1887

Abstract

Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance () and green fluorescence (), or lactose metabolism (), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into or strains via electroporation or natural transformation. Recombinants (Gm or Lac) were obtained at frequencies ranging from 10 to 10 c.f.u. (µg DNA). Cassettes were integrated by site-specific recombination at the integron site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of -containing recombinants revealed that expression levels from the integron-associated promoter P were five- to 10-fold higher in the plasmid-borne integron In compared with the chromosomal integrons. Integration of cassettes into integrons allowed the bacteria to grow on lactose, and the gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

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2011-12-01
2019-09-16
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