A novel transposon-based method for elimination of large bacterial plasmids

Elimination or modification of large plasmids of bacteria is often an essential step in functional analysis of these replicons. However, the conventional plasmid-curing procedures such as ethidium bromide and heat treatment are insufficient in many cases. For instance, curing of the large virulence plasmid of Salmonella Enteritidis 2102 has failed when these treatments were applied. To overcome the difficulties, a two-step transposon-based curing method has been developed. First, a Tn10-based transposable unit carrying a KmR marker gene and the joined IS30 ends transposes from a replication deficient conjugative plasmid into the target replicon. Then, the inducible IS30 transposase, using the highly reactive joined IS30 ends, mediates deletions or gives rise to the loss of the target plasmid. The efficiency of the method has been monitored by the frequency of KmS colonies after induction of IS30 transposase, and it was shown that the KmS phenotype often accompanied the complete loss of the virulence plasmid or the formation of deletion derivatives. The procedure has been successfully applied also in removing the large virulence plasmid from enterotoxigenic Escherichia coli (ETEC O147), suggesting that the transposon-based method can be a useful tool for eliminating native plasmids in several bacteria.