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Integrons: mobilizable platforms that promote genetic diversity in bacteria
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Vol.15 No.7
Integrons: mobilizable platforms that promote genetic diversity in bacteria Yan Boucher1, Maurizio Labbate1, Jeremy E. Koenig2 and H.W. Stokes1 1
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia Department of Biochemistry and Molecular Biology, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, B3H 1X5, Canada
2
Integrons facilitate the capture of potentially adaptive exogenous genetic material by their host genomes. It is now clear that integrons are not limited to the clinical contexts in which they were originally discovered because »10% of bacterial genomes that have been partially or completely sequenced harbour this genetic element. This wealth of sequence information has revealed that integrons are not only much more phylogenetically diverse than previously thought but also more mobilizable, with many integrons having been subjected to frequent lateral gene transfer throughout their evolutionary history. This indicates that the genetic characteristics that make integrons such efficient vectors for the spread of antibiotic resistance genes have been associated with these elements since their earliest origins. Here, we give an overview of the structural and phylogenetic diversity of integrons and describe evolutionary events that have contributed to the success of these genetic elements. A simple structure leading to much diversity A bacterium harbouring an integron can capture and express genes found in its environment as part of small mobile elements, termed gene cassettes. Integrons were first discovered in clinical contexts, where they included short arrays of gene cassettes, the genes of which mostly encode antibiotic resistance determinants. These clinically important integrons, now known as class 1, 2 and 3 integrons, are by far the most studied and were used as a model to develop a formal structural definition for these genetic elements [1] (Box 1). These integrons share their core structure with all other integrons, which can be minimally defined as an integrase gene (intI) with a contiguous recombination site (attI). This simple core structure makes the integron an efficient platform for the site-specific integration of novel genetic material and potentially adaptive traits, without disruption to the genome of the recipient. Integrons are not rare genetic elements. After surveying all 603 completely or partially sequenced genomes, we found that 56 (9%) of them harboured integrons (Figures 1 and 2). The integrons found in these diverse organisms display as much variation in the evolutionary paths they have followed as their hosts do. This is most evident when looking at the integrons that have been more thoroughly studied. These include those frequently found in a clinical Corresponding author: Stokes, H.W. ([email protected]). Available online 12 June 2007. www.sciencedirect.com
context (mostly classes 1, 2 and 3) in addition to some of those found in bacteria recovered from natural environments (i.e. Vibrio, Pseudomonas and Xanthomonas). We briefly describe the characteristics of these well-known integrons from clinical and environmental settings before using a phylogeny of integron integrases to describe the evolutionary history that enabled these elements to succeed in a wide variety of hosts and environments. The evolution of integrons associated with antibiotic-resistance determinants are discussed in more detail, given their clinical relevance and the amount of literature available. Finally, we ask whether we can classify integrons to improve our understanding of these elements and, furthermore, briefly discuss their adaptive potential. It is important to get a firm grasp of the evolutionary processes that shaped integron diversity if we hope to understand how they can contribute to the rapid spread of novel phenotypes among bacteria (such as antibiotic resistance). The presence of integrons in close to 10% of the vast number of bacterial genome sequences now available makes it clear that these elements can no longer be perceived as an interesting oddity and that they need to be integrated into our models of microbial evolution. Variation on a theme: multiple evolutionary routes for integrons The first integrons to be described, although homologous, do not share recent common ancestry and were ascribed to different classes (1, 2 and 3) based on their respective intI gene sequences. The ‘class’ system has since been used as a loose taxonomic scheme, different classes simply representing evolutionarily divergent integrons showing substantial differences in the sequences of their intI genes (Box 2). Although integrons belonging to classes 1, 2 and 3 are not close evolutionary relatives, they nonetheless share several important features. They are contained in transposons and plasmids and found to include small arrays of up to six or so cassettes. They also share a collective genecassette pool, which is known to be small in relative terms (containing little more than 100 distinct cassettes) and almost uniquely comprising antibiotic-resistance determinants [2]. The association of these integrons with mobile elements and resistance genes has led to their rapid dispersal among various bacteria found in environments exposed to antibiotics [3,4]. Class 1, 2 and 3 integrons are most commonly embedded in diverse and highly mobile elements (e.g. various plasmids
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Box 1. What is an integron? As shown in Figure I, integrons are one part of a two-component system that capture disparate individual genes and physically link them in arrays. The first component of this system is the ‘core’ integron, which includes a gene (intI) encoding a site-specific recombinase (IntI) and a recombination site (attI). The second part of the system comprises a family of mobile elements known as gene cassettes. Individual cassettes usually consist of a single gene and a second type of recombination site. This cassette-associated recombination site comprises diverse sequence families, which all share a common structure. The original name given to these sites was 59base element (59-be), although the term attC has also been commonly used to describe them in recent years. Gene cassettes are captured by the integron via an IntI-mediated site-specific recombination reaction between the 59-be and attI sites [1,14,23,33,34]. During this reaction, IntI monomers bind preferentially to a single DNA strand of a gene cassette 59-be site. This single-stranded 59-be site adopts a folded structure to generate a double-stranded recombination site, which recombines with a double-stranded attI site that is also bound by IntI monomers [35,36]. Although it is widely assumed that a cassette promoter (Pc) found within the intI gene or attI site is likely to be a universal feature of integrons, its presence has only been confirmed for class 1 and class 3 integrons [33] and for an integron from Pseudomonas stutzeri [32]. In these integrons, cassette-associated genes lack their own promoter and rely on Pc for expression of the genes they contain. Multiple insertion events at an attI site are common and cassette-associated genes are usually inserted in the same orientation, producing tandem arrays of co-transcribed genes expressed from Pc. Although the integrase gene is usually in the opposite orientation to that of cassette-encoded genes, some integrons display an integrase in the same orientation as the genes in its associated cassette array, and these are termed inverted integrase integrons.
Figure I. The integron–gene cassette site-specific recombination system.
and transposons) and, thereby, have become broadly distributed amongst the Gram-negative bacteria [1] and even, in some cases, to Gram-positive bacteria [5,6]. Most other integron classes, however, are confined to chromosomes, the first examples of which were uncovered in the genus Vibrio [7]. Vibrio integrons are associated with large gene-cassette arrays (from 50 to >200 cassettes) and seem to have evolved mostly through vertical inheritance with limited lateral gene transfer (LGT) of the integron core [8]. The large arrays found in vibrios often contain gene cassettes encoding toxin-antitoxin systems [9]. Large arrays can be susceptible to some forms of genetic rearrangement, for example, the Vibrio fischeri ES114 array is split into two segments, one on each chromosome [10]. The extent of the Vibrio gene-cassette pool diversity is unknown, but is nonetheless orders of magnitude larger than the collective pool of cassettes found in class 1, 2 and 3 integrons. An interesting feature of some Vibrio integrons is that many of their cassettes contain no apparent open reading frame (ORF). These non protein-coding cassettes are often paralogous: a single family of such cassettes has 34 copies in Vibrio vulnificus YJ016 and 45 copies in V. vulnificus CMCP6 [11,12]. Vibrio gene cassettes are frequently transferred www.sciencedirect.com
across short phylogenetic distances but much more rarely across larger distances [11]. Such frequent short-range cassette movement has resulted in highly variable arrays in vibrios. For example, the arrays of the V. vulnificus strains CMCP6 and YJ016 show little similarity in cassette composition or order [11,12]. Integrons found in the genus Pseudomonas have also been studied in some detail. They are only present in a small subset of the many species belonging to this genus and are, therefore, likely to have been acquired by LGT late in its evolutionary history [13]. The number of cassettes Pseudomonas integrons contain varies with as little as ten gene cassettes in a strain of Pseudomonas stutzeri and >32 cassettes in Pseudomonas alcaligenes. The latter bacterium harbours multiple cassettes arrays; only one has been characterized (containing 32 cassettes), the other arrays being found either proximal or distal to that which is found in the P. alcaligenes genome [13]. Pseudomonas integrons also show large variability in the composition of their cassette arrays, with even otherwise closely related strains of a single species displaying little similarity in their cassette array content [14].
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Figure 1. Phylogenetic tree of the RpoB proteins found in all bacteria that harbour integrons. Proteobacteria are colour-coded according to the Order to which they belong. For species that harbour an integron but for which no RpoB sequence is available, a RpoB sequence from a closely related species was used as a substitute. Only species for which most of the gene-cassette array had been sequenced were included. The tree and bootstrap support values were inferred by maximum likelihood using PHYML [37].
