Enhanced specialized transduction using recombineering in mycobacterium tuberculosis

JoAnn M. Tufariello, Adel A. Malek, Catherine Vilchèze, Laura E. Cole, Hannah K. Ratner, Pablo A. González, Paras Jain, Graham F. Hatfull, Michelle H. Larsen, William R. Jacobs

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100- fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria.

Original languageEnglish (US)
JournalmBio
Volume5
Issue number3
DOIs
StatePublished - May 27 2014

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Mycobacterium tuberculosis
Mycobacteriophages
Genetic Engineering
Homologous Recombination
Mycobacterium
Bacteriophages
Libraries
Vaccines
Escherichia coli
Infection
Genes

ASJC Scopus subject areas

  • Microbiology
  • Virology

Cite this

Tufariello, J. M., Malek, A. A., Vilchèze, C., Cole, L. E., Ratner, H. K., González, P. A., ... Jacobs, W. R. (2014). Enhanced specialized transduction using recombineering in mycobacterium tuberculosis. mBio, 5(3). https://doi.org/10.1128/mBio.01179-14

Enhanced specialized transduction using recombineering in mycobacterium tuberculosis. / Tufariello, JoAnn M.; Malek, Adel A.; Vilchèze, Catherine; Cole, Laura E.; Ratner, Hannah K.; González, Pablo A.; Jain, Paras; Hatfull, Graham F.; Larsen, Michelle H.; Jacobs, William R.

In: mBio, Vol. 5, No. 3, 27.05.2014.

Research output: Contribution to journalArticle

Tufariello, JM, Malek, AA, Vilchèze, C, Cole, LE, Ratner, HK, González, PA, Jain, P, Hatfull, GF, Larsen, MH & Jacobs, WR 2014, 'Enhanced specialized transduction using recombineering in mycobacterium tuberculosis', mBio, vol. 5, no. 3. https://doi.org/10.1128/mBio.01179-14
Tufariello JM, Malek AA, Vilchèze C, Cole LE, Ratner HK, González PA et al. Enhanced specialized transduction using recombineering in mycobacterium tuberculosis. mBio. 2014 May 27;5(3). https://doi.org/10.1128/mBio.01179-14
Tufariello, JoAnn M. ; Malek, Adel A. ; Vilchèze, Catherine ; Cole, Laura E. ; Ratner, Hannah K. ; González, Pablo A. ; Jain, Paras ; Hatfull, Graham F. ; Larsen, Michelle H. ; Jacobs, William R. / Enhanced specialized transduction using recombineering in mycobacterium tuberculosis. In: mBio. 2014 ; Vol. 5, No. 3.
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