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 Mycobacterium tuberculosis, M. marinum, and M. abscessus.