Xanthomonas integrons are also well studied. Their array size is comparable to those of pseudomonads (1–22 cassettes), but the integron integrase is frequently inactivated, commonly by the insertion of a transposon. Similarly, genes within cassettes have frequently suffered from such insertions [15]. Another feature of Xanthomonas www.sciencedirect.com
integron arrays is a low degree of inter-strain variability, possibly a result of the loss of integrase activity leading to fixation of arrays [15]. The integrons described here constitute only a small portion of integron diversity (Figure 2). Despite this, it is obvious that substantial variation has occurred during
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Figure 2. Phylogenetic tree of known integron integrases (IntI). A single integron integrase is included for each bacterial species, provided that all IntI from that species cluster together in a preliminary analysis. Only species for which most of the gene-cassette array has been sequenced are included. Colour-coding is the same as that used in Figure 1. Black boxes indicate integrons that are associated with antibiotic resistance gene cassettes, with the particular cassette identified in the box. Class 1, 2 and 3
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Box 2. In search of a classification system for integrons So far, integrons have been mostly classified through the sequence similarity of their intI genes (class 1, 2, 3, SXT ICE and pRSV1) or taxonomic group to which their host belongs (i.e. Xanthomonas, Vibrio or Pseudomonas). Both of these approaches have serious limitations. The degree of nucleotide-sequence identity required to be part of an integron class has never been clearly defined in the literature, but was assumed to be similar to what is used to differentiate antibiotic resistance genes (98% nucleotide-sequence identity). We can now recognize many groups of integrons that are evolutionarily related but between their intI genes share variable degrees of sequence similarity (Xanthomonas, Vibrio, Pseudomonas). Trying to define integron groups such as the latter through an association with a particular lineage can also be misleading. For example, the integrons found in Vibrio fischeri species are clearly divergent from other Vibrio integrons but would be classified in one group if taxonomic affiliation was used as the main criteria. A naturalistic classification scheme based on phylogeny might be the best solution, avoiding some of the classification issues associated with frequent lateral gene transfer (LGT) while also considering the evolutionary history of integrons. Three broad groups of integrons can be defined based on phylogenetic considerations, which also agree with other characteristics (genetic structure, environmental distribution and/or taxonomic affiliation of the host): (i) the soil/freshwater proteobacteria group, (ii) the inverted integrase group, and (iii) the marine g-proteobacteria group. Other smaller groups, for which various characteristics are consistent with phylogeny of the integrase gene, can also be defined. The previously described Pseudomonas, Xanthomonas and Vibrio integrons types would all be valid under a phylogenybased definition, with a minor modification for the Vibrio: the V. fischeri integron type would have to be excluded from that group because of its uncertain ancestry. Class 1, 2 and 3 integron types would also be valid, as long as their definition can be broadened to include those associated with different types of mobile elements and association with any type of gene cassettes.
integron evolution, yielding a range of features conserved in specific integron lineages. In summary, the degree of mobility, phylogenetic distribution, frequency in a given taxonomic group, amount of coding content in cassettes, diversity and size of the gene-cassette pool, number of associated cassettes, rate of gene-cassette acquisition or loss, and degree of cassette paralogy in the array are all variables that enable integrons to change and adapt to different functional niches in various host organisms. Integrons have been frequently mobilized throughout their evolutionary history If the phylogenetic tree of integron integrases (IntI) is compared with the tree obtained for a molecular marker believed to be a good approximation of an ‘organismal’ phylogeny, such as the RNA polymerase subunit B gene (rpoB) [16], it becomes clear the history of IntI is not one of simple vertical inheritance (Figures 1 and 2). The many statistically supported inconsistencies between the IntI and RpoB trees points to several events of LGT of integrons across a wide range of phylogenetic distances. Moreover, out of the 50 different species that harbour an integron and for which a complete or partial genome sequence is available, 30 have transposon and/or recombinase genes
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located within their gene-cassette array or flanking their integron. Three main evolutionary groups of integrons are evident from the phylogeny of their integrase genes, two of which have already been identified in a previous study [17] (Figure 2). The first is mostly composed of proteobacterial integrons from freshwater and soil environments and includes class 1 and class 3 integrons (the ‘soil/freshwater proteobacteria’ group). The second group is composed of the integrons of marine g-proteobacteria and includes class 2 integrons in addition to those found in vibrios on the sulfamethoxazole- and trimethoprim-resistant (SXT) integrative and conjugative element (ICE) and pRSV1 plasmid (the marine g-proteobacteria group). The third group, identified here for the first time, is well-supported statistically and includes bacteria from a variety of taxonomic groups but has one distinctive feature: all integrons in this group harbour an integron integrase gene in the same orientation as the genes encoded by their associated cassette arrays (i.e. the attI site is found at the 30 end of the integrase gene). These can be defined as the inverted integrase group (Box 1). It is obvious that LGT has had an important role in the shaping of all of these groups. For example, many close relatives of Pseudomonas harbour an integron (e.g. Congregibacter, Saccharophagus, Marinobacter, Oceanospirillum, Reinekea and Oceanobacter) (Figure 1). However, the integrons of these relatives belong to the marine group, whereas the Pseudomonas integrons belong to the soil/freshwater group. Similarly, Xanthomonas and Nitrosococcus integrons, instead of clustering with other g-proteobacteria such as Vibrionales or Alteromonadales, are more closely related to their b-proteobacterial homologs (Figure 2). The monophyletic group of integrons with inverted integrases includes representatives from multiple bacterial phyla: Proteobacteria, Planctomycetes, Chlorobi, Spirochaetes and possibly Cyanobacteria (the lack of cassettes makes it difficult to say if genuine integrons are present in this last phylum). This indicates multiple lateral transfers across large phylogenetic distances (Figure 2). The marine group possibly has a single origin because it is composed of three related groups of g-proteobacteria: the Vibrionales, Alteromonadales and Pseudomonadalesrelated marine g-proteobacteria (Figure 2). However, none of these three groups is monophyletic within the marine clade, indicating LGT between members of that clade. For example, one of the Marinobacter aquaeolei integrons clusters with Shewanella denitrificans, whereas all other Shewanella integrons cluster with the relative of M. aquaeolei, Oceanobacter sp. RED65 (Figure 2). Even the vibrios, a group for which integrons seem to have followed evolutionary lines similar to their host chromosomes, might have originally acquired this genetic element through LGT. In the RpoB tree, vibrios are sister taxa to the monophyletic Alteromonadales, the latter group including the genera Shewanella, Pseudoalteromonas, Alteromonas and Psychromonas. In the IntI tree, the vibrios cluster with
integrons can contain multiple antibiotic resistance genes. The accession number of each integron integrase is in parentheses next to the taxon name of its host and the number of gene cassettes associated with it is in brackets. The tree and bootstrap support values were inferred by maximum likelihood using PHYML.
