In vivo integron integrase (inti1) abundance affects the type and amount of recombination products differentially during the growth cycle
Shearer, Julia Elaine Servatius
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The integron integrase IntI1 mediates the transfer of antibiotic resistance gene cassettes between plasmid-carried integrons by two mechanisms, integration and excision or the formation and resolution of cointegrates. Cointegrates can be isolated from cells expressing integrase under the natural promoter Pint, but cassette capture has never been experimentally observed without excess integrase. Historically, integrase activity has been measured after conjugation or transformation of its recombinant products to a recipient cell. We constructed an integron capture vector pICV8 that acquires and donates antibiotic resistance gene cassettes in strains that overexpress IntI1 and detected recombination products in cells by PCR without conjugative transfer of recombinant plasmids. Qualitative PCR revealed that pICV8 readily forms cointegrates in vivo in strains expressing integrase under its natural promoter on a low copy number plasmid, though cassette capture products are not detected without excess integrase. Using dilution PCR, we report the first measurements of the time- dependence of formation of integron intracellular products. Integrase-mediated recombination products were quantified over time using dilution PCR in low and high integrase strains. Only cointegrates were detectable in strains expressing integrase under a natural promoter, and the simple acquisition of gene cassettes could only be seen in strains overexpressing integrase. Recombination products in both the low and high integrase strains increased in late log to early stationary phase. attC x attI recombination has been reported to occur 10 times more frequently than attI x attI crossovers, but, unexpectedly, we detected more attI x attI than attC x attI recombination products in log phase for both strains. However, the high integrase strain showed increased attC recombination in stationary phase, consistent with the earlier observations on integrase crossover preferences. Thus, the intracellular abundance of IntI1 affects the amount and type of recombination events, especially toward the end of log phase.