Integrons: past, present, and future

Abstract

Integrons are versatile gene acquisition systems commonly found in bacterial genomes. They are ancient elements that are a hot spot for genomic complexity, generating phenotypic diversity and shaping adaptive responses. In recent times, they have had a major role in the acquisition, expression, and dissemination of antibiotic resistance genes. Assessing the ongoing threats posed by integrons requires an understanding of their origins and evolutionary history. This review examines the functions and activities of integrons before the antibiotic era. It shows how antibiotic use selected particular integrons from among the environmental pool of these elements, such that integrons carrying resistance genes are now present in the majority of Gram-negative pathogens. Finally, it examines the potential consequences of widespread pollution with the novel integrons that have been assembled via the agency of human antibiotic use and speculates on the potential uses of integrons as platforms for biotechnology.

FIG 1

Integron structure. The basic integron platform consists of the following: intI, a gene for the integron integrase; Pc, an integron-carried promoter; attI, the integron-associated recombination site; and gene cassettes, sequentially inserted into an array via recombination between attI and the cassette associated-recombination sites, attC. (A) Gene cassettes normally contain a single open reading frame (ORF) (arrow) expressed from the Pc promoter. In some integrons, Pc lies between intI and attI. (B) Cassettes with two ORFs, no ORF, or an ORF in the reverse direction are known. In some genera, intI is transcribed in the same direction as the gene cassettes. (C) Gene cassettes may also contain internal promoters.

FIG 2

Acquisition of gene cassettes. Integrons acquire new gene cassettes by recombination between the attC of a circular cassette and the attI site of the integron. This inserts incoming cassettes at a position proximal to the integrase gene and its embedded promoter. Cassette arrays can expand by repeated cassette acquisition, but cassettes can also be excised as closed circles by attI × attC or attC × attC recombination.

FIG 3

Structure of gene cassettes and associated recombination sites. (A) A single gene cassette is shown in linear form, inserted into a cassette array. From left to right, the salient features are as follows: the conserved recombination site, GTTRRY, with the vertical arrow showing the recombination point; the start codon and open reading frame encoded by the cassette; and the attC site, containing integrase binding domains R″, L″, L′, and R′. (B) Detailed structure of a single attC site. These elements have partially palindromic sequences (labeled with letters), such that R″ can pair with R′ and L″ can pair with L′, thus forming a stable cruciform structure recognized by integron integrases. An extra base, labeled with an asterisk in L″, ensures correct orientation and insertion of cassettes into the array. Between the terminal palindromic regions is a region that varies in length (16 to 109 nucleotides [nts]) and sequence between different cassettes. This region is also capable of forming a stable secondary structure, and the lack of sequence conservation suggests that structure, rather than sequence, is important for recognition. (C) An attI site from a class 1 integron. The attI1 site also has L and R elements, with the conserved recombination point G↓TTRRRY. The attI of class 1 integrons also has two direct repeats, DR1 and DR2, but these are not known from the attI sites of other integron classes.

FIG 4

Conserved sequence boundaries of chromosomal class 1 integrons. Schematic maps of a chromosomal class 1 integron as found in betaproteobacteria and after its insertion into a Tn402 transposon are shown. Symbols are as Fig. 1 and 2. Additional features: IRi and IRt are the 25-bp terminal inverted repeats of the Tn402 transposon, and the tni module contains genes involved in Tn402 transposition activity. Both the left- and right-hand boundaries of the class 1 integron demonstrate precise sequence breakpoints. Sequences in the top alignment, showing the left-hand boundary, include relevant regions from the chromosomal class 1 integron from Hydrogenophaga PL2G6 (accession no. EU327989) (A), the chromosomal class 1 integron from Aquabacterium PL1F5 (accession no. EU327988) (B), the chromosomal class 1 integron from Acidovorax MUL2G8 (accession no. DQ372710) (C), the chromosomal class 1 integron from Imtechium PL2H3 (accession no. EU327990) (D), the IncP-1 beta multiresistance plasmid pB8, which also carries Tn402 (accession no. AJ863570) (E), plasmid R751 from Enterobacter aerogenes, which carries Tn402, and a clinical-type class 1 integron contained within this transposon (accession no. U67194) (F), Tn6008 from Enterobacter cloacae, which carries sequence typical of Tn402-like transposons up to the CGGCC motif shared with class 1 integrons but carries no class 1 integron sequences beyond that point (accession no. EU316185) (G), and a bla_VIM-1 clinical class 1 integron from Pseudomonas aeruginosa VR-143/97 that has an ISPa7 insertion element inserted at the Tn402/class 1 integron boundary (accession no. Y18050) (H).

FIG 5

Model for the origin and subsequent divergence of the mobile class 1 integrons that are now common in Gram-negative pathogens. (A) The common ancestor of all clinical class 1 integrons was a member of a diverse group of class 1 integrons located on the chromosomes of Betaproteobacteria. (B and C) This chromosomal class 1 integron was captured by the Tn402 transposon (B) to generate a transposon/integron hybrid carrying the qacE cassette, encoding resistance to disinfectants (C). (D) A gene for resistance to sulfonamides, sul1, was then captured, deleting part of the qacE cassette and thus generating the 3′ conserved segment (3′-CS). (E) Deletions and insertions involving tni generated Tn402 transposition-incompetent integrons, while acquisition of further antibiotic resistance cassettes took place, expanding the range of antibiotic resistance phenotypes conferred by integrons. (F) Acquisition of new cassettes continued, and the Tn402-integron hybrid moved onto diverse plasmids and other transposons, such as the Tn21 family. These events generated further diversity and accelerated the penetration of class 1 integrons into a wide variety of pathogens and commensals.

FIG 6

Role of resistance gene pollution in generating novel, complex DNA elements. (A) A typical class 1 integron from human-pathogenic or commensal bacteria. This type of DNA element commonly pollutes aquatic environments. It consists of inverted DNA repeats IRi and IRt, the class 1 integron integrase gene intI1, and a gene cassette, aadA2, which confers streptomycin resistance. The 3′ conserved segment consists of fused genes for disinfectant and sulfonamide resistance (qacEΔ/sul1), ORF5, and the remnants of genes encoding transposition functions (tniΔ). (B and C) In an aquatic environment, such an integron was modified by acquiring a novel gene cassette encoding two methionine sulfoxide reductases (msrB and msrA) (B) and replacing the inverted repeats IRi and IRt with miniature inverted-repeat transposable elements (MITEs) (C). (D) This event generated a compound MITE/integron element. (E) Mobility conferred by the MITEs allowed insertion of the compound integron into a genomic island. (F) This genomic island moved into at least three different species of the genus Acinetobacter, carrying the integron with it. Consequently, resistance determinants released from human waste streams may interact with gene cassettes and mobile DNA elements in aquatic ecosystems to generate new combinations of potential virulence genes in environmental bacteria. The presence of these bacteria in food items provides a readily accessible route for contamination of the food chain and the emergence of novel, virulent pathogens.