IntI2 integron integrase in Tn7

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.     

Intracellular Steady-State Concentration of Integron Recombination Products Varies with Integrase Level and Growth Phase

IntI1 mediates the recombination of antibiotic-resistant gene cassettes between different integrons in the same cell, facilitating the persistence and dissemination of these genes. Historically, integrase activity has been measured by conjugating recombinant products from donor cells overexpressing integrase and quantifying them in recipient cells. Here we report the first measurements of the steady-state intracellular abundance of integrase-mediated recombination products in strains expressing natural or high IntI1 levels. Recombination products in both high and natural integrase strains increased markedly through late log phase and continued to rise in stationary phase in the high integrase strain, but declined in the natural expression strain. Simple acquisition of gene cassettes was seen only in strains expressing high integrase; in strains with natural integrase levels, only cointegrates between the two integron-bearing plasmids were detectable at all growth stages. Unexpectedly, more attI × attI than attC × attI recombination products were seen in log phase for both strains; however, in stationary phase, the high integrase strain increased attC recombination, consistent with earlier observations of integrase crossover site preferences. Thus, direct quantification of the steady-state concentration of recombination products reveals that the integrase's intracellular concentration affects the amount and type of recombination events in a growth-phase-dependent manner.     

Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites

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.     

Epidemiology and molecular mechanism of integron-mediated antibiotic resistance in <Emphasis Type="Italic">Shigella</Emphasis>

Integrons are gene capture and expression systems that are characterized by the presence of an integrase gene. This encodes an integrase, a recombined site, and a promoter. They are able to capture gene cassettes from the environment and incorporate them using site-specific recombination. The role of integrons and gene cassettes in the dissemination of multidrug resistance in Gram-negative bacteria is significant. In Shigella species, antimicrobial resistance is often associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes. Multiple and complex expression regulation mechanisms involving mobile genetic elements in integrons have been developed in the evolution of Shigella strains. Knowledge of the epidemiology and molecular mechanisms of antimicrobial resistance in this important pathogen is essential for the implementation of intervention strategies. This review was conducted to introduce the structures and functions of integrons in Shigella species and mechanisms that control integron-mediated events linked to antibiotic resistance.     

Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.Integrons are genetic elements that capture and rearrange genes that are contained within mobile gene cassettes by a mechanism of site-specific recombination mediated by an integrase (3). Several types of integron integrases have been described for clinical and environmental bacteria; classes 1, 2, and 3 integron integrases (1, 10, 11) and VchIntIA (17) and IntI9 (12) integrases are the only ones that are associated with antibiotic resistance genes. Some of these integrases were found exclusively on plasmids (IntI2*) (11) or on chromosomes (VchIntIA) (17), while others were found in both genetic contexts (IntI1) (7, 8, 20, 21). The efficiency of integron integrases to carry out cassette excision varies from one integrase to another and also depends on the structure and sequence of the attC sites located at both ends of the gene. IntI1 is generally the most active integrase, followed by IntI3. IntI2*179E and SonIntIA are less active but appear to tolerate more variation in attC sites. These enzymes could serve as models for determining important residues responsible for high levels of activity, using mutagenesis to substitute consensus residues and assaying for gain of function.Class 1 integrons, carrying the intI1 integrase gene, are generally associated with mobile elements, such as plasmids and Tn21-like transposons, and are most frequently found in clinical isolates (18). They are found mainly among gram-negative bacteria and especially among enterobacteria and pseudomonads (14). Class 1 integrons have also been found in some gram-positive bacteria, such as Enterococcus, Staphylococcus, and Corynebacterium (6). The clinical-type class 1 integrons (7) consist of two conserved regions and a variable region in which resistance genes are inserted in the form of cassettes (Fig. ​(Fig.1A).1A). These integrons were clearly derived from a structure related to Tn402, as they share many characteristics associated with this type of transposon (21). The common ancestor of clinical-type class 1 integrons was possibly a member of an integron pool that was acquired by diverse Betaproteobacteria (7). This hypothesis is based on the recent isolation of several new class 1 integron integrases from environmental DNA samples which are not associated with antibiotic resistance genes or with Tn402-like transposons (7, 8, 21).Open in a separate windowFIG. 1.(A) General structure of clinical-type class 1 integrons. Cassettes are inserted in the variable region of integrons by a site-specific recombinational mechanism. The attI1 and attC sites are shown by tiling and diagonal black lines, respectively, and promoters are denoted by P1, P2, P3, and P. Genes are as follows: intI1, integrase gene; qacEΔ1, antiseptic resistance gene; sul1, sulfonamide resistance gene; orf5, gene of unknown function. (B) Representation of the chromosomal integron of S. amazonensis SB2BT. The attI_Sam and attC sites are shown by a black box and horizontal black lines, respectively. Genes are as follows: SamintIA, integrase gene; orf, open reading frame gene.Class 2 integrons, carrying the intI2* integrase pseudogene, are present on Tn7 transposons and their derivatives (11). The intI2* gene encodes an integrase identical to 46% with IntI1, but its reading frame was interrupted by an early termination codon. The activity of this protein is restored when the stop codon at position 179 is replaced by a glutamate codon (11). Recently, two new intI2 genes were identified within integrons found in Providencia stuartii (2) and Escherichia coli (16). The sequences of these genes are not interrupted; position 179 is occupied by a glutamine codon, and the genes apparently code for functional enzymes. These intI2 genes each differ from intI2* of Tn7 at five positions (2, 16).Class 3 integrons, characterized by the presence of the intI3 gene, have been found in Serratia marcescens AK9373, in Klebsiella pneumoniae FFUL 22K isolated in Portugal, in four strains of Pseudomonas putida isolated in Japan, and more recently, in Delftia acidovorans C17 and Delftia tsuruhatensis A90 (1, 4, 19, 23). The IntI3 integrase has 61% identity with IntI1.The class 4 integron, with VchintIA, is an integron carried by the small chromosome of Vibrio cholerae O:1 569B (17). This integron contains more than 216 open reading frames (ORFs) coding for proteins of unknown functions associated with V. cholerae repetitive DNA sequence (VCR) elements to form 179 cassettes, and occupies about 3% of the bacterial genome.In recent years, the draft genomes of various environmental strains led to the identification of more than 100 new integron integrases. Among these, the SonintIA and NeuintIA integrase genes have been found, respectively, in genomes of Shewanella oneidensis MR-1 and Nitrosomonas europaea and shown to be active in cassette excision and integration (5, 13). Shewanella amazonensis SB2BT is an environmental gram-negative gammaproteobacterium that plays an important role in the bioremediation of contaminated metals and radioactive wastes (22). The U.S. Department of Energy Joint Genome Institute sequenced its 4.3-Mbp genome (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000507","term_id":"119765642","term_text":"CP000507"}}CP000507). The genome encodes an integron integrase, SamIntIA, which is 64.8% identical to SonIntIA and 60.2% identical to IntI2* but only 46.9% identical to VchIntIA and 44.6% to IntI1. A sequence alignment of SamIntIA, SonIntIA, and IntI2* indicates that they are closely related, especially in the N-terminal and the C-terminal regions.Several residues of SamIntIA differed from a consensus alignment of active integron integrases. We wished to determine whether SamIntIA is active, compare its activity to that of SonIntIA and of IntI2*179E, and determine whether the alteration of certain residues affects its excision activity.     

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination

An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attl site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59-base-element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non-specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.     

Incidence of Extended-Spectrum β-Lactamases and Characterization of Integrons in Extended-Spectrum β-Lactamase-producing Klebsiella pneumoniae Isolated in Shantou, China

This study is concerned with the level of antibiotic resistance of extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae, isolated in Shantou, China, and its mechanism. Seventy- four non-repetitive clinical isolates of K. pneumoniae producing ESBLs were isolated over a period of 2 years. Antibiotic susceptibility, carried out by Epsilometer test, showed that most of the isolates were multiresistant. Polymerase chain reaction showed that, among the several types of β-lactamases, SHV was the most prevalent, TEM was the second most prevalent, and CTX-M was the least prevalent. Sixty-nine isolates were positive for integrase gene IntI1, but no IntI2 or IntI3 genes were found. The variable region of class 1 integrons were amplified and further identified by sequencing. Thirteen different gene cassettes and 11 different cassette combinations were detected. Dfr and aadA cassettes were predominant and cassette combinations dfrA12, orfF and aadA2 were most frequently found. No gene cassettes encoding ESBLs were found. Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.     

The IntI1 Integron Integrase Preferentially Binds Single-Stranded DNA of the attC Site

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.     

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

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.     

Efficiency of recombination reactions catalyzed by class 1 integron integrase IntI1

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.     

Recombination activity of a distinctive integron-gene cassette system associated with Pseudomonas stutzeri populations in soil

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.     

Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency

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.     

