The emergence and spread of antibiotic resistance among bacterial pathogens is the most striking exam- ple of evolution that has been observed in bacteria over the past six decades. Indeed, antimicrobial resist- ance has been an impediment to effective infectious disease therapy for as long as antibiotics have been used. Single-drug-resistance phenotypes were not entirely unforeseen, as was demonstrated in early lab- oratory studies. By contrast, multidrug resistance was not anticipated, because the co-appearance of multiple mutations conferring resistance was considered to be beyond the evolutionary potential of a given bacterial population. However, only six years after the intro- duction (and massive production) of streptomycin, tetracycline and chloramphenicol, isolates of Shigella dysenteriae that were simultaneously resistant to each of these antibiotics, and to sulphonamides, were identified1. At this time, it also became clear that the emergence of multidrug-resistant strains could not be attributed to mutation alone. In the 1970s, multidrug resistance was determined in many cases to be associ- ated with transmissible plasmids, and the importance of integrons in the acquisition of resistance genes was only recognized much later, in the late 1980s (REF. 2.) It is clear, however, that integrons contributed to the initial outbreaks of multidrug resistance in the 1950s, as indicated by the involvement of Tn21, an integron- containing transposon, in the resistance phenotype propagated by plasmid NR1 (R100)3. Given the time scale of the development of multidrug-resistant Shigella strains, it is clear that bacteria were prepared for such a challenge and had already evolved the appropriate genetic tools, including integrons. What is an integron? Integrons are assembly platforms that incorporate exogenous open reading frames (ORFs) by site-specific recombination and convert them to functional genes by ensuring their correct expression. All integrons character- ized to date are composed of three key elements necessary for the capture of exogenous genes: a gene (intI) encod- ing an integrase belonging to the tyrosine-recombinase family a primary recombination site (attI) and an out- ward-orientated promoter (Pc) that directs transcription of the captured genes4. Integron-encoded integrases can recombine discrete units of circularized DNA5 known as gene cassettes in a RecA-independent manner. Integration occurs downstream of the resident Pc promoter at the attI site, allowing expression of the genes in the cassette (FIG. 1). All integron-inserted gene cassettes identified share spe- cific structural characteristics and generally contain a single gene and an imperfect inverted repeat at the 3��� end of the gene called an attC site (or 59-base element). The attC sites are a diverse family of nucleotide sequences that function as recognition sites for the site-specific integrase. They vary in length from 57 bp to 141 bp6, and their nucle- otide sequence similarities are primarily restricted to the boundaries, which contain conserved sequences known as the R������ sequence (RYYYAAC, where R is a purine and Y is a pyrimidine) and the R��� sequence7 (GTTRRRY, where the point of recombination is between the G and the T bases) (FIG. 1). When integrons were first described, the definition of an integron suggested that the integron element itself was a mobile DNA element2. This assumption stemmed from the fact that the first integrons to be characterized were located in transposons. In these examples, the Unit�� Plasticit�� du G��nome Bact��rien- CNRS URA 2171, Department G��nomes et G��n��tique, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, France. e-mail:mazel@pasteur.fr doi:10.1038/nrmicro1462 Transposon A mobile DNA element that can relocate within the genome of its host. Integrons: agents of bacterial evolution Didier Mazel Integrons are assembly platforms ��� DNA elements that acquire open reading frames embedded in exogenous gene cassettes and convert them to functional genes by ensuring their correct expression. They were first identified by virtue of their important role in the spread of antibiotic-resistance genes. More recently, our understanding of their importance in bacterial genome evolution has broadened with the discovery of larger integron structures, termed superintegrons. These DNA elements contain hundreds of accessory genes and constitute a significant fraction of the genomes of many bacterial species. Here, the basic biology of integrons and superintegrons, their evolutionary history and the evidence for the existence of a novel recombination pathway is reviewed. REVIEWS 608 | AUGUST 2006 | VOLUME 4 www.nature.com/reviews/micro �� 2006 Nature Publishing Group

