Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

Vladimir Pelicic, Mary Jackson, Jean Marc Reyrat, William R. Jacobs, Brigitte Gicquel, Christophe Guilhot

Research output: Contribution to journalArticle

368 Scopus citations

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°C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 106 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.

Original languageEnglish (US)
Pages (from-to)10955-10960
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number20
DOIs
StatePublished - Sep 30 1997

ASJC Scopus subject areas

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