Plasmid DNA transfer in Mycobacterium smegmatis involves novel DNA rearrangements in the recipient, which can be exploited for molecular genetic studies.

Abstract

The establishment of molecular genetic techniques is essential for development of new treatments for mycobacterial infections. To this end, we recently described a novel DNA transfer process that occurs in the model mycobacterial organism Mycobacterium smegmatis. This transfer system is most like conjugal DNA transfer in that it requires two viable parents, is DNAse resistant and occurs between distinct donor and recipient strains. Cis-acting sequences called bom, which confer transferability, are distinct from the prototypical oriT sites of conjugative plasmids, as they occur at multiple locations in the chromosome and require RecA in the recipient to mediate plasmid recircularization. Here, we show that a plasmid containing two of these bom regions can undergo several fates in the recipient cell, each of which require recipient recombination functions. The products of plasmid transfer that we observed provide further insights toward a model for DNA transfer. Furthermore, we have taken advantage of the recombination events that occur in the recipient to develop simple procedures for capturing, or replacing specific segments of the recipient chromosome. To demonstrate the potential of the system, we describe the capture and deletion of 25 kb of the M. smegmatis chromosome, and targeted-allele exchange of the recipient recB and recD genes. Using these transfer-mediated rearrangements, we demonstrate that homology with the recipient chromosome and RecB, but not RecD, are essential for DNA transfer.