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Recombineering in Mycobacterium tuberculosis

Nature Methods volume 4, pages 147–152 (2007)Cite this article

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.

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Figure 1: Mycobacteriophage Che9c encodes RecE- and RecT-like proteins.
Figure 2: Targeted allelic exchange of the M. smegmatis leuD locus.
Figure 3: Allelic exchange of the M. smegmatis groEL1 gene.
Figure 4: Targeted gene replacement of the M. tuberculosis groEL1 locus.

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Acknowledgements

We thank M. Scanlon for technical support, T. Hsu and W. Jacobs, Jr. for plasmids (pYUB854, p0004S, p0004S:leuB, p0004S:leuD), A. Schwacha and M. Bochman for help with DNA binding studies, J. Flynn and colleagues for help with M. tuberculosis manipulations, A. Ojha for comments on the manuscript and W. Jacobs Jr. for discussions. This work was support by US National Institutes of Health grant AI067649.

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Authors and Affiliations

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

    Julia C van Kessel & Graham F Hatfull

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  1. Julia C van Kessel
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Correspondence to Graham F Hatfull.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Biochemical properties of Che9c gp60 and gp61.

Supplementary Fig. 2

Recombineering frequencies are dependent on the length of DNA homology.

Supplementary Fig. 3

Full-length blots and gels.

Supplementary Table 1

Plasmids used in recombineering.

Supplementary Table 2

Oligonucleotides used in recombineering studies.

Supplementary Methods

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van Kessel, J., Hatfull, G. Recombineering in Mycobacterium tuberculosis. Nat Methods 4, 147–152 (2007). https://doi.org/10.1038/nmeth996

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