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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: 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.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

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).
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pone-0001315-g002: 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).

Mentions: To obtain a sufficient quantity for enzyme purification, overexpression of the IntI1 protein was performed in the BL21 E. coli bacterial strain at 25°C for 4 hours after 1 mM IPTG induction. At higher temperature, most of the protein remained in the insoluble fraction, reflecting the high insolubility of the protein previously observed [9], [16]. Extraction in the presence of 500 mM NaCl and 0.25% Triton X-100 allowed us to obtain a highly soluble enzyme. The soluble fraction was used for nickel-affinity chromatography purification. As shown in figure 2A, a protein displaying a good level of purity was obtained in the 250–350 mM imidazole fractions. The major protein band of 40 kDa apparent molecular weight reacted with anti-His monoclonal antibodies, thereby confirming its nature (figure 2B).


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001315-g002: 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).
Mentions: To obtain a sufficient quantity for enzyme purification, overexpression of the IntI1 protein was performed in the BL21 E. coli bacterial strain at 25°C for 4 hours after 1 mM IPTG induction. At higher temperature, most of the protein remained in the insoluble fraction, reflecting the high insolubility of the protein previously observed [9], [16]. Extraction in the presence of 500 mM NaCl and 0.25% Triton X-100 allowed us to obtain a highly soluble enzyme. The soluble fraction was used for nickel-affinity chromatography purification. As shown in figure 2A, a protein displaying a good level of purity was obtained in the 250–350 mM imidazole fractions. The major protein band of 40 kDa apparent molecular weight reacted with anti-His monoclonal antibodies, thereby confirming its nature (figure 2B).

Bottom Line: 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.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