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Psychromonas, Pseudoalteromonas and Alteromonas to the exclusion of the Shewanella, making the Alteromonadales polyphyletic. This strongly indicates LGT of integrons between the Vibrionales and Alteromonadales. The vibrios are likely to have been the recipient of this LGT, given their internal branching position with regards to Alteromonadales and a single origin for all of them, although with one possible exception. Indeed, the V. fischeri integrons do not cluster with other Vibrio homologues (Figure 2). However, a lack of statistical support means that a distinction between the possibility that it acquired its integron from another bacterial group (the Pseudomonales-related marine g–proteobacteria) or that it has been through a rapid evolutionary divergence cannot be made. Multiple independent origins for integrons carrying antibiotic-resistance determinants The first integrons to be discovered – class 1 integrons – now account for a substantial proportion of multi-drugresistant nosocomial infections [2]. Several other classes of integrons associated with antibiotic-resistance genes, although much less common than their class 1 relatives, have also been identified. These include class 2 and class 3 integrons, which, like class 1, are closely associated with specific transposons and are found mostly in clinical isolates. They each harbour only a small subset of the resistance cassettes found in class 1 integrons. The integron class associated with the SXT ICE of some V. cholerae [18] and the one linked to the pRSV1 plasmid of V. salmonicida [11] have only been found associated with a single resistance gene cassette (dfrA1). All of these integrons, to some extent, share a genecassette pool that includes many antibiotic-resistance determinants. Despite their sharing of gene cassettes, evolutionary analysis of their respective intI genes indicates independent origins for each of these five classes of clinically relevant integrons. Indeed, close relatives of these five classes of mobilized integrons associated with resistance determinants (class 1, class 2, class 3, SXT and pRSV1) can be found in the genomes of environmental isolates (Figure 2). Some unusual class 1 integrons have been isolated from bacteria found in freshwater sediments [19]. These were undoubtedly related to the better known class 1 integrons found in clinical isolates, sharing 99% or more nucleotidesequence identity in their intI genes and attI sites. However, they are unlike any other class 1 integron because they lack evidence of association with the Tn402 transposition system. Also, although cassettes are present, they do not contain antibiotic-resistance genes. The almost identical nucleotide sequences of the Tn402-associated class 1 integrons and these novel relatives indicate that the latter could be part of an environmental pool of class 1 integrons from which the former originated [19]. The integron from the Vibrio salmonicida pRSV1 plasmid carries a dfrA1 trimethoprim-resistance cassette, the presence of which is probably related to the massive use of trimethoprim in fish farms. This integron is associated with a cassette-encoded IS4-type transposon and also has a close relative bearing no antibiotic-resistance determinants. Pseudoalteromonas haloplanktis TAC125 carries www.sciencedirect.com
an integron on its chromosome that is 99% identical to the pRSV1 integron but harbours unrelated gene cassettes. The integrons are both flanked by the same acyl-carrier protein gene (98% identical), which strongly indicates a common origin. The pRSV1 integron originates from diseased fish of Norwegian coastal waters, whereas P. haloplanktis was isolated from coastal Antarctic waters – two very distant geographical locations. The other three classes of mobilized integrons carrying antibiotic-resistance determinants – class 2, class 3 and SXT ICE – are also strongly related to specific groups of integrons found in environmental microbes, although more distantly. Class 2 integron integrases cluster with integrases of Shewanella species and Oceanobacter sp. (60% amino acid identity) (Figure 2). Class 3 integrases share a recent common ancestor with their class 1 homologues but are more closely related to an integrase from an Acidovorax species isolated from freshwater sediments (69% identity). The integron found in the SXT ICE conjugative element is clearly related to those of the marine organisms Shewanella denitrificans and Marinobacter aquaeolei (55% identity). The origins of these five classes of mobilized integrons bearing resistance cassettes are clearly distinct, both phylogenetically (with high statistical support) and environmentally. This indicates that many integrons found in natural environments have the capacity to carry antibiotic-resistance gene cassettes. If an integron becomes associated with a mobile element that has a wide range of hosts (such as most transposons), it greatly enhances its dispersion potential. This does not seem to be a rare event because it has occurred many independent times in the evolution of integrons (as illustrated by the five different cases presented here). Although integron mobilization can greatly facilitate resistance-cassette dispersal, the latter can still occur without it. Indeed, resistance cassettes have also been found in the non-mobilized chromosomal integrons of Saccharophagus degradans (aacC-A7) [20] and Vibrio cholerae (catB9, blaP7, blaP9) [21–23]. Can we categorize or classify integrons? Since the discovery of integrons harbouring integrase genes with divergent sequences, informal categorization of these elements, based on a variety of criteria, has been taking place (Box 2). Only one scheme, however, has been openly discussed in the literature [17,24–26]. This scheme refers to integrons as ‘mobile’ (also sometimes termed multi-resistant integrons) or ‘super’ (also referred to as chromosomal integrons). ‘Mobile’ integrons are defined as being associated with mobile DNA elements (transposons or plasmids) and antibiotic-resistance genes in addition to having a small array size and substantial heterogeneity in the sequence of their cassettes 59-be sites [17]. ‘Mobile’ integrons would include class 1, 2 and 3 integrons, the integron embedded in the SXT ICE of some V. cholerae and the integron found on the V. salmonicida pRSV1 plasmid (Figure 2). However, both class 1 and pRSV1 integrons have extremely close relatives that do not fit the definition of ‘mobile’ integrons, bringing this definition into question. The Azoarcus sp. MUL2G9 integron is found on the chromosome and is not
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associated with antibiotic-resistance genes or Tn402 transposition genes, but yet shares 99–100% sequence identity (across intI and attI) to Tn402-asssociated class 1 ‘mobile’ integrons [19]. Even more at odds with the current definition of ‘mobile’ integron is the similarity between the integrons found on the V. salmonicida pRSV1 plasmid and the P. haloplanktis TAC125 chromosome (99% sequence identity across intI and attI). The P. haloplanktis integron does not carry antibiotic-resistance genes and is clearly part of a phylogenetic group of Alteromonadales integrons that would qualify as ‘super’ integrons, its closest neighbour (excluding the pRSV1 integron) being the Alteromonas macleodii chromosomal integron associated with >30 cassettes but no mobile element (Figure 2). The following characteristics have frequently been used to describe a ‘super’ integron [17]: (i) a chromosomal location; (ii) many associated gene cassettes; (iii) a high degree of sequence identity between the attC (or 59-be) sites of these cassettes; and (iv) a mostly linear descent within a given group (i.e. little or no evidence of LGT of the integron core). These characteristics should be represented in the archetypical examples of this category: the Xanthomonas, Pseudomonas and Vibrio integrons. First, although all of these examples have their integrons located on chromosomes, it is not a distinctive feature given that the majority of integrons (regardless of their possible association with transposons) are chromosomal. This makes such a characteristic of little use when trying to define a category of integron. Second, the number of cassettes associated with an integron cannot be used to categorize these elements because it is a variable feature, which is not necessarily correlated with phylogeny
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(Figure 2). This cassette number can differ markedly even between two strains of the same species: Xanthomonas campestris strain 85–10 harbours three cassettes, whereas strain ATCC33913 has 22 [15]. Using the value of 20 cassettes cited as a minimum for a ‘super’ integron [17], one of the X. campestris strains harbours a ‘super’ integron, whereas the other does not. Third, degree of identity between the 59-be sites of an integron does tend to be higher for integrons with large cassette arrays, but this difference is not always statistically significant and some notable exceptions exist. For example, one of the M. aquaeolei integrons harbours 28 cassettes, the 59-be sites of which share 44 13% nucleotide identity, one of the lowest values for any integron. P. alcaligenes (>32 cassettes, 75 14% identity), S. degradans (73 cassettes, 71 19% identity), V. vulnificus CMCP6 (211 cassettes, 70 10% identity) and A. macleodii (>30 cassettes, 72 12% identity) have 59-be sites sharing sequence identities not substantially different from some classic ‘mobile’ integrons, such as the class 1 integrons found on plasmids pSC138 (six cassettes, 69 15% identity) or R46 (four cassettes, 65 18%). The distinction between ‘mobile’ and ‘super’ integrons, in terms of the degree of identity shared by 59-be found in a given genome, originated from the comparison of V. cholerae (average of 82 14% identity) and class 1 (62 11%) 59-be sites [7]. As outlined, such a distinction is not as clear-cut when considering a greater diversity of integrons. Finally, although some ‘super’ integrons might evolve mainly by vertical inheritance for a certain time, all seem to have been involved in lateral transfer at some point in their evolutionary history. For example, the Vibrio integrons are
