Integrons and gene cassettes: hotspots of diversity in bacterial genomes

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.     

Bacterial resistance evolution by recruitment of super-integron gene cassettes

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.     

Non-palindromic attI sites of integrons are capable of site-specific recombination with one another and with secondary targets

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.     

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.     

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.     

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.     

Insertion sequences in the IS1111 family that target the attC recombination sites of integron-associated gene cassettes

Members of the recently identified IS 1111 family differ from the majority of insertion sequences (IS) in that they target specific sites in an orientation-specific manner. However, the way in which target selection is achieved is not known. ISKpn4 is representative of a new subgroup of the IS 1111 family whose members are found in the attC sites (59-be) of the gene cassettes associated with integrons. The transposases of this subgroup are closely related (over 75% identity), confirming that closely related IS usually share a common target. However, among more distant relatives encoding a transposase <45% identical to those of the ISKpn4 group, one IS, ISPa25, was found that also targets attC sites. It appears that the targeting determinant of the ISKpn4 group has become associated with a transposase gene from a different group, and this allowed us to localize the region that is likely to be required for target selection to a long noncoding region found downstream of the transposase gene in all IS 1111 family members. This region may determine an RNA used to guide the IS to its specific target.     

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.     

Integron integrase binds to bulged hairpin DNA

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.     

Comparative study of class 1 integron and Vibrio cholerae superintegron integrase activities

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.     

DNA complexes obtained with the integron integrase IntI1 at the attI1 site.

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.     

Identification and analysis of integrons and cassette arrays in bacterial genomes

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome.     

Gene cassettes and cassette arrays in mobile resistance integrons

Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.     

Mobile gene cassettes and DNA integration elements

Integrons are unique natural systems for capturing and spreading the antibiotic resistance genes among Gram-negative bacteria. Gene transfer into small genomes and into plasmids is via site-specific recombination. Integrons act as receptors of antibiotic resistance cassettes. There are known more than 50 cassettes conferring resistance to beta-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptomycin, and other antibiotics. The structure of integrons and of gene cassettes are described and the mechanisms of capture, mobilization, and expression of cassettes considered.     

Integron cassette insertion: a recombination process involving a folded single strand substrate

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.