Efficiency of recombination reactions catalyzed by class 1 integron integrase IntI1.
Ontology highlight
ABSTRACT:
SUBMITTER: Collis CM
PROVIDER: S-EPMC95170 | biostudies-literature | 2001 Apr
REPOSITORIES: biostudies-literature
Publications
Efficiency of recombination reactions catalyzed by class 1 integron integrase IntI1.
Collis C M CM Recchia G D GD Kim M J MJ Stokes H W HW Hall R M RM
Journal of bacteriology 20010401 8
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 ...[more]
Similar Datasets
Project description: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 x 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 x attC recombination versus attC x attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.
| S-EPMC2095811 | biostudies-other
Project description: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.
| S-EPMC2117344 | biostudies-literature
Project description:Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.
| S-EPMC9090394 | biostudies-literature
Project description:Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution.
| S-EPMC4438328 | biostudies-literature
Project description:Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.
| S-EPMC134885 | biostudies-literature
Project description:Gene cassettes are short, monogenic DNA elements that translocate between integrons through site-specific excision and integration. These events require that an integron-coded tyrosine recombinase forms a reactive complex with two sites, at least one of which belongs to the attC class. An attC site can be divided into two pairs of short repeats flanking a palindromic central region. The nucleotide sequence of attC among different cassettes varies extensively, implying that the site contains a structural recognition determinant with low sequence constraints. Oligonucleotides representing many different sequence modifications in either strand of the site were examined for integrase binding by using an electrophoresis mobility shift assay. The inner repeats, a central triplet and two single-nucleotide asymmetries in the site had the strongest influence on binding strength and strand choice. Our data show that the recombinase binds to a bulged hairpin in attC and that the hairpin distortion due to bulging could define the appropriate orientation of the otherwise symmetrical site. This is consistent with the strong bias for binding of recombinase to the bottom-strand oligonucleotides in vitro. Moreover, it was observed that the mobility-shifted complexes persisted under protein-denaturing assay conditions, indicating that a covalent link is indeed formed between integrase and DNA. Upon substitution of the presumed DNA-attacking residue, Y312, with a phenylalanine, DNA binding remained but there was no covalent linkage.
| S-EPMC506814 | biostudies-literature
Project description:Integrons capture gene cassettes by using a site-specific recombination mechanism. As only one class of integron and integron-determined site-specific recombination system has been studied in detail, the properties of a second class, the only known class 3 integron, were examined. The configuration of the three potentially definitive features of integrons, the intI3 gene, the adjacent attI3 recombination site, and the P(c) promoter that directs transcription of the cassettes, was similar to that found in the corresponding region (5' conserved segment) of class 1 integrons. The integron features are flanked by a copy of the terminal inverted repeat, IRi, from class 1 integrons on one side and a resolvase-encoding tniR gene on the other, suggesting that they are part of a transposable element related to Tn402 but with the integron module in the opposite orientation. The IntI3 integrase was active and able to recognize and recombine both known types of IntI-specific recombination sites, the attI3 site in the integron, and different cassette-associated 59-be (59-base element) sites. Both integration of circularized cassettes into the attI3 site and excision of integrated cassettes were also catalyzed by IntI3. The attI3 site was localized to a short region adjacent to the intI3 gene. Recombination between a 59-be and secondary sites was also catalyzed by IntI3, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.
| S-EPMC135066 | biostudies-literature
Project description:There is little information about environmental contamination with antibiotic resistance genes (ARG) in Sub-Saharan Africa, home to about 1 billion people. In this study we measured the abundance of three genes (sul1, sul2, and intI1) used as indicators of environmental contamination with ARGs in the sediments of four urban wetlands in southwestern Nigeria by qPCR. In addition, we characterised the variable regions of class 1 integrons in sulfamethoxazole/trimethoprim (SMX/TRI)-resistant bacteria isolated from the wetlands by PCR and DNA sequencing. The indicator ARGs were present in all wetlands with mean absolute copy numbers/gram of sediment ranging between 4.7x106 and 1.2x108 for sul1, 1.1x107 and 1x108 for sul2, and 5.3x105 and 1.9x107 for intI1. The relative abundances (ARG/16S rRNA copy number) ranged from about 10-3 to 10-1. These levels of ARG contamination were similar to those previously reported for polluted environments in other parts of the world. The integrase genes intI1 and intI2 were detected in 72% and 11.4% SMX/TRI-resistant isolates, respectively. Five different cassette array types (dfrA7; aadA2; aadA1|dfrA1; acc(6')lb-cr|arr3|dfrA27; arr3|acc(6')lb-cr|dfrA27) were detected among 34 (59.6%) intI1-positive isolates. No gene cassettes were found in the nine intI2-positive isolates. These results show that African urban ecosystems impacted by anthropogenic activities are reservoirs of bacteria harbouring transferable ARG.
| S-EPMC6264143 | biostudies-literature
Project description:As a major concern in public health, methicillin-resistant staphylococci (MRS) still remains one of the most prevalent pathogens that cause nosocomial infections throughout the world and has been recently labeled as a "super bug" in antibiotic resistance. Thus, surveillance and investigation on antibiotic resistance mechanisms involved in clinical MRS strains may raise urgent necessity and utmost significance. As a novel antibiotic resistance mechanism, class 1 integron has been identified as a primary source of antimicrobial resistance genes in Gram-negative organisms. However, most available studies on integrons had been limited within Gram-negative microbes, little is known for clinical Gram-positive bacteria. Based on series studies of systematic integrons investigation in hundreds of staphylococci strains during 2001-2006, this review concentrated on the latest development of class 1 integron in MRS isolates, including summary of prevalence and occurrence of class 1 integron, analysis of correlation between integron and antibiotic resistance, further demonstration of the role integrons play as antibiotic determinants, as well as origin and evolution of integron-associated gene cassettes during this study period.
| S-EPMC3136644 | biostudies-literature
Project description:Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1) in raw sewage (RS) and activated sludge (AS). The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS) and 92% (RS) of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS) as well as for Enhydrobacter (RS). The activated sludge process decreased (55%) the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.
| S-EPMC4482650 | biostudies-literature