Figure 3. Functional distribution for 1677 gene-cassette-encoded proteins from the Vibrio genera. These represent >90% of all known gene-cassette sequences. Functional annotation was performed using the COG database [38]. Proportions of proteins belonging to each broad COG functional category and subcategory are presented. The number of proteins belonging to each subcategory is indicated in parentheses next to its one-letter abbreviation. Information storage: J, translation, ribosome-structure biogenesis; K, transcription; L, replication recombination and repair. Cellular processes: D, cell-cycle control, cell division and chromosome partitioning; O, posttranslational modification, protein turnover, chaperones; M, cell-wall, membrane or envelope biogenesis; P, inorganic ion transport and metabolism. Metabolism: G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; (I) lipid transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism. Finally, the hypothetical conserved broad category has two sub-categories: R, general function prediction only; S, function unknown. www.sciencedirect.com
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likely to have originated by LGT from Alteromonadales, and integrons from the latter group itself have been involved in multiple LGT events across a range of phylogenetic distances (Figure 2). Both the Pseudomonas and Xanthomonas integrons, which cluster with b-proteobacteria in the IntI tree, are likely to have originated from an LGT event. Therefore, association with a given lineage is not an intrinsic property of ‘super’ integrons, but more of a transient stage in their evolution. The original description of integrons as ‘mobile’ or ‘super’ was based on a limited number of sequenced integrons, which resulted in an incomplete picture of their diversity. When looking at all integrons now known in an evolutionary context, most of the characteristics used to define those two categories become much less distinctive because many integrons possess features that would place them somewhere inbetween the ‘mobile’ and the ‘super’. Furthermore, given the multiple independent origins for ‘mobile’ integrons carrying antibiotic-resistance determinants, it is unlikely that they would share unique biochemical characteristics (recombinational processes) identifying them as a cohesive group distinct from other integrons such as those found in vibrios. Functional diversity of gene-cassette-encoded proteins: the key to the adaptive success of integrons Excluding those with antibiotic-resistance functions, which comprise <10% of the total known pool, the majority of proteins encoded within gene cassettes have not been functionally annotated: 65% of such proteins have no known homologues (taxonomically restricted genes) and 13% have only proteins of unknown function as homologues (Figure 3). Despite this, a wide range of functions is predicted for the 22% of cassette-associated proteins that do have characterized homologues, the most prevalent including virulence, DNA modification, phage-related functions, toxin-antitoxin systems and acetyltransferase [8,17]. Few cassette-encoded proteins have had their function experimentally determined, but even the small sample available indicates important diversity: restriction or methylation system, sulfate-binding, lipase, polysaccharide biosynthesis and dNTP pyrophosphohydrolase [27–29]. The broad functional diversity encoded in the gene-cassette metagenome indicates that the presence of an integron could contribute substantially to the adaptive potential of a bacterium. One common resident of large cassette arrays is the toxin-antitoxin system. This is a system that is hypothesized to prevent loss of DNA, most commonly plasmids, from cells because loss of this gene pair is commonly lethal to a cell. The presence of cassettes containing this system in large arrays might be a mechanism for stabilizing them and minimizing their loss [9,30]. Concluding remarks and future perspectives The integron is a common genetic element found in 10% of bacteria, including representatives from a wide diversity of phyla and environments. Only a simple core structure is required for a functional integron (the intI gene and the cognate attI integration site). Despite the conservation of its core structure, many characteristics of this element can vary enormously between evolutionary lineages. These include the number of associated cassettes, rate of cassette www.sciencedirect.com
insertion or excision, diversity of the cassette gene pool and the functional role of the latter. Nonetheless, integrons with similar characteristics have evolved multiple times independently, such as the integrons types associated with mobile elements and carrying small cassette arrays encoding antibiotic-resistance determinants. Although not mobile elements themselves, integrons have been frequently transferred among bacteria throughout their evolutionary history. Because it has only recently become clear that integrons have a broad range of associated functions in addition to a high frequency and wide dispersal in nature, several important aspects of integron biology have yet to be fully explored (Box 3). One of these aspects is the genetic regulation of such cassette-encoded genes, especially for integrons harbouring larger arrays. Integrons with few cassettes could rely solely on a Pc promoter that is proximal or internal to attI for expression of cassette genes, as is the case for most cassette genes in class 1 integrons. Those with longer arrays, however, must either only express the cassette-encoded genes proximal to the integrase (through a Pc promoter) or have other promoters located along their cassette arrays. Little evidence is currently available to distinguish between these two possibilities. Although we know that the gene-cassette pool to which environmental integrons have access must be large and diverse [31], we do not know its true extent and whether it is shared by all integrons or divided into smaller gene pools to which only specific integrons have access. Looking at the overlap in gene-cassette content of the integrons from bacteria with different degrees of phylogenetic relatedness could answer part of this question. Also, although we know that most integrons evolve faster than other parts of their host’s genomes, we do not know the exact difference in evolutionary rates. This could be measured by examining several closely related organisms. The relative ratio between the evolutionary rates of an integron and the rest of its host genome is likely to vary across bacterial diversity, but in some cases could be very high given that strains of the same species often have completely different arrays
Box 3. Outstanding questions in regards integrons generally Are all cassette-encoded proteins expressed in large cassette arrays? Do integrons associated with such arrays contain a Pc promoter in their intI or attI and/or harbour other promoters within their gene cassettes? What is the rate of gene-cassette acquisition or loss compared with that of other genomic gene? Do gene cassettes lacking detectable ORFs have a functional role in integrons? What is the range of functions encoded by the gene-cassette pool? Do most ORFs found in gene cassettes encode functional proteins? Is there one large gene-cassette pool shared by all integrons or is this pool subdivided into smaller ones that do not interact much with each other? What range of 59-be (attC) site sequences can various groups of integrons integrases recombine? Are there any accessory proteins involved in increasing the efficiency of integron integrases in particular lineages of bacteria? What is the mechanism responsible for gene-cassette creation?
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(e.g. V. vulnificus and P. stutzeri) [12,32]. By increasing the rate of LGT of its host and the gene pool to which it has access, integrons can have direct impact on the adaptability of the bacterial populations with which they are associated. The frequent mobilization of these genetic elements and their stable maintenance in various microbial lineages underscores this adaptive value. Acknowledgements We thank Olga Zhaxybayeva for her assistance in compiling statistics on integrons. Aspects of this work have been supported by the Australian Research Council, the Australian National Health and Medical Research Council, the Canadian Institutes for Health Research and the Macquarie University Centre for Microbial Functional Networks.
References 1 Hall, R.M. et al. (1999) Mobile gene cassettes and integrons in evolution. Ann. N. Y. Acad. Sci. 870, 68–80 2 Recchia, G.D. and Hall, R.M. (1995) Gene cassettes: a new class of mobile element. Microbiology 141, 3015–3027 3 Levings, R.S. et al. (2005) The genomic island SGI1, containing the multiple antibiotic resistance region of Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely distributed in other S. enterica serovars. J. Bacteriol. 187, 4401–4409 4 Summers, A.O. (2006) Genetic linkage and horizontal gene transfer, the roots of the antibiotic multi-resistance problem. Anim. Biotechnol. 17, 125–135 5 Nandi, S. et al. (2004) Gram-positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter. Proc. Natl. Acad. Sci. U. S. A. 101, 7118–7122 6 Shi, L. et al. (2006) Unnoticed spread of class 1 integrons in grampositive clinical strains isolated in Guangzhou, China. Microbiol. Immunol. 50, 463–467 7 Mazel, D. et al. (1998) A distinctive class of integron in the Vibrio cholerae genome. Science 280, 605–608 8 Rowe-Magnus, D.A. et al. (2003) Comparative analysis of superintegrons: engineering extensive genetic diversity in the Vibrionaceae. Genome Res. 13, 428–442 9 Szekeres, S. et al. (2007) Chromosomal toxin–antitoxin loci can diminish large-scale genome reductions in the absence of selection. Mol. Microbiol. 63, 1588–1605 10 Ruby, E.G. et al. (2005) Complete genome sequence of Vibrio fischeri: a symbiotic bacterium with pathogenic congeners. Proc. Natl. Acad. Sci. U. S. A. 102, 3004–3009 11 Boucher, Y. et al. (2006) Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio. BMC Evol. Biol., DOI: 10.1186/1471-2148-6-3 (http://www.biomedcentral.com) 12 Chen, C.Y. et al. (2003) Comparative genome analysis of Vibrio vulnificus, a marine pathogen. Genome Res. 13, 2577–2587 13 Vaisvila, R. et al. (2001) Discovery and distribution of super-integrons among pseudomonads. Mol. Microbiol. 42, 587–601 14 Holmes, A.J. et al. (2003) Recombination activity of a distinctive integron-gene cassette system associated with Pseudomonas stutzeri populations in soil. J. Bacteriol. 185, 918–928 15 Gillings, M.R. et al. (2005) Integrons in Xanthomonas: a source of species genome diversity. Proc. Natl. Acad. Sci. U. S. A. 102, 4419–4424 16 Case, R.J. et al. (2007) The 16S rRNA and rpoB genes as molecular markers for microbial ecology. Appl. Environ. Microbiol. 73, 278– 288
