Site-specific recombination of the cassettes and pseudocassettes embedded in the class 2 integron In2-8.

MARÍA SOLEDAD RAMÍREZ; MARÍA PAULA QUIROGA ; CECILIA QUIROGA; DANIELA CENTRÓN

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

The Tn7::In2-8 transposon, described in A. baumannii multiresistant isolates, contains an unusual class 2 integron array. This integron has the particularity that downstream the stop codon of the catB2 gene a sequence with 100% identity to the attI2 was found. A nonelucidated mechanism might be involved in the acquisition of the catB2 pseudocassette. In view of the uncommon features of this class 2 integron, the integrase-mediated site-specific recombination of the cassettes and pseudocassettes embedded in the In2-8 were studied. The cassettes and pseudocassettes were cloned in order to use them for in vivo recombination assays. E. coli cells harboring the intI1 gene and the plasmid that served as substrate were cultured in the presence of IPTG, and plasmid DNA was isolated. Specific PCR primers were used to identify plasmid species where excision has occurred. We observed that the IntI1 excised the attC-catB2(DattI2)- dfrA1-attC cassette as a module, although it was shown that IntI1 could also excised attC-catB2(DattI2) and (DattI2)-dfrA1-attC. We also found the excision of aadA1, dfrA1, sat2, aadB and orfX. We demonstrated that the IntI1 recognized all the cassettes and pseudocassettes embedded in the In2::8, showing that the DattI2 could be used as a substrate to mediate the specific recombiantion reaction.