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

Dubois V, Debreyer C, Litvak S, Quentin C, Parissi V - PLoS ONE (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Microbiology and Pathogenicity (MCMP), UMR 5097-CNRS, University Victor Segalen Bordeaux 2, Bordeaux, France.

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.

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Related in: MedlinePlus

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.
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pone-0001315-g001: 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.

Mentions: Thus, we sought to produce and purify an active recombinant integrase from a class 1 integron previously isolated from a clinical strain of Pseudomonas aeruginosa [18] see figure 1. Then we set up an in vitro recombination assay to characterize its biochemical properties. Using this new assay, we show that IntI1 possesses an in vitro recombination activity on both attI1 and attC but with different efficiencies, consistent with its differential affinity for each DNA element. This new in vitro assay of IntI1 recombination activity allows further functional analysis of the protein.


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

Dubois V, Debreyer C, Litvak S, Quentin C, Parissi V - PLoS ONE (2007)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2117344&req=5

pone-0001315-g001: 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.
Mentions: Thus, we sought to produce and purify an active recombinant integrase from a class 1 integron previously isolated from a clinical strain of Pseudomonas aeruginosa [18] see figure 1. Then we set up an in vitro recombination assay to characterize its biochemical properties. Using this new assay, we show that IntI1 possesses an in vitro recombination activity on both attI1 and attC but with different efficiencies, consistent with its differential affinity for each DNA element. This new in vitro assay of IntI1 recombination activity allows further functional analysis of the protein.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Microbiology and Pathogenicity (MCMP), UMR 5097-CNRS, University Victor Segalen Bordeaux 2, Bordeaux, France.

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.

Show MeSH
Related in: MedlinePlus