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Nat Methods. 2007 Feb;4(2):147-52. Epub 2006 Dec 17.

Recombineering in Mycobacterium tuberculosis.

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Pittsburgh Bacteriophage Institute and Department of Biological Sciences, 376 Crawford Hall, 4249 Fifth Ave., University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.


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.

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