Abstract
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 (n}{}{{att}}{{{C}}_{{rm{bs}}}}) and top (n}{}{{att}}{{{C}}_{{rm{ts}}}}) strands of the paradigmatic attCaadA7 site. We found for both sequences two structures—a straight, canonical hairpin and a kinked hairpin. Remarkably, the recombination-preferred n}{}{{att}}{{{C}}_{{rm{bs}}}} predominantly formed the straight hairpin, while the n}{}{{att}}{{{C}}_{{rm{ts}}}} 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 n}{}{{att}}{{{C}}_{{rm{ts}}}}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.
Highlights
Bacteria possess an impressive capability of adapting to environmental stresses
To characterize the secondary structure of the attCbs DNA hairpin, it was tethered between two glass microspheres of 1 and 2 m diameter through two 860-bp-long DNA handles (Figure 1D)
By separating the two silica beads from each other, force was applied to the molecular construct, causing the stretching of the DNA handles, which eventually triggered unfolding of the attC hairpin
Summary
Bacteria possess an impressive capability of adapting to environmental stresses. They are known to develop antibiotic multi-resistance by horizontal gene transfer. Upon SOS response, the integron tyrosine-recombinase IntI recognizes folded, single-stranded DNA hairpins called attC sites, which flank the open reading frame in cassettes [3,4] (Figure 1A). To ensure correct expression of the protein-coding sequences from these cassettes, the orientation of the inserted DNA sequence has to be tightly controlled with respect to the cassette promoter Pc. Bouvier et al and Nivina et al have identified important structural elements of attC sites, which ensure the correct orientation through the strand-selectivity of IntI for the bottom strand (attCbs) relative to the top strand (attCts) [5,6]. A recent study has reported that a nucleotide skew induced by UCS and VTS stabilizes the folded bottom strand,
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