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17 Mazel, D. (2006) Integrons: agents of bacterial evolution. Nat. Rev. Microbiol. 4, 608–620 18 Hochhut, B. et al. (2001) Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob. Agents Chemother. 45, 2991–3000 19 Stokes, H.W. et al. (2006) Class 1 integrons potentially predating the association with Tn402-like transposition genes are present in a sediment microbial community. J. Bacteriol. 188, 5722–5730 20 Elbourne, L.D. and Hall, R.M. (2006) Gene cassette encoding a 3-Naminoglycoside acetyltransferase in a chromosomal integron. Antimicrob. Agents Chemother. 50, 2270–2271 21 Petroni, A. et al. (2004) CARB-9, a carbenicillinase encoded in the VCR region of Vibrio cholerae non-O1, non-O139 belongs to a family of cassette-encoded b-lactamases. Antimicrob. Agents Chemother. 48, 4042–4046 22 Melano, R. et al. (2002) New carbenicillin-hydrolyzing b-lactamase (CARB-7) from Vibrio cholerae non-O1, non-O139 strains encoded by the VCR region of the V. cholerae genome. Antimicro Agents Chemother. 46, 2162–2168 23 Rowe-Magnus, D.A. et al. (2002) Bacterial resistance evolution by recruitment of super-integron gene cassettes. Mol. Microbiol. 43, 1657–1669 24 Hall, R.M. et al. (2007) What are superintegrons? Nat. Rev. Microbiol. 5, DOI: 10.1038/nrmicro1462-c1 and author’s reply DOI: 10.1038/ nrmicro1462-c2 (www.nature.com) 25 Hall, R.M. and Stokes, H.W. (2004) Integrons or super integrons? Microbiology 150, 3–4 26 Rowe-Magnus, D.A. et al. (1999) Super-integrons. Res. Microbiol. 150, 641–651 27 Rowe-Magnus, D.A. et al. (2001) The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc. Natl. Acad. Sci. U. S. A. 98, 652–657 28 Smith, A.B. and Siebeling, R.J. (2003) Identification of genetic loci required for capsular expression in Vibrio vulnificus. Infect. Immun. 71, 1091–1097 29 Robinson, A. et al. Structural genomics of the bacterial mobile metagenome. In Structural Proteomics: High throughput methods (Guss, J.M., Huber, T. and Kobe, B. eds), Humana Press (in press) 30 Budde, P.P. et al. (2007) Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae. J. Bacteriol. 189, 491–500 31 Michael, C.A. et al. (2004) Mobile gene cassettes: a fundamental resource for bacterial evolution. Am. Nat. 164, 1–12 32 Coleman, N.V. and Holmes, A.J. (2005) The native Pseudomonas stutzeri strain Q chromosomal integron can capture and express cassette-associated genes. Microbiology 151, 1853–1864 33 Collis, C.M. et al. (2002) Characterization of the class 3 integron and the site-specific recombination system it determines. J. Bacteriol. 184, 3017–3026 34 Leon, G. and Roy, P.H. (2003) Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea. J. Bacteriol. 185, 2036–2041 35 Bouvier, M. et al. (2005) Integron cassette insertion: a recombination process involving a folded single strand substrate. EMBO J. 24, 4356– 4367 36 MacDonald, D. et al. (2006) Structural basis for broad DNA-specificity in integron recombination. Nature 440, 1157–1162 37 Guindon, S. and Gascuel, O. (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 38 Tatusov, R.L. et al. (2003) The COG database: an updated version includes eukaryotes. BMC Bioinformatics, 4, 41, DOI: 10.1186/14712105-4-41 (www.biomedcentral.com)
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Integrons: mobilizable platforms that promote genetic diversity in bacteria Yan Boucher1, Maurizio Labbate1, Jeremy E. Koenig2 and H.W. Stokes1 1
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW 2109, Australia Department of Biochemistry and Molecular Biology, Dalhousie University, 5850 College Street, Halifax, Nova Scotia, B3H 1X5, Canada
2
Integrons facilitate the capture of potentially adaptive exogenous genetic material by their host genomes. It is now clear that integrons are not limited to the clinical contexts in which they were originally discovered because »10% of bacterial genomes that have been partially or completely sequenced harbour this genetic element. This wealth of sequence information has revealed that integrons are not only much more phylogenetically diverse than previously thought but also more mobilizable, with many integrons having been subjected to frequent lateral gene transfer throughout their evolutionary history. This indicates that the genetic characteristics that make integrons such efficient vectors for the spread of antibiotic resistance genes have been associated with these elements since their earliest origins. Here, we give an overview of the structural and phylogenetic diversity of integrons and describe evolutionary events that have contributed to the success of these genetic elements. A simple structure leading to much diversity A bacterium harbouring an integron can capture and express genes found in its environment as part of small mobile elements, termed gene cassettes. Integrons were first discovered in clinical contexts, where they included short arrays of gene cassettes, the genes of which mostly encode antibiotic resistance determinants. These clinically important integrons, now known as class 1, 2 and 3 integrons, are by far the most studied and were used as a model to develop a formal structural definition for these genetic elements [1] (Box 1). These integrons share their core structure with all other integrons, which can be minimally defined as an integrase gene (intI) with a contiguous recombination site (attI). This simple core structure makes the integron an efficient platform for the site-specific integration of novel genetic material and potentially adaptive traits, without disruption to the genome of the recipient. Integrons are not rare genetic elements. After surveying all 603 completely or partially sequenced genomes, we found that 56 (9%) of them harboured integrons (Figures 1 and 2). The integrons found in these diverse organisms display as much variation in the evolutionary paths they have followed as their hosts do. This is most evident when looking at the integrons that have been more thoroughly studied. These include those frequently found in a clinical Corresponding author: Stokes, H.W. ([email protected]). Available online 12 June 2007. www.sciencedirect.com
context (mostly classes 1, 2 and 3) in addition to some of those found in bacteria recovered from natural environments (i.e. Vibrio, Pseudomonas and Xanthomonas). We briefly describe the characteristics of these well-known integrons from clinical and environmental settings before using a phylogeny of integron integrases to describe the evolutionary history that enabled these elements to succeed in a wide variety of hosts and environments. The evolution of integrons associated with antibiotic-resistance determinants are discussed in more detail, given their clinical relevance and the amount of literature available. Finally, we ask whether we can classify integrons to improve our understanding of these elements and, furthermore, briefly discuss their adaptive potential. It is important to get a firm grasp of the evolutionary processes that shaped integron diversity if we hope to understand how they can contribute to the rapid spread of novel phenotypes among bacteria (such as antibiotic resistance). The presence of integrons in close to 10% of the vast number of bacterial genome sequences now available makes it clear that these elements can no longer be perceived as an interesting oddity and that they need to be integrated into our models of microbial evolution. Variation on a theme: multiple evolutionary routes for integrons The first integrons to be described, although homologous, do not share recent common ancestry and were ascribed to different classes (1, 2 and 3) based on their respective intI gene sequences. The ‘class’ system has since been used as a loose taxonomic scheme, different classes simply representing evolutionarily divergent integrons showing substantial differences in the sequences of their intI genes (Box 2). Although integrons belonging to classes 1, 2 and 3 are not close evolutionary relatives, they nonetheless share several important features. They are contained in transposons and plasmids and found to include small arrays of up to six or so cassettes. They also share a collective genecassette pool, which is known to be small in relative terms (containing little more than 100 distinct cassettes) and almost uniquely comprising antibiotic-resistance determinants [2]. The association of these integrons with mobile elements and resistance genes has led to their rapid dispersal among various bacteria found in environments exposed to antibiotics [3,4]. Class 1, 2 and 3 integrons are most commonly embedded in diverse and highly mobile elements (e.g. various plasmids
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Box 1. What is an integron? As shown in Figure I, integrons are one part of a two-component system that capture disparate individual genes and physically link them in arrays. The first component of this system is the ‘core’ integron, which includes a gene (intI) encoding a site-specific recombinase (IntI) and a recombination site (attI). The second part of the system comprises a family of mobile elements known as gene cassettes. Individual cassettes usually consist of a single gene and a second type of recombination site. This cassette-associated recombination site comprises diverse sequence families, which all share a common structure. The original name given to these sites was 59base element (59-be), although the term attC has also been commonly used to describe them in recent years. Gene cassettes are captured by the integron via an IntI-mediated site-specific recombination reaction between the 59-be and attI sites [1,14,23,33,34]. During this reaction, IntI monomers bind preferentially to a single DNA strand of a gene cassette 59-be site. This single-stranded 59-be site adopts a folded structure to generate a double-stranded recombination site, which recombines with a double-stranded attI site that is also bound by IntI monomers [35,36]. Although it is widely assumed that a cassette promoter (Pc) found within the intI gene or attI site is likely to be a universal feature of integrons, its presence has only been confirmed for class 1 and class 3 integrons [33] and for an integron from Pseudomonas stutzeri [32]. In these integrons, cassette-associated genes lack their own promoter and rely on Pc for expression of the genes they contain. Multiple insertion events at an attI site are common and cassette-associated genes are usually inserted in the same orientation, producing tandem arrays of co-transcribed genes expressed from Pc. Although the integrase gene is usually in the opposite orientation to that of cassette-encoded genes, some integrons display an integrase in the same orientation as the genes in its associated cassette array, and these are termed inverted integrase integrons.
