a lacZ reporter gene

Division of Microbiology, Central Drug Research Institute, Lucknow 226001, India¹

Author for correspondence: Ranjana Srivastava. Tel: +91 522 220908. Fax: +91 522 223405. e-mail: root{at}cscdri.ren.nic.in

An insertion sequence of Mycobacterium fortuitum has been isolated using a trap vector following insertion in and inactivation of the lacZ reporter gene. The trap vector is a temperature-sensitive (ts) Escherichia coli–mycobacterium shuttle plasmid, pCD4, which contains ts oriM, the kanamycin-resistance gene as a selection marker and a lacZ expression cassette. The ts mutation present in pCD4 functions in mycobacteria and enables screening for transposable elements from the mycobacterial genome that disrupt the lacZ gene by screening for white colonies on X-Gal plates in both mycobacterial as well as E. coli hosts. The vector was used to isolate a novel 1·653 kb insertion sequence from M. fortuitum named IS219. IS219 duplicated host DNA at the target site, had inverted repeats at its ends and contained two ORFs on one strand. One of the predicted proteins showed homology to a putative transposase from Acetobacter pasteurianus. IS219 was present in two copies in the genome of M. fortuitum. The trap vector appears to be useful in trapping insertion sequences from different mycobacteria by screening for the disrupted LacZ phenotype.

Keywords: insertion sequence, Mycobacterium fortuitum, reporter gene, molecular trap

Abbreviations: IS, insertion sequence; Kan, kanamycin; ts, temperature sensitive

The EMBL accession number for the sequence reported in this paper is Y18875 MF018875

^a Present address: Department of Radiation Medicine, Biochemistry & Molecular Biology, Georgetown University Medical Centre, Washington DC, USA.