- Email: [email protected]
The Peril and Promise of Integrons: Beyond Antibiotic Resistance
TIMI 1766 No. of Pages 10
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Review
The Peril and Promise of Integrons: Beyond Antibiotic Resistance Timothy M. Ghaly,1,* Jemma L. Geoghegan,1 Sasha G. Tetu,2,3 and Michael R. Gillings1,3 Integrons are bacterial genetic elements that can capture, rearrange, and express mobile gene cassettes. They are best known for their role in disseminating antibiotic-resistance genes among pathogens. Their ability to rapidly spread resistance phenotypes makes it important to consider what other integronmediated traits might impact human health in the future, such as increased virulence, pathogenicity, or resistance to novel antimicrobial strategies. Exploring the functional diversity of cassettes and understanding their de novo creation will allow better pre-emptive management of bacterial growth, while also facilitating development of technologies that could harness integron activity. If we can control integrons and cassette formation, we could use integrons as a platform for enzyme discovery and to construct novel biochemical pathways, with applications in bioremediation or biosynthesis of industrial and therapeutic molecules. Integron activity thus holds both peril and promise for humans.
Highlights Integrons are DNA elements that helped drive the global antibiotic-resistance crisis. They capture and express mobile genes known as cassettes. Their activity holds both peril and promise for humans. The peril is the next wave of integronmediated traits: those beyond resistance. What other cassette-encoded traits might impact human health? Cassettes encoding virulence and novel resistance mechanisms can be recovered from environmental samples around the globe. Such cassettes should be accessible to integroncarrying pathogens anywhere on the planet.
Integron Activity Integrons are genetic elements that drive rapid adaptation in bacteria by capturing, rearranging, and expressing mobile genes known as cassettes. Their ability to capture diverse gene cassettes means that they can confer genomic plasticity on their host and facilitate rapid responses to diverse selection pressures. Integron activity is mediated by the integron-integrase gene (intI). IntI is responsible for catalysing the insertion of exogenous gene cassettes at the integron recombination site (attI; Figure 1). In general, gene cassettes are circular DNA molecules that consist of an open reading frame (ORF) and a cassette recombination site (attC) [1]. Once inserted within the integron platform, gene cassettes are generally expressed from the integron promoter (Pc) [2]. Multiple gene cassettes can accumulate within the platform to form a gene cassette array. IntI mediates the insertion, excision, and rearrangement of gene cassettes within its array. Cassettes that are distant from the Pc promoter may not be expressed [3,4], but can be excised and reinserted at the attI site where their expression is maximised [5]. Thus, many cassettes can be stored in an array to form a low-cost memory reservoir of functions for the host cell [6]. It is this activity that allows integrons to generate genomic and phenotypic diversity. Integrons are best known for their role in helping to facilitate the global antibiotic-resistance crisis. Their capture and expression of resistance genes has allowed integron-carrying cells to rapidly respond to antibiotic selection pressures [5,7]. However, the role integrons have played in disseminating antibiotic-resistance determinants is just a specialised case of their more general role in bacterial evolution [8]. Integron activity holds both peril and promise for humans. As pathogens become increasingly antibiotic resistant, selective advantages will accrue to cells that acquire the ‘next wave’ of clinically relevant traits that could affect human health, such as increased virulence, pathogenicity, or resisTrends in Microbiology, Month 2019, Vol. xx, No. xx
The promise of integrons lies in their potential use in biotechnology. Harnessing their ability for genetic rearrangement, and their diverse gene cassettes, could facilitate enzyme discovery, and the construction of novel biochemical pathways.
1
Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia 2 Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia 3 ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW 2109, Australia
*Correspondence: [email protected] (T.M. Ghaly).
https://doi.org/10.1016/j.tim.2019.12.002 © 2019 Elsevier Ltd. All rights reserved.
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Figure 1. Integron Structure and Activity. All integrons carry an integron-integrase gene (intI), an integron recombination site (attI), and a gene cassette promoter (Pc). Circular, mobile gene cassettes are inserted into a cassette array via IntImediated recombination between the attI and the attC site of the inserting cassette. Any gene cassette within an array can be excised by IntI to form a circular gene cassette, which may be reinserted at the attI site or discarded.
tance to novel antimicrobials. Integrons are almost certain to play a key role in spreading these clinically relevant traits. Thus, understanding the dynamics of integron activity has the potential to inform us of new threats linked to rapid bacterial evolution and improve our screening and mitigation approaches [9]. At the same time, such knowledge will help us to take advantage of the potential benefits of integron activity. If we can harness and manipulate integrons and their gene cassettes, there is a clear potential for their valuable applications in industry, biotechnology, agriculture, and health.
