1887

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 [ 59 base element (59-be)] upstream of promoterless [gentamicin (Gm) resistance] and (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in strain Q. Electroporation of pUS23 into Q gave ampicillin-resistant transformants, which yielded Gm green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at was detected by PCR in 8 % of Gm colonies and the frequency of integration was estimated as 2·0×10 per Q(pUS23) cell. RT-PCR confirmed integron-mediated expression of in one recombinant strain (Q23-17) and a promoter (P) was localized to the 5′ end of the 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 site. An insertion sequence (IS; IS family) was discovered in the vector backbone of the reporter plasmid integrated at and also in a pUS23 derivative recovered as a plasmid in JM109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.

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2005-06-01
2020-02-25
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