A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase IntI1 activity

Abstract

IntI1 integrase is a tyrosine recombinase involved in the mobility of antibiotic resistance gene cassettes within bacterial class 1 integrons. Recent data have shown that its recombination specifically involves the bottom strand of the attC site, but the exact mechanism of the reaction is still unclear. An efficient in vitro assay is still required to better characterize the biochemical properties of the enzyme. In this report we describe for the first time an in vitro system partially reproducing the activity of a recombinant pure IntI1. This new assay, which constitutes the only available in vitro model of recombination by IntI1, was used to determine whether this enzyme might be the sole bacterial protein required for the recombination process. Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites. However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data. The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction. Our data strongly suggest that the enzyme possesses all the functions for generating and/or recognizing this structure even in the absence of other cellular factors. Furthermore, the in vitro assay reported here constitutes a powerful tool for the analysis of the recombination steps catalyzed by IntI1, its structure-function studies and the search for specific inhibitors.

Figure 1. Schematic representation of recombinant plasmid pC23 part structure encoding the class 1 integron in P. aeruginosa Pa695 (adapted from Dubois et al., 2002, accession number AF355189).

The horizontal arrows indicate the translation orientation of the genes. The conserved core and inverse core sites are underlined and the cassette boundaries are represented by vertical arrows. The black arrowheads indicate the different primers described in materials and methods used for cloning the IntI1 gene and the different recombination and excision substrates: A: IntI1-3′-stop, B: IntI1-5′-Topo, C: attI1-LBamH1, D: attI1-RHindIII, E: C12T3bis and F: 3′CS.

Figure 2. SDS-PAGE (A) and western blot (B) analysis of protein factions containing IntI1(his)₆.

Lane M: molecular weight markers in kDa; lane 1: soluble crude extract from E. coli 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).

Figure 3. In vitro DNA binding of IntI1 with free double-stranded attI1 (A) and attC (B) recombination sites.

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.

Figure 4. In vitro DNA binding of IntI1 with free single-stranded attC (A) and attI (B) recombination sites.

Free 5′ ³²P radiolabeled ssDNA fragments containing recombination sites (0.1 pmoles) were incubated with purified IntI1 (5–10 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.

Figure 5. Comparison of IntI1 1 affinity for double-stranded (A) and single-stranded (B) recombination sites.

Filter binding assays were performed as described in materials and methods section using either double-stranded attI (attI ds) and attC (attC ds) either top (top) or bottom (bot) strand of attI and attC. Percentages of substrate retained on filters are shown. Values are the mean±standard deviation (error bars) of three independent experiments.

Figure 6. In vitro recombination catalyzed by IntI1 at attI1 and attC sites.

Reactions were performed for 90 min in the presence of purified IntI1 (5 or 10 pmoles), 0.1 to 0.2 pmoles of either pGEM-T-attI1 or pGEM-T-attC (pattI1 and pattC in the figure) and 0.1 pmoles free 5′ ³²P radiolabeled recombination sites under standard conditions described in materials and methods. Products were loaded on 1% agarose gel and autoradiographied (A). F: free recombination sites, RP: recombination products. The recombination products were quantified and the percentage of recombination versus the amount of IntI in pmoles was plotted (B).

Figure 7. In vitro recombination catalyzed by wild type, R146E and R286K mutated IntI1.

Reactions were performed for 90 min in the presence of purified enzyme (5 pmoles), 0.1 pmoles of linear radiolabeled recombination sites attI1 or attC and 0.1 pmoles of pGEM-T-attI1 under standard conditions described in materials and methods section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.

Figure 8. In vitro recombination catalyzed by wild type IntI1 in presence of double- or single-stranded substrates.

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 attI1 or attC and 0.1 pmoles of pGEM-T-attI1 or pGEM-T-attC under standard conditions described in materials and methods section. Products were loaded on 1% agarose gel and autoradiographied. F: free recombination sites, RP: recombination products.

Figure 9. Effect of cations (A), salt (B) and detergent (C) on in vitro recombination catalyzed by IntI1.

Recombination reactions were performed in the presence of 2 pmoles purified IntI1, 0.1 pmoles free attI1^* and 0.1 pmoles pGEM-T-attI1 and different concentrations of MgCl₂, MnCl₂ NaCl and detergent as indicated. Recombination products were quantified with DNAJ software and are shown on the graphs as the percentage of recombinant product versus the total substrate.