Integrons and gene cassettes: a genetic construction kit for bacteria

Peter M. Bennett

Bristol Centre for Antimicrobial Research and Evaluation (BCARE), University of Bristol, Department of Pathology and Microbiology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK

The battle between humans and microbes has been waged since time immemorial. For a brief period, following the discovery and development of the main groups of antibiotics, mankind appeared to gain the upper hand against the legion of bacteria responsible for much morbidity and mortality among humans and their domestic animals, but the advantage was illusory, because the bacteria were quietly and efficiently evolving and acquiring resistance genes and resistance gene arrays that provided them with protection against the pharmacopoeia of antibiotics deployed to contain them.

Bacteria constantly surprise us and no more so than within the arena of antibiotic resistance. Bacteria have access, in principle, to a large selection of resistance genes scattered throughout the bacterial kingdom and mechanisms have evolved to reassort these genes, moving them genetically from one DNA molecule to another and physically from one bacterial cell to another. Indeed, bacteria can be considered to have access to a comprehensive genetic engineering tool kit that provides the potential to remodel and to mix and match resistance genes according to requirements. Our experience over the last 20–30 years indicates that this tool kit is in frequent use.

Genetic mobility involves a variety of mechanisms that have evolved to move DNA sequences from one cell to another and from one DNA molecule to another. 1 The former type involves conjugation, transformation and transduction mechanisms, all of which have been demonstrated to be important in the movement of resistance genes among clinically important bacteria. The latter systems comprise different recombination mechanisms: (i) classical recombination, which is RecA-dependent and requires extensive homology between the recombining DNA molecules; this mechanism has been largely responsible for the development of the penicillin-binding protein gene mosaics in penicillin-resistant Streptococcus pneumoniae and penicillin-resistant Neisseria gonorrhoeae; (ii) transposition, which normally involves a discrete transposable element and requires no homology between the recombining sequences; many drug resistance transposons have been described in both Gram-positive and Gram-negative bacteria; (iii) site-specific recombination, which involves recombination between short homologous sequences mediated by recombination enzymes that are specific for the particular recombination site and which achieves directed insertion of a resistance gene or genes.1 This last mechanism has been shown to be involved in the dissemination of resistance genes via elements termed integrons and gene cassettes,2 the latest genetic structures to be added to the list of elements involved in the spread of drug resistance genes.

An integron (Figure 1) is defined as a genetic element that possesses a site, attI, at which additional DNA, in the form of gene cassettes, can be integrated by site-specific recombination, and which encodes an enzyme, integrase, that mediates these site-specific recombination events. Gene cassettes are discrete genetic elements that may exist as free, circular, non-replicating DNA molecules when moving from one genetic site to another,3 but which are normally found as linear sequences that constitute part of a larger DNA molecule, such as a plasmid or bacterial chromosome. Gene cassettes normally contain only a single gene and an additional short sequence, called a 59 base element, that functions as a specific recombination site.4 Accordingly, the cassettes are small, normally of the order of 500–1000 bp. The genes carried on gene cassettes usually lack promoters and are expressed from a promoter on the integron. 5 ,6 In rare instances a cassette may carry two genes; these exceptions are likely to have been generated by the fusion of two individual cassettes, which at one time were side-by-side, the double gene cassette being generated by a deletion that removed sequences on either side of the joint boundary, including the 59 base element that was located at the end of the first gene, i.e. the one that reads towards where the joint boundary was.7



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Figure. Class 1 integron structure (see text for explanation).

