Structural heterogeneity of attC integron recombination sites revealed by optical tweezers

A predominant tool for adaptation in Gram-negative bacteria is the functional genetic platform called integron. Integrons capture and rearrange promoterless gene cassettes in a unique recombination process involving the recognition of folded single-stranded DNA hairpins—so-called attC sites—with a strong preference for the attC bottom strand. While structural elements have been identified to promote this preference, their mechanistic action remains incomplete. Here, we used high-resolution single-molecule optical tweezers (OT) to characterize secondary structures formed by the attC bottom ([Formula: see text]) and top ([Formula: see text]) strands of the paradigmatic attC(aadA7) site. We found for both sequences two structures—a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred [Formula: see text] predominantly formed the straight hairpin, while the [Formula: see text] preferentially adopted the kinked structure, which exposes only one complete recombinase binding box. By a mutational analysis, we identified three bases in the unpaired central spacer, which could invert the preferred conformations and increase the recombination frequency of the [Formula: see text] in vivo. A bioinformatics screen revealed structural bias toward a straight, canonical hairpin conformation in the bottom strand of many antibiotic resistance cassettes attC sites. Thus, we anticipate that structural fine tuning could be a mechanism in many biologically active DNA hairpins.