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.
Pelicic
V
V
Unité de Génétique Mycobactèrienne, Institut Pasteur, Paris, France.
Reyrat
J M
JM
Gicquel
B
B
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