The Peril The peril of integrons is the next wave of cassette-encoded activity. As pathogens become increasingly antibiotic resistant, capturing gene cassettes that increase virulence or pathogenicity may provide additional selective advantages. In clinical settings where antibiotic resistance is now commonplace, pathogens that can acquire an increased capacity for immune evasion or host transmission will have a significant advantage over other pathogenic strains. There is evidence that this is already occurring [10], and that integrons will continue to interfere with human attempts to harness or control bacterial growth. At the core of this issue is understanding the diversity and dissemination of gene cassettes, and the selection pressures that drive their relative abundances. Sampling the Diversity of the Gene Cassette Metagenome Integrons are present in diverse bacteria, occurring in about 17% of sequenced bacterial genomes [11,12]. Any bacterium carrying an integron has access to a vast pool of genetic diversity held within the cassette metagenome [13,14]. Gene cassettes are extraordinarily diverse and ubiquitous. They have been recovered from every environment surveyed, including soils, aquatic 2
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sediments, animal and plant microbiomes, aquatic biofilms, marine and freshwater, and deepsea hydrothermal vents [13,15–19]. Recent sequencing of gene cassettes from different soils estimates that between 4000 and 18 000 different gene cassettes occur in every 0.3 g of surface soil [13]. Further, this high richness turns over rapidly across short spatial scales, with cassette similarity between any two points dropping to between 0.1% and 10% at distances of as little as 100 m [13]. Capture of gene cassettes within an integron cassette array relies on the highly conserved palindromic structure of attC sites, rather than a specific DNA sequence motif. Consequently, evolutionary distinct classes of integrons from different Phyla could potentially capture any gene cassette from this cassette metagenome [11]. Together, these factors suggest that the global gene cassette metagenome is an extensive, shared resource that can be exploited by diverse bacteria. The cassette metagenome can be rapidly sampled by integrons, enabled, at least in part, by the atypical mechanism of cassette insertion. Integrase-mediated cassette insertion results in the formation of a single-stranded recombination structure, which when resolved via replication generates two different integron variants [20] (Figure 2). Cassette insertion involves recombination between the double-stranded attI site of the integron and the single-stranded attC site of the inserting cassette [21,22] (Figure 2). The resulting recombination structure produces an asymmetrical Holliday junction, which is resolved after replication of the complete molecule [20] (Figure 2). As a result, the incoming gene cassette will only be inserted into one of the two daughter molecules. Replication of the recombinogenic strand resolves the Holliday junction, resulting in the integration of the gene cassette, while replication of the alternate strand generates the original substrate prior to the recombination event (Figure 2). Replicative resolution thus generates integron diversity between daughter molecules. In turn, this allows direct competition between the two daughter lineages, one with the newly inserted
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Figure 2. Replicative Resolution of Gene Cassette Insertion [20]. Insertion of gene cassettes involves the recombination between attI and only one strand of the attC. This generates an atypical Holliday junction, which is resolved after replication (dotted black arrows; lagging strand not shown) of the complete recombinant molecule. Replicative resolution generates one daughter molecule with the newly inserted gene cassette and one without. Abbreviations: ds, double stranded; ss, single stranded.
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cassette and one without. Integrons can therefore sample any incoming gene cassette, while also maintaining the original phenotype, which will predominate if the new cassette profile reduces overall host fitness. Ultimately, this promotes rapid sampling of the cassette metagenome without any disruption to successful cassette arrays. The same mechanism is likely to be involved in cassette excision [20]. This implies that the excision and reinsertion of a cassette can result in gene duplication. The excision of a gene cassette will generate one daughter molecule that still carries the excised cassette after replication. Consequently, reinsertion of this cassette can give rise to an integron that carries two copies of this gene [20]. Gene duplication has long been recognised as a driving force in evolution [23,24]. It provides the raw material for the evolution of new gene functions, where mutations can accumulate in one of the gene copies. These mutations can lead to the diversification of gene cassettes, and the generation of altered or novel functions. Indeed, we see multiple families of gene cassettes that share high sequence homology spanning the whole ORF and attC site [25,26]. This diversity may be, at least in some part, a product of gene duplication events. The dynamics of cassette insertion and excision may help explain the diversification of integron cassette arrays, and the gene cassettes themselves. The Remarkable Success of Antibiotic-Resistance Gene Cassettes Given the huge diversity of the cassette metagenome, it is important that we try to predict which cassette-encoded traits might be selected in current and future clinical settings. Before we examine future traits, however, we should first ask: Why have antibiotic-resistance cassettes been so successful in disseminating across the globe? More specifically, why is there a disproportionately large number of resistance cassettes carried by ‘mobile integrons’? At least five classes of integrons have become embedded within mobile DNA elements, and these events appear to have occurred in the recent past [8,27,28]. Their mobility allows rapid movement across broad phylogenetic boundaries, and consequently they gain access to a significant portion of the cassette metagenome. Indeed, cassettes associated with mobile integrons appear to have been collected from multiple genomic backgrounds. This can be inferred by the inconsistent codon usage in their cassette ORFs, and the considerable sequence diversity in their attC sites [11]. By contrast, chromosomal integrons often have cassette arrays with highly similar attC sites [16,29–31]. These homologous attC sites are species specific and cluster according to host topology [29,31], indicating that chromosomal integrons generally recruit gene cassettes of intraspecific origin, while mobile integrons disperse between species and can sample cassettes with diverse origins. Despite the heterogenous origins of cassettes associated with mobile integrons, they exhibit remarkably homogenous functionality, with the majority of cassettes conferring resistance to antibiotics [6,25]. Even outside clinical settings, the cassette arrays of mobile integrons regularly consist of antibiotic-resistance genes [10,32–34]. Resistance genes are under strong positive selection imposed by the heavy use of antibiotics in medicine and agriculture [35]. However, an important further explanation for their overrepresentation is that resistance cassettes are particularly good examples of single-gene/single-phenotype entities, which do not have to be integrated into metabolic networks and therefore have minimal disturbance to the existing genome [36]. In general, the successful integration of heterologous genes into a new host depends on their interaction with specific components of gene regulatory networks and host physiology [37,38]. In mobile integrons, heterologous cassettes are much more likely to be disruptive to host fitness. 4
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This may explain why the majority of cassettes associated with mobile integrons encode antibioticmodifying enzymes that function without significant cellular interactions [25]. It is therefore clear that resistance cassettes of mobile integrons do not proportionately represent all mechanisms of resistance. Rather, these cassettes largely encode β-lactamases, acetyltransferases, and nucleotidyl transferases [25]. By contrast, genes conferring antibiotic resistance through regulatory mechanisms are rarely observed within mobile integrons [25]. This should become a key factor when considering which traits mobile integrons might disseminate under future selection regimes. Predicting Future Traits of Clinical Relevance Predicting cassette-encoded traits that may be harmful to human health is a formidable task, given that more than 80% of sequenced gene cassettes encode proteins of unknown functions [13,39,40]. However, since these diverse gene cassettes can be targeted by PCR [13,41], they can be cloned into a surrogate host. Despite their relatively low abundance in environmental DNA, their functions could be assessed via a functional metagenomic approach [42,43]. In particular, environmental compartments could be systematically screened for cassettes that encode clinically relevant functions, including novel antimicrobial-resistance mechanisms, virulence, and pathogenicity. Particular focus should be given to traits that promote colonisation and invasion of the human host [7]. These may comprise a broad range of functional traits such as biofilm formation, signalling, cell surface adhesion, secretion, stress responses, detoxification, or nutrient acquisition. Importantly, these traits could be screened by first targeting environments known to be reservoirs of antibiotic-resistance genes, such as hospital effluents and pharmaceutical waste [44]. These environments likely represent the same selection pressures applied in clinical settings, and are thus ideal sites to detect novel cassette-encoded functions that might affect human health. In addition to screening cassettes based on function, we can also screen cassettes based on sequence homology [45]. For this, we should begin by examining the more prevalent genes in the cassette metagenome. There appear to be a subset of cassettes that are ubiquitous across the globe [13]. Cassettes sequenced from different soils across Australia and Antarctica revealed almost 600 cassettes that were universally present [13]. Furthermore, some of these cassettes have also been recovered in independent studies from various locations across Australia, Canada, and France [14,19,40,46]. Remarkably, the presence of these cassettes appears to be independent of environment type, having been recovered from a variety of soils, freshwater sediment, and tar pond sludge. The ubiquity of these cassettes suggests that their functions warrant further investigation, particularly because they occur in diverse environments, and their prevalence suggests that they would be the most accessible cassettes for mobile integrons to capture and express. Among this set of ubiquitous cassettes are a suite which encodes a diverse range of putative virulence proteins [13]. These include permeases, dihydrolipoyl dehydrogenase, peptidase, nucleotidyl transferase, glycosyl transferase, N-acetyltransferases, iron-sulphur proteins, a VWA-containing protein, and an XRE family transcriptional regulator. In addition, there are several cassette-encoded proteins potentially involved in phage resistance, including endonucleases, methylases, and a CRISPR–Cas3 protein [13]. This raises some concerns over the future use of phage therapy as an alternative to antibiotic treatment, since integrons appear to have ready access to phage-resistance genes. In addition, there have been a number of previously identified gene cassettes that encode virulence proteins, such as lipocalins [47], surface polysaccharides [48], enterotoxins [49], isochorismatases [39], lipases [14,29], and methionine sulfoxide reductases [10]. Together, these findings highlight the role that integron gene cassettes may play in the acquisition of novel phenotypes of significance for future clinical settings.
