Positive selection of allelic exchange mutants in Mycobacterium bovis BCG.
sacB expression is lethal to mycobacteria in the presence of sucrose. It can therefore serve as 1 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.
Unité de Génétique Mycobactèrienne, Institut Pasteur, Paris, France.
V
V
Pelicic
J M
JM
Reyrat
B
B
Gicquel
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D009163:Mycobacterium bovis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012641:Selection, Genetic; D013395:Sucrose / Q000494:pharmacology*; D014169:Transformation, Bacterial; D014510:Urease / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase; D014510:Urease