title: Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors.
abstract: The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.
authors.affiliation: Unité de Génétigue Mycobactérienne, CNRS URA 1300, Institut Pasteur, Paris, France. vpelicic@pasteur.fr
authors.forename: V
authors.initials: V
authors.lastname: Pelicic
authors.forename: J M
authors.initials: JM
authors.lastname: Reyrat
authors.forename: B
authors.initials: B
authors.lastname: Gicquel
mesh_terms: D000483:Alleles; D005822:Genetic Vectors; D006602:Hexosyltransferases / Q000378:metabolism*; D016296:Mutagenesis; D009161:Mycobacterium / Q000235:genetics*; D013395:Sucrose / Q000378:metabolism*
chemical_list: D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase