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

Abstract

SummaryThe 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.

Key experimental evidence of chromosomal DNA transfer among selected tuberculosis-causing mycobacteria

“…In M. smegmatis, ESX-1 and RecA are required in the recipient for DNA transfer to occur, the latter plausibly for homologous recombination events. An M. smegmatis recA donor mutant led to a higher rate of gene conversion in the recipient, likely caused by unrepaired DNA breaks in the donor and, hence, more potential DNA substrates to be transferred (13). For tubercle bacilli, previous genome-based in silico analyses suggested that the analyzed M. canettii strains contained M. canettii J and M. canettii K-derived recombination tracts, thus proposing that these two strains were efficient donors (11).…”

“…An interesting observation in this respect was made for the nonvirulent, fast-growing model organism Mycobacterium smegmatis, which shows an unconventional conjugal DNA transfer (12,13), distinct from classical plasmid-mediated DNA transfer described for other bacteria (14,15). M. smegmatis transconjugants typically display highly mosaic genomes reminiscent of products of eukaryotic meiosis, composed of multiple donor-derived sequence segments that are distributed without apparent regional bias across the genome.…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…These mycolic acid layers are exceptionally thick and form a highly ordered lipid bilayer membrane that is covalently attached to layers of complex sugars and the murein cell wall. In view of this novel cell envelope composition, it is perhaps not surprising that M. smegmatis conjugatively transfers DNA independently of classical T4SS components (98,277). Initial screens for mutations affecting DNA transfer initially failed to identify any transferdefective mutations but did result in the isolation of a number of hyperconjugative mutations.…”

The RD1 virulence locus ofMycobacterium tuberculosisregulates DNA transfer inMycobacterium smegmatis

“…A comprehensive understanding of the biology of this organism is critical for the identification of novel drug targets, the development of vaccines, and determining how it evades the host immune system; this requires the development of basic molecular techniques to determine the genetic and biochemical basis of pathogenesis and drug resistance (2,3). Our recent efforts have been focused on the characterization of a conjugation system and its application for use in mycobacterial genetics (4)(5)(6)(7).…”