IntI1 integrase efficiently mediates recombination involving the blages-1 unusual cassette.

QUIROGA MP, FONSECA E, VICENTE AC, CERQUETTI MC, CENTRÓN D.

Congreso; XLVII Reunión Anual de la Sociedad Sociedad Argentina de Investigaciones Bioquímcas y Biología Molecular; 2011

Within a Pseudomonas aeruginosa colistin-only-sensitive isolate, was found a class 1 integron with the cassette array orf126-blaGES-1-aac(6’)-Ib. The blaGES-1 unusual cassette has the particularity that downstream the stop codon of the blaGES-1 gene there is a sequence of 11 nucleotides with 100% identity to the attI1, instead of a typical attC recombination site. The aim of this study was to determine if the IntI1 integrase mediates site-specific recombination of the orf126 cassette or the blaGES-1 unusual cassette. The orf126- blaGES-1 array was cloned in order to use it for in vivo recombination assays. E. coli cells harboring the plasmid that served as substrate and another containing the intI1 gene alone or also the attI1, were cultured in the presence of IPTG, and plasmid DNA was isolated. Specific PCR primers were used to identify plasmid species where excision or insertion has occurred. We observed that the IntI1 excised the attI1-orf126-attC-blaGES-1(attI1) structure as a module as well as separated units and inserts them at the attI1. We demonstrated that the IntI1 recognized the orf126 cassette and the blaGES-1 unusual cassette, showing that an unusual cassette harboring a attI1 could be used by IntI1 as a substrate to mediate the specific not only excision but also insertion reactions, demonstrating its functionality.