Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue

Summary Plasmids have been described in almost all bacterial species analysed and have proven to be essential genetic tools. In many bacteria these extrachro- mosomal DNAs are cryptic with no known markers or function, which makes their characterization and genetic exploitation extremely difficult. Here we describe a system that will allow the rescue of any cir- cular DNA (plasmid or phage) using an in vitro trans- position system to deliver both a selectable marker (kanamycin) and an Escherichia coli plasmid origin of replication. In this study, we demonstrate the rescue of four cryptic plasmids from the opportunistic pathogen Mycobacterium avium. To evaluate the host range of the rescued plasmids, we have examined their ability to be propagated in Mycobacterium smegmatis and Mycobacterium bovis BCG, and their compatibility with other mycobacterial plasmids. In addition, we use a library of transposon insertions to sequence one plasmid, pVT2, and to begin a genetic analysis of plasmid genes. Using this approach, we identified a putative conjugative relaxase, suggesting this myco- bacterial plasmid is transferable, and three genes required for plasmid establishment and replication.