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. 1997 Sep 30;94(20):10955-60.
doi: 10.1073/pnas.94.20.10955.

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

V Pelicic  1 , M Jackson, J M Reyrat, W R Jacobs Jr, B Gicquel, C Guilhot
Affiliations

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

V Pelicic et al. Proc Natl Acad Sci U S A. .

Abstract

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39 degrees C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 10(6) mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

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Figures

Figure 1
Figure 1
Design of ts-sacB vectors for positive selection of rare genetic events (pPR27 is shown as an example). Only single restriction sites that can be used for the subsequent cloning of a transposon or a mutant allele are shown.
Figure 2
Figure 2
Southern blot analysis of representative M. tuberculosis::Tn5368 clones and expected schematic hybridization patterns for a transposition mutant. Five mutants were picked at random (clones 1–5). M. tuberculosis 103 DNA (WT) was included as a control and as expected showed no hybridization signal. Genomic DNA was digested with BamHI or XhoI and probed for hybridization with pPR32, a vector consisting of the Tn5368 transposon cloned into the BamHI site of pPR23. Molecular masses are indicated in kilobases.
Figure 3
Figure 3
Sequences of cloned insertion sites for several M. tuberculosis and M. bovis BCG transposition mutants. Approximately 500 bp of DNA flanking the transposon was sequenced using IS1096 outward primers α and β, but only DR are shown. Imperfectly repeated nucleotides are underlined.
Figure 4
Figure 4
Southern blot analysis of M. tuberculosis purC mutants and expected schematic pattern of hybridization for an allelic exchange mutant. Five auxotrophic mutants were picked at random (clones 1–5). Chromosomal DNA was digested with BamHI and probed for hybridization with the pMJ103 vector. M. tuberculosis 103 DNA was included as a control (WT). Molecular masses are indicated in kilobases.
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References

    1. Bloom B R, Murray C J L. Science. 1992;257:1055–1064. - PubMed
    1. Falkow S. Rev Infect Dis. 1988;10:S274–S276. - PubMed
    1. Jacobs W R., Jr Immunobiology. 1992;184:147–156. - PubMed
    1. Arruda S, Bomfim G, Knights R, Huima-Byron T, Riley L W. Science. 1993;261:1454–1457. - PubMed
    1. Pascopella L, Collins F M, Martin J M, Lee M H, Hatfull G F, Stover C K, Bloom B R, Jacobs W R., Jr Infect Immunol. 1994;62:1313–1319. - PMC - PubMed

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