Figure I. The integron–gene cassette site-specific recombination system.
and transposons) and, thereby, have become broadly distributed amongst the Gram-negative bacteria [1] and even, in some cases, to Gram-positive bacteria [5,6]. Most other integron classes, however, are confined to chromosomes, the first examples of which were uncovered in the genus Vibrio [7]. Vibrio integrons are associated with large gene-cassette arrays (from 50 to >200 cassettes) and seem to have evolved mostly through vertical inheritance with limited lateral gene transfer (LGT) of the integron core [8]. The large arrays found in vibrios often contain gene cassettes encoding toxin-antitoxin systems [9]. Large arrays can be susceptible to some forms of genetic rearrangement, for example, the Vibrio fischeri ES114 array is split into two segments, one on each chromosome [10]. The extent of the Vibrio gene-cassette pool diversity is unknown, but is nonetheless orders of magnitude larger than the collective pool of cassettes found in class 1, 2 and 3 integrons. An interesting feature of some Vibrio integrons is that many of their cassettes contain no apparent open reading frame (ORF). These non protein-coding cassettes are often paralogous: a single family of such cassettes has 34 copies in Vibrio vulnificus YJ016 and 45 copies in V. vulnificus CMCP6 [11,12]. Vibrio gene cassettes are frequently transferred www.sciencedirect.com
across short phylogenetic distances but much more rarely across larger distances [11]. Such frequent short-range cassette movement has resulted in highly variable arrays in vibrios. For example, the arrays of the V. vulnificus strains CMCP6 and YJ016 show little similarity in cassette composition or order [11,12]. Integrons found in the genus Pseudomonas have also been studied in some detail. They are only present in a small subset of the many species belonging to this genus and are, therefore, likely to have been acquired by LGT late in its evolutionary history [13]. The number of cassettes Pseudomonas integrons contain varies with as little as ten gene cassettes in a strain of Pseudomonas stutzeri and >32 cassettes in Pseudomonas alcaligenes. The latter bacterium harbours multiple cassettes arrays; only one has been characterized (containing 32 cassettes), the other arrays being found either proximal or distal to that which is found in the P. alcaligenes genome [13]. Pseudomonas integrons also show large variability in the composition of their cassette arrays, with even otherwise closely related strains of a single species displaying little similarity in their cassette array content [14].
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Figure 1. Phylogenetic tree of the RpoB proteins found in all bacteria that harbour integrons. Proteobacteria are colour-coded according to the Order to which they belong. For species that harbour an integron but for which no RpoB sequence is available, a RpoB sequence from a closely related species was used as a substitute. Only species for which most of the gene-cassette array had been sequenced were included. The tree and bootstrap support values were inferred by maximum likelihood using PHYML [37].
Xanthomonas integrons are also well studied. Their array size is comparable to those of pseudomonads (1–22 cassettes), but the integron integrase is frequently inactivated, commonly by the insertion of a transposon. Similarly, genes within cassettes have frequently suffered from such insertions [15]. Another feature of Xanthomonas www.sciencedirect.com
integron arrays is a low degree of inter-strain variability, possibly a result of the loss of integrase activity leading to fixation of arrays [15]. The integrons described here constitute only a small portion of integron diversity (Figure 2). Despite this, it is obvious that substantial variation has occurred during
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Figure 2. Phylogenetic tree of known integron integrases (IntI). A single integron integrase is included for each bacterial species, provided that all IntI from that species cluster together in a preliminary analysis. Only species for which most of the gene-cassette array has been sequenced are included. Colour-coding is the same as that used in Figure 1. Black boxes indicate integrons that are associated with antibiotic resistance gene cassettes, with the particular cassette identified in the box. Class 1, 2 and 3
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Box 2. In search of a classification system for integrons So far, integrons have been mostly classified through the sequence similarity of their intI genes (class 1, 2, 3, SXT ICE and pRSV1) or taxonomic group to which their host belongs (i.e. Xanthomonas, Vibrio or Pseudomonas). Both of these approaches have serious limitations. The degree of nucleotide-sequence identity required to be part of an integron class has never been clearly defined in the literature, but was assumed to be similar to what is used to differentiate antibiotic resistance genes (98% nucleotide-sequence identity). We can now recognize many groups of integrons that are evolutionarily related but between their intI genes share variable degrees of sequence similarity (Xanthomonas, Vibrio, Pseudomonas). Trying to define integron groups such as the latter through an association with a particular lineage can also be misleading. For example, the integrons found in Vibrio fischeri species are clearly divergent from other Vibrio integrons but would be classified in one group if taxonomic affiliation was used as the main criteria. A naturalistic classification scheme based on phylogeny might be the best solution, avoiding some of the classification issues associated with frequent lateral gene transfer (LGT) while also considering the evolutionary history of integrons. Three broad groups of integrons can be defined based on phylogenetic considerations, which also agree with other characteristics (genetic structure, environmental distribution and/or taxonomic affiliation of the host): (i) the soil/freshwater proteobacteria group, (ii) the inverted integrase group, and (iii) the marine g-proteobacteria group. Other smaller groups, for which various characteristics are consistent with phylogeny of the integrase gene, can also be defined. The previously described Pseudomonas, Xanthomonas and Vibrio integrons types would all be valid under a phylogenybased definition, with a minor modification for the Vibrio: the V. fischeri integron type would have to be excluded from that group because of its uncertain ancestry. Class 1, 2 and 3 integron types would also be valid, as long as their definition can be broadened to include those associated with different types of mobile elements and association with any type of gene cassettes.
integron evolution, yielding a range of features conserved in specific integron lineages. In summary, the degree of mobility, phylogenetic distribution, frequency in a given taxonomic group, amount of coding content in cassettes, diversity and size of the gene-cassette pool, number of associated cassettes, rate of gene-cassette acquisition or loss, and degree of cassette paralogy in the array are all variables that enable integrons to change and adapt to different functional niches in various host organisms. Integrons have been frequently mobilized throughout their evolutionary history If the phylogenetic tree of integron integrases (IntI) is compared with the tree obtained for a molecular marker believed to be a good approximation of an ‘organismal’ phylogeny, such as the RNA polymerase subunit B gene (rpoB) [16], it becomes clear the history of IntI is not one of simple vertical inheritance (Figures 1 and 2). The many statistically supported inconsistencies between the IntI and RpoB trees points to several events of LGT of integrons across a wide range of phylogenetic distances. Moreover, out of the 50 different species that harbour an integron and for which a complete or partial genome sequence is available, 30 have transposon and/or recombinase genes
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located within their gene-cassette array or flanking their integron. Three main evolutionary groups of integrons are evident from the phylogeny of their integrase genes, two of which have already been identified in a previous study [17] (Figure 2). The first is mostly composed of proteobacterial integrons from freshwater and soil environments and includes class 1 and class 3 integrons (the ‘soil/freshwater proteobacteria’ group). The second group is composed of the integrons of marine g-proteobacteria and includes class 2 integrons in addition to those found in vibrios on the sulfamethoxazole- and trimethoprim-resistant (SXT) integrative and conjugative element (ICE) and pRSV1 plasmid (the marine g-proteobacteria group). The third group, identified here for the first time, is well-supported statistically and includes bacteria from a variety of taxonomic groups but has one distinctive feature: all integrons in this group harbour an integron integrase gene in the same orientation as the genes encoded by their associated cassette arrays (i.e. the attI site is found at the 30 end of the integrase gene). These can be defined as the inverted integrase group (Box 1). It is obvious that LGT has had an important role in the shaping of all of these groups. For example, many close relatives of Pseudomonas harbour an integron (e.g. Congregibacter, Saccharophagus, Marinobacter, Oceanospirillum, Reinekea and Oceanobacter) (Figure 1). However, the integrons of these relatives belong to the marine group, whereas the Pseudomonas integrons belong to the soil/freshwater group. Similarly, Xanthomonas and Nitrosococcus integrons, instead of clustering with other g-proteobacteria such as Vibrionales or Alteromonadales, are more closely related to their b-proteobacterial homologs (Figure 2). The monophyletic group of integrons with inverted integrases includes representatives from multiple bacterial phyla: Proteobacteria, Planctomycetes, Chlorobi, Spirochaetes and possibly Cyanobacteria (the lack of cassettes makes it difficult to say if genuine integrons are present in this last phylum). This indicates multiple lateral transfers across large phylogenetic distances (Figure 2). The marine group possibly has a single origin because it is composed of three related groups of g-proteobacteria: the Vibrionales, Alteromonadales and Pseudomonadalesrelated marine g-proteobacteria (Figure 2). However, none of these three groups is monophyletic within the marine clade, indicating LGT between members of that clade. For example, one of the Marinobacter aquaeolei integrons clusters with Shewanella denitrificans, whereas all other Shewanella integrons cluster with the relative of M. aquaeolei, Oceanobacter sp. RED65 (Figure 2). Even the vibrios, a group for which integrons seem to have followed evolutionary lines similar to their host chromosomes, might have originally acquired this genetic element through LGT. In the RpoB tree, vibrios are sister taxa to the monophyletic Alteromonadales, the latter group including the genera Shewanella, Pseudoalteromonas, Alteromonas and Psychromonas. In the IntI tree, the vibrios cluster with
integrons can contain multiple antibiotic resistance genes. The accession number of each integron integrase is in parentheses next to the taxon name of its host and the number of gene cassettes associated with it is in brackets. The tree and bootstrap support values were inferred by maximum likelihood using PHYML.
