Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates

V. Balasubramanian, Martin S. Pavelka, Stoyan S. Bardarov, Jean Martin, Torin Weisbrod, Ruth A. Mcadam, Barry R. Bloom, William R. Jacobs

Research output: Contribution to journalArticle

125 Citations (Scopus)

Abstract

Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations. Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M. tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful. In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M. tuberculosis. Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328. The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes. A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli. A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion. Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M. tuberculosis. The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M. tuberculosis-based vaccines.

Original languageEnglish (US)
Pages (from-to)273-279
Number of pages7
JournalJournal of Bacteriology
Volume178
Issue number1
StatePublished - Jan 1996

Fingerprint

Cosmids
Mycobacterium tuberculosis
Genetic Recombination
Mycobacterium
Genes
Digestion
Tuberculosis Vaccines
Kanamycin Resistance
Kanamycin
Electroporation
Homologous Recombination
DNA Restriction Enzymes
Mycobacterium bovis
Leucine
Mutagenesis
Libraries
Alleles
Genome
Escherichia coli
Mutation

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

Balasubramanian, V., Pavelka, M. S., Bardarov, S. S., Martin, J., Weisbrod, T., Mcadam, R. A., ... Jacobs, W. R. (1996). Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. Journal of Bacteriology, 178(1), 273-279.

Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. / Balasubramanian, V.; Pavelka, Martin S.; Bardarov, Stoyan S.; Martin, Jean; Weisbrod, Torin; Mcadam, Ruth A.; Bloom, Barry R.; Jacobs, William R.

In: Journal of Bacteriology, Vol. 178, No. 1, 01.1996, p. 273-279.

Research output: Contribution to journalArticle

Balasubramanian, V, Pavelka, MS, Bardarov, SS, Martin, J, Weisbrod, T, Mcadam, RA, Bloom, BR & Jacobs, WR 1996, 'Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates', Journal of Bacteriology, vol. 178, no. 1, pp. 273-279.
Balasubramanian V, Pavelka MS, Bardarov SS, Martin J, Weisbrod T, Mcadam RA et al. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. Journal of Bacteriology. 1996 Jan;178(1):273-279.
Balasubramanian, V. ; Pavelka, Martin S. ; Bardarov, Stoyan S. ; Martin, Jean ; Weisbrod, Torin ; Mcadam, Ruth A. ; Bloom, Barry R. ; Jacobs, William R. / Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. In: Journal of Bacteriology. 1996 ; Vol. 178, No. 1. pp. 273-279.
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