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

RAMíREZ MARíA SOLEDAD; QUIROGA MARíA PAULA; BELLO HELIA; MáRQUEZ CAROLINA; CENTRóN DANIELA

Roma

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

European Society of Clinical Microbiology and Infectious Diseases

The transposon Tn7::In2-8, contains an unusual class 2 integron array consisting in sat2-aadB-catB2(DattI2)-dfrA1-sat2-aadA1-orfX-ybfA-ybfB-ybgA. This integron has the particularity that downstream the stop codon of the catB2 gene, and corresponding to a rearrangement at the inverted core site (ICS), the expected attC site has been replaced by a 244 bp DNA region with 100% identity to the attI2 class 2 integron recombination site, converting the catB2 gene cassette in an unusual gene cassette, indicated as attC-catB2-(DattI2). In addition, another partial sequence of the attI (13 bp) is found located downstream the orfX gene. We wonder, from a functional perspective, which cassettes and/or unusual cassettes could be mobilized at high frequencies by the type 1 integrase. We performed in vivo recombination assays of all potential mobile elements found in the class 2 integron array 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, were done by inducing expression of intI1 with 0.5 mM isopropyl-â-d-thiogalactopyranoside (IPTG) followed by incubation at 37°C for 3 hs in L-broth containing the appropriate antibiotics. Plasmids were extracted from 5 ml of the culture, with the Miniprep kit (Qiagen) and used for the detection of the excision events by PCR  using the appropriate primer pair in 40 colonies per mating which were repeated three times in independent assays. We observed variable values of excision for the gene cassettes found in In2-8, and, although the region containing attC-catB2(DattI2)-dfrA1-attC is recognized by the integrase IntI1 as a fused gene cassette, the unusual attC-catB2-DattI2 gene cassette was found to be the most efficient substrate for the IntI1 recombinase. To confirm excision of the unusual attC-catB2-(DattI2) cassette, specific primers were designed to detect the circularized gene cassette generated after the cassette excision. Two amplification products were obtained from this reaction, one of them of 450 bp and the other of 250 bp. Sequence analyses of both products revealed to different sites recognized by the IntI1 to mediate the attC-catB2-(DattI2) 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, the one found in the 250 bp amplification product, 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 functional unusual mobile element. We observed the excision of the orfX, the ybfB, and the complete structure orfX-ybfA-ybfB-ybgA. The high frequency of excision, circularization and insertion of the attC-catB2-DattI2 array evidences the plasticity of recombination of the system mediated by the type 1 integrase gene. We observed variable values of excision for the gene cassettes found in In2-8, and, although the region containing attC-catB2(DattI2)-dfrA1-attC is recognized by the integrase IntI1 as a fused gene cassette, the unusual attC-catB2-DattI2 gene cassette was found to be the most efficient substrate for the IntI1 recombinase. To confirm excision of the unusual attC-catB2-(DattI2) cassette, specific primers were designed to detect the circularized gene cassette generated after the cassette excision. Two amplification products were obtained from this reaction, one of them of 450 bp and the other of 250 bp. Sequence analyses of both products revealed to different sites recognized by the IntI1 to mediate the attC-catB2-(DattI2) 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, the one found in the 250 bp amplification product, 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 functional unusual mobile element. We observed the excision of the orfX, the ybfB, and the complete structure orfX-ybfA-ybfB-ybgA. The high frequency of excision, circularization and insertion of the attC-catB2-DattI2 array evidences the plasticity of recombination of the system mediated by the type 1 integrase gene.