Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

• DOI: 10.1073/pnas.94.20.10955
• Publication Date: 2002-07-26T14:31:44Z
• Authors: Vladimir Pelicic; Mary Jackson; Jean-Marc Reyrat; William R. Jacobs; Brigitte Gicquel; Christophe Guilhot
• Keywords: Insertional mutagenesis; Transposon mutagenesis

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