Enhanced specialized transduction using recombineering in mycobacterium tuberculosis

Jo Ann 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 journalArticlepeer-review

18 Scopus citations


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)
Article numbere01179-14
Issue number3
StatePublished - May 27 2014

ASJC Scopus subject areas

  • Microbiology
  • Virology


Dive into the research topics of 'Enhanced specialized transduction using recombineering in mycobacterium tuberculosis'. Together they form a unique fingerprint.

Cite this