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We have seen how proficiently integrons, particularly class 1 integrons, can disseminate resistance genes across the globe. The formation and subsequent spread of this class of integron is largely due to its association with diverse mobile elements, coupled with sustained anthropogenic selection pressures [28,50,51]. Indeed, millions to billions of copies of class 1 integrons now exist in every gram of faeces from humans and agricultural livestock, with up to 1023 copies being shed into the environment every day [52]. This remarkable abundance places class 1 integrons in an ideal position to sample the diversity of the cassette metagenome at any location on the planet. Given the presence of clinically relevant genes in the worldwide pool of gene cassettes, any selection that favours the acquisition of such genes is likely to rapidly promote the fixation of such gene transfers. These traits highlight the peril of integron activity and should be considered when developing novel antimicrobials or alternate infection-treatment strategies.
The Promise The promise of integrons lies in their potential applications in industry, medicine, biotechnology, and synthetic biology. Here we highlight two promising avenues of research. The first is screening the cassette metagenome for industrially relevant enzymes. The second is controlling integron activity and gene cassette formation for the purpose of genome manipulation. Gene Cassettes as a Resource for Enzyme Discovery Enzymes are crucial for the development of environmentally sustainable industrial processes, and the vast majority of enzymes used in industry are of microbial origin [53]. Therefore, prospecting for microbial enzymes is a key step in the advance of biotechnology and industry. Here, the greatest benefit is likely to be had by focussing on novel proteins that are encoded by rare genes, thereby tapping into the hitherto unsampled repertoire of microbial functions. The overrepresentation of novel proteins and protein folds within the cassette metagenome [13,54] highlights the potential of cassette screening as an efficient method for discovery of novel functions. Furthermore, the high local richness and rapid spatial turnover of gene cassettes [13] suggest that repeated functional screening projects would substantially increase the discovery of diverse enzymatic activities. As mentioned previously, amplified gene cassette libraries can be cloned in a surrogate host, allowing high-throughput screening based on function. The vast repertoire of cassetteencoded functions can therefore be examined. Gene cassettes are an important component of bacterial adaptation [6]. In particular, they help bacteria adapt to specific niches and help generate ecotypes [55–57]. This suggests that the cassette metagenome collectively encodes a vast number of proteins that could have industrially useful activities. In polluted sediments, for example, cassettes are known to encode diverse enzymes related to the biodegradation of xenobiotic compounds [40,55]. Functional screening of gene cassettes in areas exposed to intense selection pressures or from harsh environmental conditions could lead to the discovery of novel enzymes that can be used for industrial purposes. We might also be able to impose artificial selection pressures to prospect for genes that encode functions of interest [58]. We have already seen this occur naturally, exemplified by the global prevalence of antibiotic-resistance gene cassettes. Resistance cassettes only constitute a tiny proportion of the cassette metagenome in environmental compartments [13,40,56]. However, intense antibiotic selection for resistance cassettes dramatically increased their distribution and abundance, highlighting the potential for directed evolution to recover genes of interest. Artificial selection could expose a vast number of functionally desirable genes that otherwise occur at vanishingly low abundance and thus remain undetected. Given the size and diversity of the cassette metagenome, an untold number of functions await discovery. 6
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Integron Manipulation To control integron activity is to control gene acquisition, rearrangement, and expression within an in vivo system. Such attempts have already been made. First, integrons have been used to recruit chromosomal gene cassettes onto plasmid vectors for downstream applications [59]. Second, an integron-mediated cloning technique has been developed. Here, synthetic and natural gene cassettes were introduced into cells, resulting in their acquisition by pre-existing integrons [60]. This allows an alternate method for molecular cloning that does not rely on a vector or antibiotic selection. Finally, the natural integron-integrase activity can be manipulated to select optimal arrangement and content of genes within biological pathways [61]. Proof-of-concept has been demonstrated via the construction and optimisation of a tryptophan biosynthesis pathway [62]. Here, Bikard et al. [62] delivered tryptophan biosynthesis genes, along with regulatory elements, as individual gene cassettes in a synthetic integron. Upon induction of integronmediated recombination, thousands of gene cassette combinations were generated overnight. A subset of these gene arrangements were found to enhance tryptophan biosynthesis by an order of magnitude relative to their natural gene arrangement [62]. Such applications have huge potential for