Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

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 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.

In vivo transposition of mariner -based elements in enteric bacteria and mycobacteria

“…Plasmid pMycoMar was introduced into M. smegmatis mc 2 155 by electroporation applying conditions described (21). After overnight recovery in 7H9 broth at 30°C, cells were plated on LuriaBertani medium plates containing kanamycin at either 30°C (to determine the total number of transformants) or 39°C (to select for insertion mutants).…”

“…E. coli strains DH5␣ pir, SM10 pir (18), and BW20767 (19) and M. smegmatis strain mc 2 155 (20) were maintained by standard methods. Plasmids pPR23 (21) and pBMML2S were maintained in E. coli. Bacteriophages, including a virulent phage and mycobacteriophage D29, were maintained by standard methods.…”

Comparative and functional genomics of the Mycobacterium tuberculosis complex a aThis review is based on the 2002 Marjory Stephenson Prize Lecture delivered by the author at the 150th Meeting of the Society for General Microbiology, 9 April 2002.

“…Do specific mycobacterial factors determine the outcome? Answers to these questions will be provided by functional genomics and, in recent years, there have been spectacular advances in gene replacement technology (Bardarov et al, 1997 ;Hinds et al, 1999 ;Parish & Stoker, 2000 ;Pelicic et al, 1997). It is now relatively straightforward to construct knockout mutants although this remains a lengthy process owing to the slow growth of tubercle bacilli.…”

galK-based suicide vector mediated allelic exchange in Mycobacterium abscessus

“…We sought to develop an efficient suicide vector system for allelic exchange in M. abscessus. The key to this is a suitable counterselectable marker, such as sacB (conferring sucrose sensitivity), rpsL (conferring streptomycin susceptibility in a streptomycin-resistant background) or galK (conferring 2-deoxygalactose susceptibility) [14,[27][28][29]. Others have shown that sacB does not work in M. abscessus, and the rpsL system requires a pre-existing streptomycin resistance mutation in the choromosomal copy of rpsL, which complicates strain construction [16,23,29].…”