Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates

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.

The dynamics of repeated elements: Applications to the epidemiology of tuberculosis

“…One potential source of persistence is horizontal transfer of the element, which if frequent enough, may stave off extinction. Although our model of this force is simplistic, the preliminary conclusion is that horizontal transfer in TB is too rare for it to increase the mean copy numbers to observed levels (21,22) [for discussion of low conjugation rates in E. coli see Condit (31), Condit et al (32), and Levin and Lenski (33)]. Therefore, in the case of IS6110, the answer to the problem of persistence lies elsewhere.…”

Disruption of IcsP, the major Shigella protease that cleaves IcsA, accelerates actin‐based motility

“…E. coli strains were grown in L broth, and S. flexneri strains were grown in tryptic soy broth. Where appropriate, This study (Meissner et al, 1987) 2764 F ¹ (cI857 b2 red␤3 S7 )⌬(gpt-proA)62 leuB1 glnV44 ara-14 galK2 lacY1 hsdS20 rpsL20 xyl-5 mtl-1 recA13 (Jacobs et al, 1993) (Balasubramanian et al, 1996) antibiotics were added to the following final concentrations: ampicillin, 100 g ml ¹1 ; chloramphenicol, 25 g ml ¹1 ; kanamycin, 30 g ml ¹1 ; and tetracycline, 12.5 g ml ¹1 . PtK2 (potoroo kidney epithelial) cells used for motility studies were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) with low glucose and 10% fetal calf serum.…”

“…A genomic library of S. flexneri DNA was constructed essentially as described previously (Balasubramanian et al, 1996). Genomic DNA from S. flexneri wild-type strain 2457T, which includes both chromosomal and virulence plasmid DNA, was partially digested with Sau 3AI and was ligated to BamHI arms of pYUB328, which had been previously prepared by digestion of pYUB328 with XbaI, dephosphorylation of the XbaI ends and digestion with BamHI.…”

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis

“…Another method for creating mutants is allelic exchange mutagenesis. Recently, low frequency allelic exchange was demonstrated in bacteria of the M. tuberculosis complex, again using a suicide delivery vector (9,10), and new protocols allowing easier detection of allelic exchange mutants also have been developed (11)(12)(13). However, as for transposon mutagenesis, detection of very rare allelic exchange events is hindered by low transformation efficiencies and high frequencies of illegitimate recombination.…”