optimising known biochemical pathways and refining synthetic organisms with engineered genomes [63]. The genes involved in these pathways, however, must already be identified, limiting the usefulness of this strategy to construct entirely novel pathways. As will be discussed later, we might take better advantage of the integron system by controlling the de novo formation of gene cassettes (Box 1). Controlling this process in vivo could allow the sampling of unknown genes and, ultimately, the creation of entirely new biochemical pathways. Potential Future Applications of the Integron System Fully realising the promise of integrons involves controlling integrase activity and manipulating the de novo creation of gene cassettes. The former is regulated by the bacterial SOS response [64] and can be artificially induced. However, the mechanism for the latter still awaits discovery (Box 1). Control of this system would be invaluable for synthetic biology. Presumably, the cassette metagenome is populated by genes recruited from the bacterial pangenome. Determining this process and understanding what genes can be ‘sampled’ by this mechanism would allow better control of their transformation into functional gene cassettes. Box 1. Gene Cassette Genesis How do gene cassettes arise? This is widely regarded as the single most important unanswered question relating to integron biology [6]. Answering this question is the key to fully realising the promise of integron activity. The leading hypothesis for this process involves reverse transcription of an mRNA ancestor molecule [65,66]. This suggestion is based on the fact that gene cassettes generally lack a promoter, and are composed of a single ORF with minimal non-coding DNA. The hypothesised mechanism is catalysed by group IIC–attC introns, which are ribonucleoprotein complexes that possess both self-splicing and reverse-transcription activity. They have an affinity for inserting adjacent to stem–loop motifs, such as transcriptional terminators or attC sites [67–70]. The proposed model involves two independent intron insertion events. One intron inserting at a solitary attC site, and the other at the transcriptional terminator of a gene of interest. Homologous recombination between the two intron copies would create a gene–intron–attC intermediate. Once this intermediate is transcribed, the intron would self-splice and reverse transcribe the gene–attC RNA template to form a DNA gene cassette. This model, however, lacks experimental validation, and has been disputed [6]. The primary criticism is that this model does not account for the creation of cassettes that have promoters, such as toxin–antitoxin cassettes, as the promoter region cannot be present in the mRNA transcript. Group IIC–attC introns have been shown, at least in vitro, to also possess DNA-dependent polymerase activity [65], which could potentially explain the presence of promoters in such cassettes. However, an exact model for how this might occur has not been presented, nor has any explanation of why this would occur so infrequently. Uncovering the mechanism of gene cassette genesis is of considerable importance.
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The diversity of the cassette metagenome [13] suggests that, at the least, a large number of bacterial genes can naturally act as templates for cassette formation. By manipulating this system, we might be able to expand the range of genes that can be turned into cassettes and increase the rate at which this occurs. Further, by controlling integron-integrase activity, newly formed cassettes could then be acquired, rearranged, and expressed within a synthetic integron platform (Figure 3). By carefully targeting an environment of interest, or by applying artificial selection, we could increase the likelihood of capturing novel genes with desirable functions. Such a strategy might allow sampling of a significant proportion of the protein universe and could ultimately be used to build entirely new biochemical pathways for bioremediation or biosynthesis of industrial or therapeutic molecules. The strength of such an application lies in its capacity to construct industrially valuable operons that encode completely novel protein folds without any prior knowledge of their functions.
Concluding Remarks Integrons have played a significant role in the spread of antibiotic resistance across the globe [25]. This phenomenon has been driven by the sustained selection pressures imposed by human antimicrobial use in agriculture and medicine [7]. As pathogens become increasingly antibiotic resistant, we must consider which bacterial traits will arise from the next wave of anthropogenic selection. In particular, these might include increased virulence or pathogenicity, as well as resistance to novel antimicrobial strategies. It is almost certain that integrons will play a significant role in the acquisition of such traits. Pre-emptive efforts to identify the candidate genes that are likely to be involved would allow us to better avoid further perils of integron-mediated bacterial evolution.
Outstanding Questions How are gene cassettes formed de novo? What conditions influence the rate of cassette genesis? Do cassettes encode traits, other than resistance, that are relevant in clinical settings? Are these traits likely to be mobilisable by integron platforms? How can we screen cassettes for useful activities? How can we efficiently sample environmental gene cassettes for novel pathway construction?
