Transformation of mycobacterial species using hygromycin resistance as selectable marker

1 MRC Tuberculosis and Related Infections Unit, RPMS, Hammersmith Hospital, Ducane Road, London W12 OHS, UK 2 National Institute of Immunology, Shahid Jeet Singh Marg, New Delhi 110 067, India 3 Department of Medical Microbiology, University College London Medical School, London W1P 7PN, UK ABSTRACT Summary: Electroporation with shuttle plasmids carrying a kanamycin resistance gene as a selectable marker failed to generate transformants in two mycobacterial species currently being used in human vaccine trials ( Mycobacterium w and Mycobacterium vaccae). In contrast, efficient transformation [10 3 -10 5 transformants (µg DNA) –1 ] was obtained using novel vectors with selection based on expression of resistance to hygromycin. The hygromycin resistance vector was also found to be more efficient than kanamycin resistance vectors for transformation of Mycobacterium smegmatis and Mycobacterium bovis BCG . The hygromycin resistance vector was used to overexpress superoxide dismutase of Mycobacterium tuberculosis in M. vaccae in a form suitable for detailed structural analysis. The potential use of this approach for generation of novel recombinant mycobacterial vaccines is discussed. Author for correspondence: Douglas B. Young. Tel: +44 71 723 1252. Fax: +44 71 262 6299. Keywords: mycobacteria, transformation, electroporation, hygromycin resistance Present address: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati Medical Center, Cincinnati, Ohio, USA. Present address: Department of Medical Microbiology, St '1s Hospital Medical School, Norfolk Place, London W2 1PG, UK.