 
Integrons were discovered as the result of systematic examination of resistance plasmids and transposons that carry overlapping sets of resistance genes but that otherwise are unrelated. 5 ,8 ,9 ,10 Although only identified as discrete, defined structures relatively recently, integrons and their associated components, gene cassettes, are known to have been constituents of the first resistance plasmids reported in the mid 1960s. That apparently unrelated transposons and resistance plasmids of different incompatibility (Inc) groups might possess limited regions of sequence homology at their resistance gene loci, even though the resistance genes may be different, was first suggested as a result of comparisons of restriction endonuclease mapping data. 5 ,8 Subsequently, comparisons of the nucleotide sequences flanking the resistance genes on transposon Tn21, on plasmid R100 (an IncFII type plasmid), and on a number of broad host-range R plasmids, notably R46 (IncN), R388 and Sa (both IncW) and R751 (IncP) revealed that the immediate nucleotide contexts of several of the resistance genes were very closely related, even though the resistance genes were different. 4 ,11 Resistance gene loci commonly comprise two highly conserved regions flanking a variable region, within which are located the resistance genes. The homologous sequences may flank either a single resistance gene or a set of two or more resistance genes and are referred to as the 5'-conserved region (5'-CR), since these sequences are found upstream from (i.e. preceding) the resistance gene(s), and the 3'-conserved region (3'-CR), which is found downstream of (i.e. following) the resistance gene(s). The 5'-CR is approximately 1.4 kb long, while the 3'-CR is 2 kb.4

Sequencing analysis also revealed that immediately downstream of each resistance gene there was a copy of what appeared to be essentially the same short sequence which, because of its size, was called the 59 base element.4 In fact, these short sequences are not identical, but each has a core sequence that is readily identified. The punctuation of sets of resistance genes by copies of the 59 base element indicated that blocks of resistance genes had been assembled by stepwise accretion of single resistance genes by a common mechanism. The association of a short specific sequence, i.e. a copy of a 59 base element, with each resistance gene suggested that site-specific recombination, as used by bacteriophage lambda to integrate into the Escherichia coli chromosome, was involved in the construction of resistance gene arrays. This interpretation was given substantial credibility when sequence analysis of the 5'-CR copy on Tn21 identified a gene encoding a protein predicted to be a member of the lambda integrase family.12 Further analysis of this gene, intI, and its product confirmed that IntI is indeed a site-specific recombinase and that one of its substrates is a copy of a 59 base element present on a gene cassette. The second substrate is the cassette receptor, or integration, site, attI, on the integron. 13 ,14 ,15 ,16

Integration of bacteriophage lambda into the E. coli chromosome, a step leading to lysogeny, involves a circular form of the bacteriophage DNA and two specific short nucleotide sequences, one (attP) on the phage genome and the other (attB) on the bacterial chromosome. 1 A single recombinational crossover within a short core sequence common to attP and attB, mediated by the bacteriophage-encoded enzyme integrase, integrates lambda, as a linear sequence, into the bacterial chromosome. A reversal of this recombination, mediated also by integrase plus another bacteriophage function, called excisionase (encoded by the bacteriophage gene xis), using the hybrid recombination sites generated by the integration event, excises the phage genome precisely from the chromosome and releases it as the original circular DNA molecule. This frees bacteriophage lambda to replicate independently of the bacterial chromosome and marks the transition from lysogeny to lytic phage growth. Integration of lambda into and excision from the E. coli chromosome represent the paradigm for site-specific recombination.1 The acquisition and loss of gene cassettes by integrons is an exact parallel, except that both forward and reverse reactions are mediated by the same enzyme, IntI, and that gene cassettes do not replicate as independent DNA molecules.

Three distinct classes of integrons, each of which shows the distinctive features of an integron (integrase gene, gene cassette receptor site and a promoter for genes carried on the cassettes) have been reported. 2 Class 1 accommodates the majority of integrons found in clinical isolates to date and members of this group were those originally classified as integrons. These elements constitute the most intensively studied integrons and are the only group for which gene cassette movement has been demonstrated experimentally. Class 2 includes an integron system found on transposon Tn7 and related elements. The integron on Tn7 possesses three integrated gene cassettes adjacent to a defective integrase gene, and lacks the 3'-CR. Class 3 is represented by a single example.

