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

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 [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the Cl in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded Gm R green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attl was detected by PCR in 8% of Gm(R) colonies and the frequency of attl integration was estimated as 2.0 x 10(-8) per P. stutzeri Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter (P-c) was localized to the 5' end of the intl gene. 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 the attl site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attl and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.