A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase

Abstract

Integron In2 integrase (IntI1)-mediated site-specific recombination between two primary sites occurs at a high frequency, while that between a primary and a secondary site occurs at frequencies around 10,000 times lower. Secondary sites consist of a pentanucleotide with only two fully conserved residues (GWTMW). The analysis of IntI1-mediated recombinants in the plasmid pOX38 revealed the existence in this plasmid of a site used at a frequency intermediate between those of primary and secondary sites. Analysis of this site showed two potentially relevant structural features: first, a set of two consensus pentanucleotides, separated by 5 bp and in opposite orientations, forming what will be called a double site; and second, a longer sequence with some extent of sequence symmetry with the double site at its 3' end. A recombinant plasmid, pSU18P, containing a double site was constructed. Examination of R388-pSU18P recombinants showed that double sites were used preferentially over single pentanucleotides by IntI1. Comparisons of the nucleotide sequences of known 59-bp elements showed that in most cases there was a double site at each element end. Mutagenesis of the F hot spot was carried out to make it look more like the consensus 59-bp element. The improved sites showed recombination frequencies and specificities almost comparable to those observed at IntI1 primary sites.