Worldwide Prevalence of Class 2 Integrases outside the Clinical Setting Is Associated with Human Impact

An intI-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (≤98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised ∼70 to 100% (n = 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%; n = 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the Enterobacteriaceae, we coupled PCR assays targeted at intI and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to Firmicutes (Clostridium and Bacillus) and Bacteroidetes (Chitinophaga and Sphingobacterium) dominated a low-G+C fraction (∼40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (≤98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.Integrons are genetic modules described in pathogenic and commensal bacteria that confer the ability to capture and express promoterless DNA units, called gene cassettes, which encode a variety of adaptive functions including antibiotic resistance (9, 42, 64). The acquisition of gene cassettes occurs through a site-specific recombination mechanism catalyzed by an integron-encoded integrase (IntI). The integrative recombination reaction occurs primarily between an integron receptor site (attI) and a cassette-associated sequence known as the attC site or 59-base element (11). However, integron integrases are able to recognize and process nonspecific secondary targets as well as attI and attC sites with a high degree of sequence variation (20, 25). This versatility facilitates the exchange of exogenous genes between different integrons through various recombination reactions (attI × attC, attI × attI, and attC × attC) that propel the adaptability and evolution of bacterial genomes (8, 11, 31, 38, 55, 58). Although integrons can be chromosomally encoded, they also may be horizontally transferred via transduction or by transposons associated with conjugative plasmids (42, 61). Three major groups (classes 1 to 3) are known to be associated with laterally transferred elements and highly prevalent in the clinical scene. In most of the cases, these have also been reported to harbor almost exclusively gene cassettes encoding antibiotic resistance functions (42). All together, these traits have led to their designation as “mobile” (9) or “clinical” (22) integrons. Although integrons have been traditionally classified according to the percent identity of the nucleotide or predicted amino acid sequence of their respective intI genes (9, 43, 71), several structural features and differences in abundance patterns have been identified which distinguish classes 1 to 3 (9, 42).Class 1 integrons are the most widely studied variant and are typically linked to replicative Tn21 transposons, which appears to contribute to their extensive distribution (48). A key feature commonly reported within the class 1 module is the presence of a highly conserved 3′ region comprised of a qacEΔ gene and a sul1 gene, which provide protection against quaternary ammonium compounds and sulfa drugs, respectively. In contrast, class 2 integrons are routinely associated with nonreplicative Tn7 transposons, are less frequently detected and, hence, remain an understudied group relative to their class 1 counterparts (42, 48, 65). Even less is known about the class 3 variants, which so far have been described in only three instances (71).Except for the identical IntI2 elements recently reported in Providencia stuartii and Escherichia coli strains isolated from beef cattle sources and the human urinary tract, respectively, all known integrases encoded by class 2 integrons are considered nonfunctional due to the presence of the ochre 179 codon (6, 40, 42). Nevertheless, it has been argued that integrons with truncated class 2 integrases might be implicated in the transfer and high prevalence of antibiotic resistance genes among clinical isolates, possibly via the in trans activities of other functional integrases or the suppression of the stop codon (27). So far, class 2 integrons have been described in association with isolates affiliated to the gamma, beta, and epsilon subdivisions of the Proteobacteria but have been more frequently reported among members of the Gammaproteobacteria group, particularly the Enterobacteriaceae (1, 14, 19, 56, 57). However, most of these studies have focused on easily culturable, aerobic bacteria or those of clinical importance, leading to the exclusion of unculturable or difficult-to-grow commensals that could be inconspicuous but important reservoirs of class 2 elements in the environment. Although the occurrence and quantification of integrons and integron-associated genes by means of molecular, culture-independent methods are being increasingly documented outside the clinical scene (18, 22, 28, 48, 49, 51, 65, 70), the estimates of the extant diversity of the integron platform in nature are still rudimentary. Likewise, further work is needed for the identification of environmental hosts of integrons commonly found in clinical strains without the bias associated with culture techniques (48).In order to provide a comprehensive view of integron integrase variation and prevalence patterns of IntI2 elements in the environment, we PCR amplified partial intI sequences from metagenomic DNA isolated from various terrestrial, marine, and deep-sea habitats exposed to various degrees of anthropogenic or fecal impact. Amplification conditions were optimized to facilitate the assessment of the frequency of IntI2-like sequences relative to that of environmental integron integrases. Additionally, since the guanine-plus-cytosine content of DNA corresponds to taxonomy (68), we coupled G+C fractionation of total DNA (4, 5, 29, 30) with PCR assays targeted at intI and 16S rRNA genes to identify potential, unconventional hosts of class 2 integrons in soil that had received swine manure.     

Cellular pathways controlling integron cassette site folding

By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single‐stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double‐stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non‐recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination.