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1.
Bouvier M  Demarre G  Mazel D 《The EMBO journal》2005,24(24):4356-4367
Integrons play a major role in the dissemination of antibiotic resistance genes among Gram-negative pathogens. Integron gene cassettes form circular intermediates carrying a recombination site, attC, and insert into an integron platform at a second site, attI, in a reaction catalyzed by an integron-specific integrase IntI. The IntI1 integron integrase preferentially binds to the 'bottom strand' of single-stranded attC. We have addressed the insertion mechanism in vivo using a recombination assay exploiting plasmid conjugation to exclusively deliver either the top or bottom strand of different integrase recombination substrates. Recombination of a single-stranded attC site with an attI site was 1000-fold higher for one strand than for the other. Conversely, following conjugative transfer of either attI strand, recombination with attC is highly unfavorable. These results and those obtained using mutations within a putative attC stem-and-loop strongly support a novel integron cassette insertion model in which the single bottom attC strand adopts a folded structure generating a double strand recombination site. Thus, recombination would insert a single strand cassette, which must be subsequently processed.  相似文献   

2.
A Gravel  B Fournier    P H Roy 《Nucleic acids research》1998,26(19):4347-4355
Integrons are genetic elements that are able to capture genes by a site-specific recombination mechanism. Integrons contain a gene coding for a lambda-like integrase that carries out site-specific recombination by interacting with two different target sites; the attI site and the palindromic sequence attC (59 base element). Cassette integrations usually involve the attI site, while cassette excisions use attC . Therefore, the integrase should bind both sites to cleave DNA and perform site-specific recombination reactions. We have used purified maltose-binding protein fused with the integrase (MBP-IntI1) and native IntI1 protein and gel retardation assays with fragments containing the complete and partial attI1 site to show formation of four complexes in this region. Chemical modification of specific nucleotides within the attI1 site was used to investigate their interference with binding of the integrase protein. We attribute IntI1 specific binding to four regions in the attI1 site and a GTTA consensus sequence is found in three of the four regions. Interference by modified guanine and thymine residues in the DNA major groove and adenine residues in the minor groove were observed, indicating that the integrase interacts with both sides of the helix. Binding of IntI1 to attC is also discussed.  相似文献   

3.
IntI1 integrase is a member of the prokaryotic DNA integrase superfamily. It is responsible for mobility of antibiotic resistance cassettes found in integrons. IntI1 protein, as well as IntI1-COOH, a truncated form containing its carboxy-terminal domain, has been purified. Electrophoretic mobility shift assays were carried out to study the ability of IntI1 to bind the integrase primary target sites attI and aadA1 attC. When using double-stranded DNA as a substrate, we observed IntI1 binding to attI but not to attC. IntI1-COOH did not bind either attI or attC, indicating that the N-terminal domain of IntI1 was required for binding to double-stranded attI. On the other hand, when we used single-stranded (ss) DNA substrates, IntI1 bound strongly and specifically to ss attC DNA. Binding was strand specific, since only the bottom DNA strand was bound. Protein IntI1-COOH bound ss attC as well as did the complete integrase, indicating that the ability of the protein to bind ss aadA1 attC was contained in the region between amino acids 109 and 337 of IntI1. Binding to ss attI DNA by the integrase, but not by IntI1-COOH, was also observed and was specific for the attI bottom strand, indicating similar capabilities of IntI1 for binding attI DNA in either double-stranded or ss conformation. Footprinting analysis showed that IntI1 protected at least 40 bases of aadA1 attC against DNase I attack. The protected sequence contained two of the four previously proposed IntI1 DNA binding sites, including the crossover site. Preferential ssDNA binding can be a significant activity of IntI1 integrase, which suggests the utilization of extruded cruciforms in the reaction mechanisms leading to cassette excision and integration.  相似文献   

4.
Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron-encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette-associated 59-be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59-be and a non-cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non-cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.  相似文献   

5.
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.  相似文献   

6.
Integrons and gene cassettes: hotspots of diversity in bacterial genomes   总被引:1,自引:0,他引:1  
Integrons are genetic units found in many bacterial species that are defined by their ability to capture small mobile elements called gene cassettes. Cassettes usually contain only one gene, potentially any gene, and an attC recombination site, and thousands of cassettes have been sequenced. A specialized IntI site-specific recombinase encoded by the integron recognizes attC and incorporates cassettes into an attI site located adjacent to the intI gene. Over 100 types of integrons have been found, most in bacterial chromosomes. They can all potentially share the same cassettes and, as recombination between attC in a cassette and an attI can occur repeatedly, an integron can contain from zero to hundreds of cassettes. Cassette arrays that are not located next to an intI gene, or solo cassettes at apparently random sites, are also seen. Hence, integrons contribute to generation of diversity in bacterial, plasmid, and transposon genomes and facilitate extensive sharing of information among bacteria.  相似文献   

7.
The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.  相似文献   

8.
Genes borne on cassettes are mobile owing to site-specific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R-plasmids. In these processes, the palindromic site, attC (59-base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross-overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross-over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5' of the cross-over point. A region 27–36 bp 5' of attI influenced recombination with attC sites only, whereas a sequence 9–14 bp 5' of the cross-over point in attI was important for recombination with both attI and attC . Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site-specific recombinase to new sequences.  相似文献   

