POSITIVE SELECTION OF ALLELIC EXCHANGE MUTANTS IN MYCOBACTERIUM-BOVISBCG

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