title: A novel transposon-based method for elimination of large bacterial plasmids.
abstract: 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 2,102 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 Km(R) 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 Km(S) colonies after induction of IS30 transposase, and it was shown that the Km(S) 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.
authors.affiliation: Veterinary Medical Research Institute of Hungarian Academy of Sciences, H-1143 Budapest, Hungária St. 21, Hungary.
authors.forename: Ariel
authors.initials: A
authors.lastname: Imre
authors.forename: Ferenc
authors.initials: F
authors.lastname: Olasz
authors.forename: János
authors.initials: J
authors.lastname: Kiss
authors.forename: Béla
authors.initials: B
authors.lastname: Nagy
mesh_terms: D003227:Conjugation, Genetic; D004261:DNA Replication; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D012093:Replicon; D012477:Salmonella enteritidis / Q000235:genetics*; D017384:Sequence Deletion; D019895:Transposases / Q000378:metabolism; D014774:Virulence
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D019895:Transposases