Induction-free recombineering for simple targeted gene-deletions in various mycobacteria

Gene deletion is a valuable tool for phenotypic characterization in bacteriology, but in mycobacteria, generating deletion mutants remains a cumbersome and time-consuming process. Here, we present a modification to the widely used recombineering method in mycobacteria. By expressing the recombineering-promoting proteins from a single-copy episomal plasmid, we could use a constitutive promotor. This approach eliminates the need for induction prior to electroporation of the targeting substrate (linear DNA fragment comprised of homologous regions to the gene of interest, flanking a selection marker), and shortens the subsequent plasmid curing process. We successfully demonstrated this method in <i>Mycobacterium tuberculosis</i>, <i>M. marinum</i>, <i>and M. abscessus</i>.IMPORTANCEAlthough several techniques exist for generating gene-deletion mutants in mycobacteria, these procedures remain limited to laboratories more specialized in molecular biology. Here we present a very simplified procedure, which is a modification on a well-tried technique. Our proposed procedure makes genetic manipulation in mycobacteria more accessible to a greater number of researchers throughout the world, including those with less advanced molecular biology expertizse.