intI1 aacA4 qacF cmIA2 oxa9 sul1 qacE��� Integrase 3��� conserved segment attC sites VCR infC IF3 L35 L20 Integrase rplT intIA rpmI (130 kb, 3% of the genome, 176 cassettes) Variable region, 45���128 nucleotides RYYYAAC R������ GTTRRRY R��� 85% identical, 114���116 nucleotides RYYYAAC GTTRRRY R������ R��� attI site attC sites VCR (Vibrio cholerae repeats) a b Insertion sequence (IS) A small (2.5 kb), generally phenotypically cryptic segment of DNA that has a simple organization and is capable of insertion at multiple sites in a target DNA molecule. Examples include IS1, IS608 and IS911. Conjugative plasmid A plasmid that can move from one cell to another during the process of conjugation. SXT element Vibrio cholerae-derived integrating and conjugative element (also referred to as a conjugative transposon or constin). Compound transposon A segment of DNA flanked by two similar insertion sequences, in direct or inverted orientations. Examples include Tn5 and Tn10. transposition of the integrons did not depend on the activity of the integron-encoded integrase, which only mobilizes the gene cassettes within integrons. However, with the discovery of other types of integron, either car- ried by transposons or present as immobile components of bacterial genomes, the definition of an integron has evolved towards the definition outlined at the start of this section. All integrons can be divided into two distinct subsets: the mobile integrons, which are linked to mobile DNA elements and are primarily involved in the spread of antibiotic-resistance genes and the superintegrons. Mobile integrons At present, five classes of mobile integrons are known to have a role in the dissemination of antibiotic-resistance genes. These classes have been historically defined based on the sequence of the encoded integrases, which show 40���58% identity. All five classes are physically linked to mobile DNA elements, such as insertion sequences (ISs), transposons and conjugative plasmids, all of which can serve as vehicles for the intraspecies and interspecies transmission of genetic material. Three classes of mobile integrons are ���historical��� classes that are involved in the multiple-antibiotic-resistance phenotype8. Class 1 inte- grons are associated with functional and non-functional transposons derived from Tn402 (REFS 9,10) that can be embedded in larger transposons, such as Tn21. Class 2 integrons are exclusively associated with Tn7 deriva- tives9,11, and class 3 integrons are thought to be located in a transposon12 inserted in as-yet-uncharacterized plasmids13���15. The other two classes of mobile integrons, class 4 and class 5, have been identified through their involvement in the development of trimethoprim resist- ance in Vibrio species one (class 4) is a component of a subset of SXT elements found in Vibrio cholerae16, and the other (class 5) is located in a compound transposon carried on a plasmid in Vibrio salmonicida (H. S��rum et al., unpublished observations). Class 1 integrons are found extensively in clinical isolates, and most of the known antibiotic-resistance- gene cassettes belong to this class. To date, and con- sidering only those cassettes that differ in nucleotide sequence by more than 5%, over 80 different gene cassettes from class 1 integrons have been described. Between them, these elements confer resistance to all known ��-lactams, all aminoglycosides, chlorampheni- col, trimethoprim, streptothricin, rifampin, erythro- mycin, fosfomycin, lincomycin and antiseptics of the quaternary-ammonium-compound family (reviewed in REFS 17,18). By contrast, only six different resist- ance cassettes have been found that are associated with class 2 integrons19,20. This reduction in diversity is probably owing to the fact that the gene encoding the integrase in class 2 integrons contains a nonsense mutation in codon 179 (ochre 179), thereby yielding a truncated, non-functional protein21. Mutation of the ochre 179 codon into a glutamic-acid-encoding codon is sufficient to produce an integrase with full recom- binase activity21. However, it is not known whether the differences in cassette content in the different Tn7 derivatives are due to occasional natural suppression of the ochre 179 codon, leading to an active integrase, or due to the trans-acting recombination activity of another integrase, such as the class 1 integrase (IntI1), Figure 1 | Mobile integrons and superintegrons. Structural comparison of a ���classical��� mobile integron and the superintegron from Vibrio cholerae strain N16961. a | A schematic representation of the class 1 integron In40. The various resistance-gene cassettes carry different attC sites. The following antibiotic-resistance cassettes confer resistance to the following compounds: aacA4, aminoglycosides cmlA2, chloramphenicol oxa9, ��-lactams qacF and qacE, quarternary ammonium compounds. The sul gene, which confers resistance to sulphonamides, is not a gene cassette. b | A schematic representation of the chromosomal superintegron in V. cholerae the open reading frames are separated by highly homologous sequences, the V. cholerae repeats (VCRs). infC, encodes translation initiation factor IF3 rpmI and rplT encode ribosomal proteins L35 and L20, respectively. REVIEWS NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | AUGUST 2006 | 609 �� 2006 Nature Publishing Group