Dispersion and functional characterization of the uncommon arrangement of class 2 integron, In2-8, in Acinetobacter baumannii clinical isolates.

RAM¨ªREZ M.S., QUIROGA M.P., BELLO H., MARQUEZ C., CENTRON D.

Roma, Italia

Simposio; 8th International Symposium on the Biology of Acinetobacter.; 2010

Acinetobacter Group

IntI1 provided in trans can excise all the cassettes including the unusual attCcatB2¦¤attI2 and orfX-ybfA-ybfB-ybgA gene cassettes found in the class 2 integron array In2-8.Mar¨ªa Soledad Ram¨ªrez, Mar¨ªa Paula Quiroga, Marcelo Hern¨¢n cassini and Daniela Centr¨®n. ¡°Laboratorio de Investigacioens de Mecanismos de Resistencia a Antibi¨®ticos¡±, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina.The transposon Tn7::In2-8, contains an unusual class 2 integron array consisting in sat2-aadB-catB2(¦¤attI2)-dfrA1-sat2-aadA1-orfX-ybfA-ybfB-ybgA. This integron has the particularity that at the inverted core site of the catB2 gene, the expected attC site has been replaced by 244 bp DNA belonging to the attI2, converting catB2 in an unusual gene cassette. In addition, another partial sequence of the attI2 is found located downstream the orfX gene. We wonder, from a functional perspective, which unusual cassettes could be mobilized by the type 1 integrase. We performed in vivo recombination assays of all potential mobile elements found in In2-8 which have been cloned in pACYC184. In vivo recombination frequency assays with all the plasmids mentioned above, and the plasmid harboring the type 1 integrase (IntI1), were done and the detection of the excision events was performed by PCR using the corresponding primer pair in 40 colonies per mating which were repeated three times in independent assays.We observed variable values of excision for gene cassettes found in In2-8, and, although the region containing attC-catB2(¦¤attI2)-dfrA1-attC is recognized by IntI1 as a fused gene cassette, the unusual attC-catB2-¦¤attI2 cassette was found to be the most efficient substrate for IntI1. To confirm excision of the unusual attC-catB2-¦¤attI2 gene cassette, specific primers were designed to detect the circularized gene cassette generated after the cassette excision. Sequence analysis of both products obtained from this reaction revealed two different sites recognized by the IntI1 to mediate the attC-catB2-¦¤attI2 excision. One excision point corresponded to the G of the core site GTTAACC proposed as the G of the recombination site in the attI2 of class 2 integrons. The other recombination site, revealed that in this case the integrase recognized the G of the GTTATGA sequence localized 62 bp from the TAA of the catB2 gene. Experimental studies also revealed that the orfX-ybfA-ybfB-ybgA is a functionnal unusual mobile element. We observed the excision of the the orfX, the ybfB, and the complete structure ORFX-ybfA-ybfB-ybgA at two different points, the one previously described and another one at the inverse core site of the aadA1 gene cassette. The high frequency of excision of the attC-catB2-¦¤attI2 unusual gene cassette, as well as the excision of differents elements within the orfX-ybfA-ybfB-ybgA module, evidences the plasticity of recombination of the system mediated by IntI1.