Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology.

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 (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (Gm(R) or Lac(+)) were obtained at frequencies ranging from 10(1) to 10(6) c.f.u. (µg DNA)(-1). Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter P(C) were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY 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.

Urinary tract infections in a South American population: dynamic spread of class 1 integrons and multidrug resistance by homologous and site-specific recombination.

One hundred four bacterial strains mediating urinary tract infections in separate individuals from a Uruguayan community were isolated. Forty-six strains conferred a multidrug resistance phenotype. All 104 strains were examined for the presence of class 1, 2, and 3 integrons. Class 1 integrons were found in 21 isolates across four distinct bacterial genera. A large class 1 integron in a Klebsiella pneumoniae strain was fully sequenced and was 29,093 bp in length. This integron probably arose by homologous recombination since it was embedded in a hybrid Tn21-like transposon backbone which comprised a Tn5036-like tnp transposition module at the IRi integron end and a Tn21 mer module at the IRt integron end. The parent integron/transposon that contributed the Tn5036 module was not related to Tn1696 since the integron insertion points in the transposon backbones were 16 bases apart. Examination of the other 20 class 1 integron-containing strains revealed further evidence of genetic exchange. This included a strain that possessed a Tn5036 module at the IRt end but not at the IRi end and another that possessed a tnp module beyond IRi that was a hybrid of Tn21 and Tn5051 and that is presumed to have arisen by site-specific recombination. This study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance in a community.

Recombination between the dfrA12-orfF-aadA2 cassette array and an aadA1 gene cassette creates a hybrid cassette, aadA8b.

Homologous recombination between closely related gene cassettes, such as aadA1 and aadA2, which are 89% identical, can create hybrid cassettes and hybrids of existing cassette arrays. A new cassette array, dfrA12-orfF-aadA8b, which was created by such a recombination event occurring within the aadA2 cassette in the dfrA12-orfF-aadA2 array, has been identified.