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Psychromonas, Pseudoalteromonas and Alteromonas to the exclusion of the Shewanella, making the Alteromonadales polyphyletic. This strongly indicates LGT of integrons between the Vibrionales and Alteromonadales. The vibrios are likely to have been the recipient of this LGT, given their internal branching position with regards to Alteromonadales and a single origin for all of them, although with one possible exception. Indeed, the V. fischeri integrons do not cluster with other Vibrio homologues (Figure 2). However, a lack of statistical support means that a distinction between the possibility that it acquired its integron from another bacterial group (the Pseudomonales-related marine g–proteobacteria) or that it has been through a rapid evolutionary divergence cannot be made. Multiple independent origins for integrons carrying antibiotic-resistance determinants The first integrons to be discovered – class 1 integrons – now account for a substantial proportion of multi-drugresistant nosocomial infections [2]. Several other classes of integrons associated with antibiotic-resistance genes, although much less common than their class 1 relatives, have also been identified. These include class 2 and class 3 integrons, which, like class 1, are closely associated with specific transposons and are found mostly in clinical isolates. They each harbour only a small subset of the resistance cassettes found in class 1 integrons. The integron class associated with the SXT ICE of some V. cholerae [18] and the one linked to the pRSV1 plasmid of V. salmonicida [11] have only been found associated with a single resistance gene cassette (dfrA1). All of these integrons, to some extent, share a genecassette pool that includes many antibiotic-resistance determinants. Despite their sharing of gene cassettes, evolutionary analysis of their respective intI genes indicates independent origins for each of these five classes of clinically relevant integrons. Indeed, close relatives of these five classes of mobilized integrons associated with resistance determinants (class 1, class 2, class 3, SXT and pRSV1) can be found in the genomes of environmental isolates (Figure 2). Some unusual class 1 integrons have been isolated from bacteria found in freshwater sediments [19]. These were undoubtedly related to the better known class 1 integrons found in clinical isolates, sharing 99% or more nucleotidesequence identity in their intI genes and attI sites. However, they are unlike any other class 1 integron because they lack evidence of association with the Tn402 transposition system. Also, although cassettes are present, they do not contain antibiotic-resistance genes. The almost identical nucleotide sequences of the Tn402-associated class 1 integrons and these novel relatives indicate that the latter could be part of an environmental pool of class 1 integrons from which the former originated [19]. The integron from the Vibrio salmonicida pRSV1 plasmid carries a dfrA1 trimethoprim-resistance cassette, the presence of which is probably related to the massive use of trimethoprim in fish farms. This integron is associated with a cassette-encoded IS4-type transposon and also has a close relative bearing no antibiotic-resistance determinants. Pseudoalteromonas haloplanktis TAC125 carries www.sciencedirect.com
an integron on its chromosome that is 99% identical to the pRSV1 integron but harbours unrelated gene cassettes. The integrons are both flanked by the same acyl-carrier protein gene (98% identical), which strongly indicates a common origin. The pRSV1 integron originates from diseased fish of Norwegian coastal waters, whereas P. haloplanktis was isolated from coastal Antarctic waters – two very distant geographical locations. The other three classes of mobilized integrons carrying antibiotic-resistance determinants – class 2, class 3 and SXT ICE – are also strongly related to specific groups of integrons found in environmental microbes, although more distantly. Class 2 integron integrases cluster with integrases of Shewanella species and Oceanobacter sp. (60% amino acid identity) (Figure 2). Class 3 integrases share a recent common ancestor with their class 1 homologues but are more closely related to an integrase from an Acidovorax species isolated from freshwater sediments (69% identity). The integron found in the SXT ICE conjugative element is clearly related to those of the marine organisms Shewanella denitrificans and Marinobacter aquaeolei (55% identity). The origins of these five classes of mobilized integrons bearing resistance cassettes are clearly distinct, both phylogenetically (with high statistical support) and environmentally. This indicates that many integrons found in natural environments have the capacity to carry antibiotic-resistance gene cassettes. If an integron becomes associated with a mobile element that has a wide range of hosts (such as most transposons), it greatly enhances its dispersion potential. This does not seem to be a rare event because it has occurred many independent times in the evolution of integrons (as illustrated by the five different cases presented here). Although integron mobilization can greatly facilitate resistance-cassette dispersal, the latter can still occur without it. Indeed, resistance cassettes have also been found in the non-mobilized chromosomal integrons of Saccharophagus degradans (aacC-A7) [20] and Vibrio cholerae (catB9, blaP7, blaP9) [21–23]. Can we categorize or classify integrons? Since the discovery of integrons harbouring integrase genes with divergent sequences, informal categorization of these elements, based on a variety of criteria, has been taking place (Box 2). Only one scheme, however, has been openly discussed in the literature [17,24–26]. This scheme refers to integrons as ‘mobile’ (also sometimes termed multi-resistant integrons) or ‘super’ (also referred to as chromosomal integrons). ‘Mobile’ integrons are defined as being associated with mobile DNA elements (transposons or plasmids) and antibiotic-resistance genes in addition to having a small array size and substantial heterogeneity in the sequence of their cassettes 59-be sites [17]. ‘Mobile’ integrons would include class 1, 2 and 3 integrons, the integron embedded in the SXT ICE of some V. cholerae and the integron found on the V. salmonicida pRSV1 plasmid (Figure 2). However, both class 1 and pRSV1 integrons have extremely close relatives that do not fit the definition of ‘mobile’ integrons, bringing this definition into question. The Azoarcus sp. MUL2G9 integron is found on the chromosome and is not
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associated with antibiotic-resistance genes or Tn402 transposition genes, but yet shares 99–100% sequence identity (across intI and attI) to Tn402-asssociated class 1 ‘mobile’ integrons [19]. Even more at odds with the current definition of ‘mobile’ integron is the similarity between the integrons found on the V. salmonicida pRSV1 plasmid and the P. haloplanktis TAC125 chromosome (99% sequence identity across intI and attI). The P. haloplanktis integron does not carry antibiotic-resistance genes and is clearly part of a phylogenetic group of Alteromonadales integrons that would qualify as ‘super’ integrons, its closest neighbour (excluding the pRSV1 integron) being the Alteromonas macleodii chromosomal integron associated with >30 cassettes but no mobile element (Figure 2). The following characteristics have frequently been used to describe a ‘super’ integron [17]: (i) a chromosomal location; (ii) many associated gene cassettes; (iii) a high degree of sequence identity between the attC (or 59-be) sites of these cassettes; and (iv) a mostly linear descent within a given group (i.e. little or no evidence of LGT of the integron core). These characteristics should be represented in the archetypical examples of this category: the Xanthomonas, Pseudomonas and Vibrio integrons. First, although all of these examples have their integrons located on chromosomes, it is not a distinctive feature given that the majority of integrons (regardless of their possible association with transposons) are chromosomal. This makes such a characteristic of little use when trying to define a category of integron. Second, the number of cassettes associated with an integron cannot be used to categorize these elements because it is a variable feature, which is not necessarily correlated with phylogeny
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(Figure 2). This cassette number can differ markedly even between two strains of the same species: Xanthomonas campestris strain 85–10 harbours three cassettes, whereas strain ATCC33913 has 22 [15]. Using the value of 20 cassettes cited as a minimum for a ‘super’ integron [17], one of the X. campestris strains harbours a ‘super’ integron, whereas the other does not. Third, degree of identity between the 59-be sites of an integron does tend to be higher for integrons with large cassette arrays, but this difference is not always statistically significant and some notable exceptions exist. For example, one of the M. aquaeolei integrons harbours 28 cassettes, the 59-be sites of which share 44 13% nucleotide identity, one of the lowest values for any integron. P. alcaligenes (>32 cassettes, 75 14% identity), S. degradans (73 cassettes, 71 19% identity), V. vulnificus CMCP6 (211 cassettes, 70 10% identity) and A. macleodii (>30 cassettes, 72 12% identity) have 59-be sites sharing sequence identities not substantially different from some classic ‘mobile’ integrons, such as the class 1 integrons found on plasmids pSC138 (six cassettes, 69 15% identity) or R46 (four cassettes, 65 18%). The distinction between ‘mobile’ and ‘super’ integrons, in terms of the degree of identity shared by 59-be found in a given genome, originated from the comparison of V. cholerae (average of 82 14% identity) and class 1 (62 11%) 59-be sites [7]. As outlined, such a distinction is not as clear-cut when considering a greater diversity of integrons. Finally, although some ‘super’ integrons might evolve mainly by vertical inheritance for a certain time, all seem to have been involved in lateral transfer at some point in their evolutionary history. For example, the Vibrio integrons are
