In Vitro Recombination Catalyzed by Bacterial Class 1 Integron Integrase IntI1 Involves Cooperative Binding and Specific Oligomeric Intermediates

Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1) between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1•DNA complexes been identified. Using the in vitro strand transfer assay and a crosslink strategy we previously described we demonstrated that the single-stranded attC sequences could be generated in the absence of other bacterial proteins in addition to IntI. This suggests a possible role for this protein in stabilizing and/or generating this structure. The mechanism of folding of the active IntI•DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate. These findings provide further insight into the recombination reaction catalyzed by IntI1

<i>In vitro</i> DNA binding of IntI1 with double- or single-stranded <i>att</i>I1 (A) and <i>att</i>C (B) sites.

<p>Free 5′ ³²P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–15 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Gel shifted bands were then quantified using DNAJ software and are plotted in the figure as percentage of bound DNA. Results are the mean±standard deviation (error bars) of three independent experiments.</p

SDS-PAGE analysis of crosslinked IntI1•DNA complexes.

<p>IN (2 pmoles) was preincubated with the 5′-end radiolabeled ds<i>att</i>I1 (A), ds<i>att</i>C (B), ss<i>att</i>I1 (C, bs and ts), ss<i>attC</i> (D, bs and ts) for 30 min in the presence of AHDAP at 37°C (final volume 20 µl). Products were separated by electrophoresis on 12% SDS-PAGE gel. The gel was then dried and autoradiographed. The positions of monomers, dimers and tetramers bound to DNA were determined by comparison with the migration distance of protein weight markers (BIO-RAD) submitted to electrophoresis under the same conditions. F: free substrate.</p

Figure 1

<p>A- <i>In vitro</i> strand transfer catalyzed by IntI1 at <i>att</i>I1 and <i>att</i>C sites. Reactions were performed for 90 min in the presence of purified IntI1 (2 pmoles), 0.1 to 0.2 pmoles of either pGEM-T-<i>att</i>I1 or pGEM-T-<i>att</i>C (p<i>att</i>I1 and p<i>att</i>C in the figure) and 0.1 pmoles free 5′ ³²P radiolabeled <i>att</i>I1 sites under the standard conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005228#s4" target="_blank">materials and methods</a>. Products were loaded on 1% agarose gel run at 200 V, for 2 hours at room temperature and autoradiographied. F: free recombination sites, ST: strand transfer products. B- Inhibition of <i>in vitro</i> strand transfer activity of IntI1 by nuclease S1. Strand transfer reactions were carried out as described in A but with increasing amounts of S1 nuclease (0 to 15 pmoles). Strand transfer products were quantified using DNAJ software and are plotted in the figure as percentage of the initial substrate. Results are the mean±standard deviation (error bars) of three independent experiments.</p

Model for the cooperative binding and the <i>inti1</i> oligomers involved in the <i>in vitro</i> recombination catalyzed by IntI1.

<p>In the <i>att</i>I1×<i>att</i>I1 recombination reaction IntI1 binds both ds<i>att</i>I1 fragment as a dimer and catalyzes the strand exchange and the formation of the HJ intermediate (lanes A1 to A3). In the case of <i>att</i>I1×<i>att</i>C recombination, IntI1 binds ds<i>att</i>C only slightly (B1). Interaction with the first <i>att</i>I1 substrate led to cooperative binding to the second <i>att</i>C site (B2), allowing the recruitment of a second IntI1 dimer on the second strand of the <i>att</i>C site (B3) and the formation of the tetrameric intermediate, leading in turn to the stabilization of a ss<i>att</i>C intermediate (B4). The strand exchange between ds<i>att</i>I1 and bs<i>att</i>C can then be catalyzed (B5). Recombination activity detected in presence of two <i>att</i>C sites suggests that the initial low binding of the enzyme to this site is sufficient for triggering all the subsequent recombination steps (way C). ts: top strand, bs: bottom strand.</p

<i>In vitro</i> recombination catalyzed by wild type IntI1 in presence of double- or single-stranded substrates.

<p>Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled double-stranded (ds) or single-stranded (bottom strand: ss bot, or top strand: ss top) recombination sites <i>attI1</i> or <i>attC</i> and 0.1 pmoles of pGEM-T-<i>attI1</i> or pGEM-T-<i>attC</i> under standard conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001315#s4" target="_blank">materials and methods</a> section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.</p

SDS-PAGE (A) and western blot (B) analysis of protein factions containing IntI1(his)₆.

<p>Lane M: molecular weight markers in kDa; lane 1: soluble crude extract from <i>E. coli</i> DH5α expressing IntI1(his)₆; lane 2: non-retained fraction; lanes 3, 4, 5 and 6: fractions obtained after elution with respectively 20, 100, 250 and 350 mM imidazole. Western blot was performed using anti-(his)₆Ct antibodies (INVITROGEN).</p

<i>In vitro</i> recombination catalyzed by wild type, R146E and R286K mutated IntI1.

<p>Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled recombination sites <i>attI1</i> or <i>attC</i> and 0.1 pmoles of pGEM-T-<i>attI1</i> under standard conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001315#s4" target="_blank">materials and methods</a> section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.</p

<i>In vitro</i> DNA binding of IntI1 with free double-stranded <i>attI1</i> (A) and <i>attC</i> (B) recombination sites.

<p>Free 5′ ³²P radiolabeled dsDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (1–10 pmoles) at 4°C for 20 min before electrophoresis on 1% agarose gel run at 50 V, for 2 hours at 4°C. Arrows indicate the protein-DNA complexes and F corresponds to free recombination sites.</p