The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes

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Abstract
The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB59 base element (59-be)] upstream of promoterlessaadB[gentamicin (Gm) resistance] andgfp(green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI inPseudomonas stutzeristrain Q. Electroporation of pUS23 intoP. stutzeriQ gave ampicillin-resistant transformants, which yielded GmRgreen fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 atattIwas detected by PCR in 8 % of GmRcolonies and the frequency ofattIintegration was estimated as 2·0×10−8perP. stutzeriQ(pUS23) cell. RT-PCR confirmed integron-mediated expression ofaadBin one recombinant strain (Q23-17) and a promoter (Pc) was localized to the 5′ end of theintIgene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at theattIsite. An insertion sequence (ISPst5; IS5family) was discovered in the vector backbone of the reporter plasmid integrated atattIand also in a pUS23 derivative recovered as a plasmid inEscherichia coliJM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.