Figure 3. Functional distribution for 1677 gene-cassette-encoded proteins from the Vibrio genera. These represent >90% of all known gene-cassette sequences. Functional annotation was performed using the COG database [38]. Proportions of proteins belonging to each broad COG functional category and subcategory are presented. The number of proteins belonging to each subcategory is indicated in parentheses next to its one-letter abbreviation. Information storage: J, translation, ribosome-structure biogenesis; K, transcription; L, replication recombination and repair. Cellular processes: D, cell-cycle control, cell division and chromosome partitioning; O, posttranslational modification, protein turnover, chaperones; M, cell-wall, membrane or envelope biogenesis; P, inorganic ion transport and metabolism. Metabolism: G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; (I) lipid transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism. Finally, the hypothetical conserved broad category has two sub-categories: R, general function prediction only; S, function unknown. www.sciencedirect.com
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likely to have originated by LGT from Alteromonadales, and integrons from the latter group itself have been involved in multiple LGT events across a range of phylogenetic distances (Figure 2). Both the Pseudomonas and Xanthomonas integrons, which cluster with b-proteobacteria in the IntI tree, are likely to have originated from an LGT event. Therefore, association with a given lineage is not an intrinsic property of ‘super’ integrons, but more of a transient stage in their evolution. The original description of integrons as ‘mobile’ or ‘super’ was based on a limited number of sequenced integrons, which resulted in an incomplete picture of their diversity. When looking at all integrons now known in an evolutionary context, most of the characteristics used to define those two categories become much less distinctive because many integrons possess features that would place them somewhere inbetween the ‘mobile’ and the ‘super’. Furthermore, given the multiple independent origins for ‘mobile’ integrons carrying antibiotic-resistance determinants, it is unlikely that they would share unique biochemical characteristics (recombinational processes) identifying them as a cohesive group distinct from other integrons such as those found in vibrios. Functional diversity of gene-cassette-encoded proteins: the key to the adaptive success of integrons Excluding those with antibiotic-resistance functions, which comprise <10% of the total known pool, the majority of proteins encoded within gene cassettes have not been functionally annotated: 65% of such proteins have no known homologues (taxonomically restricted genes) and 13% have only proteins of unknown function as homologues (Figure 3). Despite this, a wide range of functions is predicted for the 22% of cassette-associated proteins that do have characterized homologues, the most prevalent including virulence, DNA modification, phage-related functions, toxin-antitoxin systems and acetyltransferase [8,17]. Few cassette-encoded proteins have had their function experimentally determined, but even the small sample available indicates important diversity: restriction or methylation system, sulfate-binding, lipase, polysaccharide biosynthesis and dNTP pyrophosphohydrolase [27–29]. The broad functional diversity encoded in the gene-cassette metagenome indicates that the presence of an integron could contribute substantially to the adaptive potential of a bacterium. One common resident of large cassette arrays is the toxin-antitoxin system. This is a system that is hypothesized to prevent loss of DNA, most commonly plasmids, from cells because loss of this gene pair is commonly lethal to a cell. The presence of cassettes containing this system in large arrays might be a mechanism for stabilizing them and minimizing their loss [9,30]. Concluding remarks and future perspectives The integron is a common genetic element found in 10% of bacteria, including representatives from a wide diversity of phyla and environments. Only a simple core structure is required for a functional integron (the intI gene and the cognate attI integration site). Despite the conservation of its core structure, many characteristics of this element can vary enormously between evolutionary lineages. These include the number of associated cassettes, rate of cassette www.sciencedirect.com
insertion or excision, diversity of the cassette gene pool and the functional role of the latter. Nonetheless, integrons with similar characteristics have evolved multiple times independently, such as the integrons types associated with mobile elements and carrying small cassette arrays encoding antibiotic-resistance determinants. Although not mobile elements themselves, integrons have been frequently transferred among bacteria throughout their evolutionary history. Because it has only recently become clear that integrons have a broad range of associated functions in addition to a high frequency and wide dispersal in nature, several important aspects of integron biology have yet to be fully explored (Box 3). One of these aspects is the genetic regulation of such cassette-encoded genes, especially for integrons harbouring larger arrays. Integrons with few cassettes could rely solely on a Pc promoter that is proximal or internal to attI for expression of cassette genes, as is the case for most cassette genes in class 1 integrons. Those with longer arrays, however, must either only express the cassette-encoded genes proximal to the integrase (through a Pc promoter) or have other promoters located along their cassette arrays. Little evidence is currently available to distinguish between these two possibilities. Although we know that the gene-cassette pool to which environmental integrons have access must be large and diverse [31], we do not know its true extent and whether it is shared by all integrons or divided into smaller gene pools to which only specific integrons have access. Looking at the overlap in gene-cassette content of the integrons from bacteria with different degrees of phylogenetic relatedness could answer part of this question. Also, although we know that most integrons evolve faster than other parts of their host’s genomes, we do not know the exact difference in evolutionary rates. This could be measured by examining several closely related organisms. The relative ratio between the evolutionary rates of an integron and the rest of its host genome is likely to vary across bacterial diversity, but in some cases could be very high given that strains of the same species often have completely different arrays
Box 3. Outstanding questions in regards integrons generally Are all cassette-encoded proteins expressed in large cassette arrays? Do integrons associated with such arrays contain a Pc promoter in their intI or attI and/or harbour other promoters within their gene cassettes? What is the rate of gene-cassette acquisition or loss compared with that of other genomic gene? Do gene cassettes lacking detectable ORFs have a functional role in integrons? What is the range of functions encoded by the gene-cassette pool? Do most ORFs found in gene cassettes encode functional proteins? Is there one large gene-cassette pool shared by all integrons or is this pool subdivided into smaller ones that do not interact much with each other? What range of 59-be (attC) site sequences can various groups of integrons integrases recombine? Are there any accessory proteins involved in increasing the efficiency of integron integrases in particular lineages of bacteria? What is the mechanism responsible for gene-cassette creation?
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(e.g. V. vulnificus and P. stutzeri) [12,32]. By increasing the rate of LGT of its host and the gene pool to which it has access, integrons can have direct impact on the adaptability of the bacterial populations with which they are associated. The frequent mobilization of these genetic elements and their stable maintenance in various microbial lineages underscores this adaptive value. Acknowledgements We thank Olga Zhaxybayeva for her assistance in compiling statistics on integrons. Aspects of this work have been supported by the Australian Research Council, the Australian National Health and Medical Research Council, the Canadian Institutes for Health Research and the Macquarie University Centre for Microbial Functional Networks.
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