The basic integron,17 designated In0, of class 1 is a structure comprising the 5'-CR joined directly to the 3'-CR with no intervening variable region, i.e. In0 lacks an integrated gene cassette (Figure 1). The essential features of the structure are the integrase gene, intI, the site for insertion of gene cassettes, attI, which is located beside intI, 3' to the gene, and a promoter, which is located within intI and which directs transcription towards the integration site, from which genes carried on cassettes are expressed. These features are all accommodated in the 5'-CR. The 3'-CR encodes resistance to sulphonamides, mediated by the sul1 gene, and has a truncated version of the detergent resistance gene qacE, qacE{Delta}1, and two open reading frames, orf5 and orf6, encoding proteins of unknown functions. Acquisition of one or more cassettes by sequential insertion at attI creates new integrons (Figure). Each unique arrangement is given a new number; for example, In2 encodes a single resistance gene, aadA1a, which specifies an aminoglycoside modifying enzyme, while In1 encodes resistance to aminoglycosides and ß-lactams mediated by the products of resistance genes aadA1b and oxa2, respectively. 17

A class 1 integron has, therefore, the following formal arrangement:

where r59b represents a resistance gene cassette and n indicates the number of gene cassettes integrated at the locus.

Class 1 integrons are found at many different sites on many unrelated plasmids, on transposons such as Tn21 and on bacterial chromosomes, implying that these elements are, or have been mobile. Given that there is little or no homology between the sites where they have been found, they probably move by transposition. However, only one integron is known to be an active transposon, Tn402. The complete sequence of this element indicates it is a class 1 integron. 18 In contrast, the majority of class 1 integrons found so far appear to be rearranged, transposition-defective derivatives of Tn402-like ancestral elements. 19

New integrons are generated by the insertion of new gene cassettes at attI, or the deletion of one or more cassettes from an existing integron. 20 ,21 Cassette insertion into an integron appears always to be into the cassette receptor site attI, while any cassette, or combinations of cassettes, can be deleted by site-specific recombination between any one 59 base element and either attI or a second 59 base element. Many resistance genes found in clinical isolates of Gram-negative bacteria are parts of gene cassettes. 7 ,22 The set of gene cassettes currently known accommodates genes that confer resistance to many different antibiotics, including aminoglycosides, ß-lactams, chloramphenicol and trimethoprim, as well as to antiseptics and disinfectants. So far more than 40 cassettes have been identified, and it is a reasonable expectation that many more will be discovered in the future. 7 ,23 All but five of the known cassettes encode resistance determinants,7 but it is not thought that this is a true reflection of the gene variety carried on these elements; rather it is likely to be a reflection of experimental selection bias due to our preoccupation with resistance genes.

The movement of gene cassettes in and out of integrons is a random process, like most other genetic rearrangements in bacteria. Which arrangements survive and which are lost is a matter of circumstance and natural selection. If the resistance combination carried by a particular integron is of advantage to the bacterium that harbours it, then that clone will be selected and that particular integron will be amplified by clone amplification. Integrons were constituents of the first resistance plasmids reported, conferring resistance to aminoglycosides, chloramphenicol and sulphonamides. One of the most recent additions to the list of drug-resistance gene cassettes is one with blaIMP, 24 ,25 a gene which encodes a metallo-ß-lactamase conferring resistance to carbapenems such as imipenem. This has been reported in many clinical isolates of Serratia marcescens from many hospitals in Japan. It is unlikely to be the last word on gene cassettes and integrons in terms of drug resistance. Watch this space!

References

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19 . Brown, H. J., Stokes, H. W. & Hall, R. M. (1996). The integrons In0, In2, and In5 are defective transposon derivatives. Journal of Bacteriology 178, 4429–37.[Abstract]

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24 . Ito, H., Arakawa, Y., Ohsuka, S., Wacharotayankun, R., Kato, N. & Ohta, M. (1995). Plasmid-mediated dissemination of the metallo-ß-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. Antimicrobial Agents and Chemotherapy 39, 824–9.[Abstract]

25 . Arakawa, Y., Murakami, M., Suzuki, K., Ito, H., Wacharotayankun, R., Ohsuka, S. et al. (1995). A novel integron-like element carrying the metallo-ß-lactamase gene blaIMP. Antimicrobial Agents and Chemotherapy 39, 1612–5.[Abstract]