title: Recombineering in Mycobacterium tuberculosis.
abstract: Genetic dissection of M. tuberculosis is complicated by its slow growth and its high rate of illegitimate recombination relative to homologous DNA exchange. We report here the development of a facile allelic exchange system by identification and expression of mycobacteriophage-encoded recombination proteins, adapting a strategy developed previously for recombineering in Escherichia coli. Identifiable recombination proteins are rare in mycobacteriophages, and only 1 of 30 genomically characterized mycobacteriophages (Che9c) encodes homologs of both RecE and RecT. Expression and biochemical characterization show that Che9c gp60 and gp61 encode exonuclease and DNA-binding activities, respectively, and expression of these proteins substantially elevates recombination facilitating allelic exchange in both M. smegmatis and M. tuberculosis. Mycobacterial recombineering thus provides a simple approach for the construction of gene replacement mutants in both slow- and fast-growing mycobacteria.
authors.affiliation: Pittsburgh Bacteriophage Institute and Department of Biological Sciences, 376 Crawford Hall, 4249 Fifth Ave., University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
authors.forename: Julia C
authors.initials: JC
authors.lastname: van Kessel
authors.forename: Graham F
authors.initials: GF
authors.lastname: Hatfull
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics; D005092:Exonucleases / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D009160:Mycobacteriophages / Q000737:chemistry / Q000235:genetics*; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000254:growth & development / Q000821:virology*; D011995:Recombination, Genetic; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D005092:Exonucleases