Integrons are genetic elements able to acquire and rearrange open reading frames (ORFs) embedded in gene cassette units and convert them to functional genes by ensuring their correct expression. They were originally identified as a mechanism used by Gram-negative bacteria to collect antibiotic resistance genes and express multiple resistance phenotypes in synergy with transposons. More recently, their role has been broadened with the discovery of chromosomal integron (CI) structures in the genomes of hundreds of bacterial species. This review focuses on the resources carried in these elements, on their unique recombination mechanisms, and on the different mechanisms controlling the cassette dynamics. We discuss the role of the toxin/antitoxin (TA) cassettes for the stabilization of the large cassette arrays carried in the larger CIs, known as superintegrons. Finally, we explore the central role played by single-stranded DNA in the integron cassette dynamics in light of the recent discovery that the integron integrase expression is controlled by the SOS response.
Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 pir and S17-1 pir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 pir and S17-1 pir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of ⌸-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.
Integrons are natural tools for bacterial evolution and innovation. Their involvement in the capture and dissemination of antibiotic-resistance genes among Gram-negative bacteria is well documented. Recently, massive ancestral versions, the superintegrons (SIs), were discovered in the genomes of diverse proteobacterial species. SI gene cassettes with an identifiable activity encode proteins related to simple adaptive functions, including resistance, virulence, and metabolic activities, and their recruitment was interpreted as providing the host with an adaptive advantage. Here, we present extensive comparative analysis of SIs identified among the Vibrionaceae. Each was at least 100 kb in size, reaffirming the participation of SIs in the genome plasticity and heterogeneity of these species. Phylogenetic and localization data supported the sedentary nature of the functional integron platform and its coevolution with the host genome. Conversely, comparative analysis of the SI cassettes was indicative of both a wide range of origin for the entrapped genes and of an active cassette assembly process in these bacterial species. The signature attC sites of each species displayed conserved structural characteristics indicating that symmetry rather than sequence was important in the recognition of such a varied collection of target recombination sequences by a single site-specific recombinase. Our discovery of various addiction module cassettes within each of the different SIs indicates a possible role for them in the overall stability of large integron cassette arrays.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank under accession nos. listed in Table 1.] Natural selection favors the evolution of strategies that increase the rate of adaptation, that is, chance favors the prepared genome (Caporale 1999). Although mutation generally causes only a very small and localized change in a cell, the transfer of genetic material involves much broader changes that may permit the organism to carry out new functions and adapt to environmental changes (Ochman et al. 2000). Integrons are exquisitely suited for this purpose. Integrons are natural cloning and expression systems that incorporate open reading frames (ORFs) and convert them to functional genes Mazel 1999, 2001). They have been expansively identified as the constituents of transferable elements responsible for the evolution of multidrug resistance among human, animal, and plant pathogenic isolates during the antibiotic era. More than 70 different antibiotic-resistance genes, covering most antimicrobials used against Gramnegative infections, have been characterized within integrons thus far (Rowe-Magnus et al. 2002a). The substantial impact of integrons on bacterial evolution is underscored by the present dilemma in the treatment of infectious disease, as the development of multiple-antibiotic resistance can often be traced to the stockpiling of resistance loci within integrons to create multiresis...
Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the ␥-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general genecapture system for bacterial innovation. T he impact of lateral gene transfer on bacterial evolution is underscored by the realization that foreign DNA can represent up to one-fifth of a given bacterial genome (1). Perhaps the most striking embodiment of its affect on microbial adaptation has been the rapid and widespread emergence of similar antibiotic-resistance profiles among phylogenetically diverse Gram-negative clinical and environmental isolates over the last half-century (2). The localization of antibiotic-resistance determinants to mobile entities such as plasmids and transposons readily explained this phenomenon (3-6). Closer examination revealed that in many cases a new type of genetic element, termed an integron, harbored the resistance determinants. Integrons are natural cloning and expression systems that incorporate open reading frames and convert them to functional genes (for review see refs. 7 and 8). The integron platform codes for an integrase (intI) that mediates recombination between a proximal primary recombination site (attI) and a secondary target called an attC site [or 59-base element (59be)]. The attC site is normally found associated with a single open reading frame (ORF), and the attC-ORF ...
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.
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-؋-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-؋-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-؋-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.
Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid (ccd F ) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain (ccd O157 ). The ccd F and the ccd O157 systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the ccd F system. We have shown that the chromosomal ccd O157 system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB O157 toxin targets the DNA gyrase, and the CcdA O157 antitoxin is degraded by the Lon protease. The ccd O157 chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of ccd F . ccd O157 is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccd F system is able to mediate postsegregational killing in an E. coli strain harboring the ccd O157 system in its chromosome. This shows that the plasmidic ccd F system is functional in the presence of its chromosomal counterpart.Toxin-antitoxin (TA) proteic systems were originally discovered on low-copy-number plasmids (for reviews on TA systems, see references 11, 22, 24, and 29). They are composed of two genes organized in an operon encoding a toxin and an antitoxin that antagonizes it. The expression of the TA genes is autoregulated at the transcriptional level; the antitoxin acts as a repressor and the toxin often as a corepressor. The antitoxin is an unstable protein degraded by an ATP-dependent protease, while the toxin is a stable protein that inhibits an essential cellular process (e.g., replication and translation). TA systems contribute to plasmid stability by a mechanism called postsegregational killing (PSK). PSK relies on the differential stabilities of the antitoxin and toxin proteins and leads to the killing of daughter bacteria that did not receive a plasmid copy at cell division (31, 50, 53).Recent computational analyses have shown that TA systems are widely represented in eubacterial and archaebacterial chromosomes, suggesting a role for horizontal gene transfer in the spread of these genes (5, 6, 38). The localization of chromosomal TA systems is quite varied. Some are localized within exogenous DNA islands like phages (relBE K-12 in the cryptic lambdoid Qin prophage of Escherichia coli MG1655) (40), transposons (relBE homolog in Tn5401 of Bacillus thuringiensis (23), and superintegrons (relBE, parDE, phd-doc, and hig...
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