Exploring the functional diversity of the cassette metagenome will also give us access to a huge genetic resource that we can exploit for enzyme discovery. Furthermore, understanding integron activity will allow us to take better advantage of potential benefits of these elements as genetic ‘pathway construction’ tools. In particular, knowledge of the formation of gene cassettes and
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Figure 3. The Use of Integrons as Platforms for Enzyme Discovery and as a Tool for Construction of Genetic Pathways. Integron gene cassettes could be created from available genes in the bacterial pangenome. By controlling this mechanism, we could increase the rate of cassette formation. We can exploit the vast genetic diversity held within the cassette metagenome to prospect for enzymes of industrial relevance. This can be achieved by a functional metagenomic approach, or by manipulating integron-integrase activity to construct novel biochemical pathways. By applying appropriate selection regimes, we could screen for traits of interest conferred by single genes, or by novel cassette operons.
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the molecular machinery involved might enable us to harness this process as a powerful molecular tool. This technology would have important applications for bioremediation, industry, agriculture, and medicine (see Outstanding Questions). Acknowledgements T.M.G. thanks Mary Ghaly for loving support, critical discussions, and for reviewing earlier drafts of the manuscript.
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44. Marathe, N.P. et al. (2019) Sewage effluent from an Indian hospital harbors novel carbapenemases and integron-borne antibiotic resistance genes. Microbiome 7, 97 45. Boulund, F. et al. (2017) Computational discovery and functional validation of novel fluoroquinolone resistance genes in public metagenomic data sets. BMC Genomics 18, 682 46. Michael, C.A. et al. (2004) Mobile gene cassettes: a fundamental resource for bacterial evolution. Am. Nat. 164, 1–12 47. Barker, A. and Manning, P.A. (1997) VlpA of Vibrio cholerae O1: the first bacterial member of the α2-microglobulin lipocalin superfamily. Microbiology 143, 1805–1813 48. Smith, A.B. and Siebeling, R.J. (2003) Identification of genetic loci required for capsular expression in Vibrio vulnificus. Infect. Immun. 71, 1091–1097 49. Ogawa, A. and Takeda, T. (1993) The gene encoding the heatstable enterotoxin of Vibrio cholerae is flanked by 123-base pair direct repeats. Microbiol. Immunol. 37, 607–616 50. Gillings, M. et al. (2018) Pollutants that replicate: xenogenetic DNAs. Trends Microbiol. 26, 975–977 51. Ghaly, T.M. and Gillings, M.R. (2018) Mobile DNAs as ecologically and evolutionarily independent units of life. Trends Microbiol. 26, 904–912 52. Zhu, Y.-G. et al. (2017) Microbial mass movements. Science 357, 1099–1100 53. Uchiyama, T. and Miyazaki, K. (2009) Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr. Opin. Biotechnol. 20, 616–622 54. Sureshan, V. et al. (2013) Integron gene cassettes: a repository of novel protein folds with distinct interaction sites. PLoS One 8, e52934 55. Elsaied, H. et al. (2011) Marine integrons containing novel integrase genes, attachment sites, attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays. ISME J. 5, 1162–1177 56. Koenig, J.E. et al. (2011) Coral-mucus-associated Vibrio integrons in the Great Barrier Reef: genomic hotspots for environmental adaptation. ISME J. 5, 962–972
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57. Elsaied, H. et al. (2014) Novel integrons and gene cassettes from a Cascadian submarine gas-hydrate-bearing core. FEMS Microbiol. Ecol. 87, 343–356 58. Kan, A. and Joshi, N.S. (2019) Towards the directed evolution of protein materials. MRS Commun. 9, 441–455 59. Rowe-Magnus, D.A. (2009) Integrase-directed recovery of functional genes from genomic libraries. Nucleic Acids Res. 37, e118 60. Gestal, A.M. et al. (2011) Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology. Microbiology 157, 3349–3360 61. Bikard, D. and Mazel, D. (2013) Shuffling of DNA cassettes in a synthetic integron. In Synthetic Biology (Polizzi, K.M. and Kontoravdi, C., eds), pp. 169–174, Humana Press 62. Bikard, D. et al. (2010) The synthetic integron: an in vivo genetic shuffling device. Nucleic Acids Res. 38, e153 63. Poluri, K.M. and Gulati, K. (2017) Expanding the synthetic protein universe by guided evolutionary concepts. In Protein Engineering Techniques: Gateways to Synthetic Protein Universe, pp. 27–59, Springer, Singapore 64. Guerin, É. et al. (2009) The SOS response controls integron recombination. Science 324, 1034 65. Léon, G. and Roy, P.H. (2009) Potential role of group IIC-attC introns in integron cassette formation. J. Bacteriol. 191, 6040–6051 66. Quiroga, C. and Centrón, D. (2009) Using genomic data to determine the diversity and distribution of target site motifs recognized by class C-attC group II introns. J. Mol. Evol. 68, 539–549 67. Centrón, D. and Roy, P.H. (2002) Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion. Antimicrob. Agents Chemother. 46, 1402–1409 68. Léon, G. and Roy, P.H. (2003) Excision and integration of cassettes by an integron integrase of Nitrosomonas europaea. J. Bacteriol. 185, 2036–2041 69. Sunde, M. (2005) Class I integron with a group II intron detected in an Escherichia coli strain from a free-range reindeer. Antimicrob. Agents Chemother. 49, 2512–2514 70. Quiroga, C. et al. (2008) The S. ma. I2 class C group II intron inserts at integron attC sites. Microbiology 154, 1341–1353