9.
10.
The site-specific recombinase IntI1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron-associated attI1 site and the 59-base element (59-be) family of sites that are associated with gene cassettes. IntI1 includes the four conserved amino acids that are characteristic of members of the integrase family, and IntI1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. IntI1 was purified as a fusion protein and shown to bind to isolated attI1 or 59-be recombination sites. Binding to attI1 was considerably stronger than to a 59-be. Binding adjacent to the recombination cross-over point was not detected. A strong IntI1 binding site within attI1 was localized by both deletion and footprinting analysis to a 14 bp region 24–37 bp to the left of the recombination cross-over point, and this region is known to be critical for recombination in vivo ( Recchia et al ., 1994 ). An imperfect (13/15) direct repeat of this region, located 41–55 bp to the left of the recombination cross-over point, contains a weaker IntI1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.  相似文献   

11.
12.
The class 1 integron integrase, IntI1, recognizes two distinct types of recombination sites, attI sites, found in integrons, and members of the 59-be family, found in gene cassettes. The efficiencies of the integrative version of the three possible reactions, i.e., between two 59-be, between attI1 and a 59-be, or between two attI1 sites, were compared. Recombination events involving two attI1 sites were significantly less efficient than the reactions in which a 59-be participated, and the attI1 x 59-be reaction was generally preferred over the 59-be x 59-be reaction. Recombination of attI1 with secondary sites was less efficient than the 59-be x secondary site reaction.  相似文献   

13.
We have found an integron-like integrase gene and an attI site in Shewanella oneidensis as part of a small chromosomal integron. We have cloned this gene and tested the ability of the integrase to excise cassettes from various integrons. Most cassettes flanked by two attC sites are readily excised, while cassettes in the "first" position, with an attI1 or attI3 site on one end, are not excised. An exception is a cassette with attI2 on one end. The attI2 site, from Tn7, has greater similarity to the attI site adjacent to the integrase of S. oneidensis than do attI1 or attI3. We cloned the attI site of S. oneidensis and observed the integration of two different cassettes. We have, therefore, demonstrated the function of this integron-like integrase.  相似文献   

14.
Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance. Diverse integrons have recently been detected directly in a range of natural environments. In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation of Pseudomonas stutzeri strain Q. Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM). 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar of P. stutzeri. Only strains Q and BAM were found to contain an integron and an associated gene cassette array. The intI and attI components of these strains showed 99 and 90% identity, respectively. The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes. The two integrons contained nonoverlapping sets of cassette-associated genes. In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (the Pseudomonas subfamily). The recombination activity of P. stutzeri integron components was tested in cointegrate assays. IntIPstQ was shown to catalyze site-specific recombination between its cognate attI site and 59-be sites from antibiotic resistance gene cassettes. While IntIPstQ did not efficiently mediate recombination between members of the Pseudomonas 59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1. We concluded that integrons present in P. stutzeri possess recombination activity and represent a hot spot for genomic diversity in this species.  相似文献   

15.
The presence of class 1, 2, and 3 integrons was investigated in four pediatric isolates of Salmonella enterica ser. Typhimurium (S. Typhimurium). A class 1 integron was detected in one S. Typhimurium strain, the only one that also showed resistance to various aminoglycoside antibiotics. This integron, called InJR06, and the aminoglycoside resistance determinants were located in pS06, a large (> or = 55 kb) conjugative plasmid. A single mobile cassette (encoding the aminoglycoside adenylyltransferase ANT(3')-Ia) was detected in the variable region of InJR06, while the architecture of the attI1 and attC sites was conserved.  相似文献   

16.
17.
Superintegrons (SIs) are chromosomal genetic elements containing assemblies of genes, each flanked by a recombination sequence (attC site) targeted by the integron integrase. SIs may contain hundreds of attC sites and intrinsic instability is anticipated; yet SIs are remarkably stable. This implies that either selective pressure maintains the genes or mechanisms exist which favour their persistence in the absence of selection. Toxin/antitoxin (TA) systems encode a stable toxin and a specific, unstable antitoxin. Once activated, the continued synthesis of the unstable antitoxin is necessary for cell survival. A bioinformatic search of accessible microbial genomes for SIs and TA systems revealed that large SIs harboured TA gene cassettes while smaller SIs did not. We demonstrated the function of TA loci in different genomic contexts where large-scale deletions can occur; in SIs and in a 165 kb dispensable region of the Escherichia coli genome. When devoid of TA loci, large-scale genome loss was evident in both environments. The inclusion of two TA loci, relBE1 and parDE1, which we identified in the Vibrio vulnificus SI rendered these environments refractory to gene loss. Thus, chromosomal TA loci can stabilize massive SI arrays and limit the extensive gene loss that is a hallmark of reductive evolution.  相似文献   

18.
19.
A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA-damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate-sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli.  相似文献   

20.
Bacterial resistance evolution by recruitment of super-integron gene cassettes   总被引:51,自引:0,他引:51  
The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI. We show that MRIs can randomly recruit genes directly from the cache of SI cassettes. By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V. cholerae SI and its precise insertion into the MRI. The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events.  相似文献   

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