PLASMID EVOLUTION BY ACQUISITION OF MOBILE GENE CASSETTES - PLASMID PIE723 CONTAINS THE AADB GENE CASSETTE PRECISELY INSERTED AT A SECONDARY SITE IN THE INCQ PLASMID RSF1010

Gene cassettes are mobile DNA elements which contain a specific recomb ination site, a 59-base element, recognized by the site-specific recom bination system of integrons. Gene cassettes are normally found insert ed at a unique site in an integron, downstream of a promoter which dir ects transcription of the cassette-associated genes. However, insertio n of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of t he aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter, The site o f insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (Int), and i t is likely that the cassette was inserted via a single Int-mediated r ecombination event between the 59-base element of a free, circular aad B cassette and a secondary site in RSF1010. The cassette-associated re combination site was inactivated by the insertion, and Int-mediated ex cision of the aadB cassette from this nan-specific location was not de tectable, indicating that the cassette is stably inserted. The movemen t of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.