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Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Vladimir Pelicic, Jean-Marc Reyrat, Brigitte Gicquel
DOI: http://dx.doi.org/10.1111/j.1574-6968.1996.tb08524.x 161-166 First published online: 1 November 1996


sacB expression is lethal to mycobacteria in the presence of sucrose. It can therefore serve as a counter-selectable marker for positive selection of gene replacement events as demonstrated in the fast-growing Mycobacterium smegmatis. With this methodology, a sucrose counter-selectable vector was used to deliver, into the Mycobacterium bovis BCG genome, an inactivated copy (ureC::Km) of the ureC gene encoding the mycobacterial urease. A two-step selection procedure on 2% sucrose allowed the positive selection of gene exchange mutants. This technique should thus be extremely useful for the genetic analysis of pathogenic mycobacteria.

  • Mycobacteria
  • Allelic exchange
  • sacB