Trends in Microbiology
Review
The Peril and Promise of Integrons: Beyond Antibiotic Resistance Timothy M. Ghaly,1,* Jemma L. Geoghegan,1 Sasha G. Tetu,2,3 and Michael R. Gillings1,3 Integrons are bacterial genetic elements that can capture, rearrange, and express mobile gene cassettes. They are best known for their role in disseminating antibiotic-resistance genes among pathogens. Their ability to rapidly spread resistance phenotypes makes it important to consider what other integronmediated traits might impact human health in the future, such as increased virulence, pathogenicity, or resistance to novel antimicrobial strategies. Exploring the functional diversity of cassettes and understanding their de novo creation will allow better pre-emptive management of bacterial growth, while also facilitating development of technologies that could harness integron activity. If we can control integrons and cassette formation, we could use integrons as a platform for enzyme discovery and to construct novel biochemical pathways, with applications in bioremediation or biosynthesis of industrial and therapeutic molecules. Integron activity thus holds both peril and promise for humans.
Highlights Integrons are DNA elements that helped drive the global antibiotic-resistance crisis. They capture and express mobile genes known as cassettes. Their activity holds both peril and promise for humans. The peril is the next wave of integronmediated traits: those beyond resistance. What other cassette-encoded traits might impact human health? Cassettes encoding virulence and novel resistance mechanisms can be recovered from environmental samples around the globe. Such cassettes should be accessible to integroncarrying pathogens anywhere on the planet.
Integron Activity Integrons are genetic elements that drive rapid adaptation in bacteria by capturing, rearranging, and expressing mobile genes known as cassettes. Their ability to capture diverse gene cassettes means that they can confer genomic plasticity on their host and facilitate rapid responses to diverse selection pressures. Integron activity is mediated by the integron-integrase gene (intI). IntI is responsible for catalysing the insertion of exogenous gene cassettes at the integron recombination site (attI; Figure 1). In general, gene cassettes are circular DNA molecules that consist of an open reading frame (ORF) and a cassette recombination site (attC) [1]. Once inserted within the integron platform, gene cassettes are generally expressed from the integron promoter (Pc) [2]. Multiple gene cassettes can accumulate within the platform to form a gene cassette array. IntI mediates the insertion, excision, and rearrangement of gene cassettes within its array. Cassettes that are distant from the Pc promoter may not be expressed [3,4], but can be excised and reinserted at the attI site where their expression is maximised [5]. Thus, many cassettes can be stored in an array to form a low-cost memory reservoir of functions for the host cell [6]. It is this activity that allows integrons to generate genomic and phenotypic diversity. Integrons are best known for their role in helping to facilitate the global antibiotic-resistance crisis. Their capture and expression of resistance genes has allowed integron-carrying cells to rapidly respond to antibiotic selection pressures [5,7]. However, the role integrons have played in disseminating antibiotic-resistance determinants is just a specialised case of their more general role in bacterial evolution [8]. Integron activity holds both peril and promise for humans. As pathogens become increasingly antibiotic resistant, selective advantages will accrue to cells that acquire the ‘next wave’ of clinically relevant traits that could affect human health, such as increased virulence, pathogenicity, or resisTrends in Microbiology, Month 2019, Vol. xx, No. xx
The promise of integrons lies in their potential use in biotechnology. Harnessing their ability for genetic rearrangement, and their diverse gene cassettes, could facilitate enzyme discovery, and the construction of novel biochemical pathways.
1
Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia 2 Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia 3 ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW 2109, Australia
*Correspondence: [email protected] (T.M. Ghaly).
https://doi.org/10.1016/j.tim.2019.12.002 © 2019 Elsevier Ltd. All rights reserved.
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Figure 1. Integron Structure and Activity. All integrons carry an integron-integrase gene (intI), an integron recombination site (attI), and a gene cassette promoter (Pc). Circular, mobile gene cassettes are inserted into a cassette array via IntImediated recombination between the attI and the attC site of the inserting cassette. Any gene cassette within an array can be excised by IntI to form a circular gene cassette, which may be reinserted at the attI site or discarded.
tance to novel antimicrobials. Integrons are almost certain to play a key role in spreading these clinically relevant traits. Thus, understanding the dynamics of integron activity has the potential to inform us of new threats linked to rapid bacterial evolution and improve our screening and mitigation approaches [9]. At the same time, such knowledge will help us to take advantage of the potential benefits of integron activity. If we can harness and manipulate integrons and their gene cassettes, there is a clear potential for their valuable applications in industry, biotechnology, agriculture, and health.
The Peril The peril of integrons is the next wave of cassette-encoded activity. As pathogens become increasingly antibiotic resistant, capturing gene cassettes that increase virulence or pathogenicity may provide additional selective advantages. In clinical settings where antibiotic resistance is now commonplace, pathogens that can acquire an increased capacity for immune evasion or host transmission will have a significant advantage over other pathogenic strains. There is evidence that this is already occurring [10], and that integrons will continue to interfere with human attempts to harness or control bacterial growth. At the core of this issue is understanding the diversity and dissemination of gene cassettes, and the selection pressures that drive their relative abundances. Sampling the Diversity of the Gene Cassette Metagenome Integrons are present in diverse bacteria, occurring in about 17% of sequenced bacterial genomes [11,12]. Any bacterium carrying an integron has access to a vast pool of genetic diversity held within the cassette metagenome [13,14]. Gene cassettes are extraordinarily diverse and ubiquitous. They have been recovered from every environment surveyed, including soils, aquatic 2
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sediments, animal and plant microbiomes, aquatic biofilms, marine and freshwater, and deepsea hydrothermal vents [13,15–19]. Recent sequencing of gene cassettes from different soils estimates that between 4000 and 18 000 different gene cassettes occur in every 0.3 g of surface soil [13]. Further, this high richness turns over rapidly across short spatial scales, with cassette similarity between any two points dropping to between 0.1% and 10% at distances of as little as 100 m [13]. Capture of gene cassettes within an integron cassette array relies on the highly conserved palindromic structure of attC sites, rather than a specific DNA sequence motif. Consequently, evolutionary distinct classes of integrons from different Phyla could potentially capture any gene cassette from this cassette metagenome [11]. Together, these factors suggest that the global gene cassette metagenome is an extensive, shared resource that can be exploited by diverse bacteria. The cassette metagenome can be rapidly sampled by integrons, enabled, at least in part, by the atypical mechanism of cassette insertion. Integrase-mediated cassette insertion results in the formation of a single-stranded recombination structure, which when resolved via replication generates two different integron variants [20] (Figure 2). Cassette insertion involves recombination between the double-stranded attI site of the integron and the single-stranded attC site of the inserting cassette [21,22] (Figure 2). The resulting recombination structure produces an asymmetrical Holliday junction, which is resolved after replication of the complete molecule [20] (Figure 2). As a result, the incoming gene cassette will only be inserted into one of the two daughter molecules. Replication of the recombinogenic strand resolves the Holliday junction, resulting in the integration of the gene cassette, while replication of the alternate strand generates the original substrate prior to the recombination event (Figure 2). Replicative resolution thus generates integron diversity between daughter molecules. In turn, this allows direct competition between the two daughter lineages, one with the newly inserted
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Figure 2. Replicative Resolution of Gene Cassette Insertion [20]. Insertion of gene cassettes involves the recombination between attI and only one strand of the attC. This generates an atypical Holliday junction, which is resolved after replication (dotted black arrows; lagging strand not shown) of the complete recombinant molecule. Replicative resolution generates one daughter molecule with the newly inserted gene cassette and one without. Abbreviations: ds, double stranded; ss, single stranded.
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cassette and one without. Integrons can therefore sample any incoming gene cassette, while also maintaining the original phenotype, which will predominate if the new cassette profile reduces overall host fitness. Ultimately, this promotes rapid sampling of the cassette metagenome without any disruption to successful cassette arrays. The same mechanism is likely to be involved in cassette excision [20]. This implies that the excision and reinsertion of a cassette can result in gene duplication. The excision of a gene cassette will generate one daughter molecule that still carries the excised cassette after replication. Consequently, reinsertion of this cassette can give rise to an integron that carries two copies of this gene [20]. Gene duplication has long been recognised as a driving force in evolution [23,24]. It provides the raw material for the evolution of new gene functions, where mutations can accumulate in one of the gene copies. These mutations can lead to the diversification of gene cassettes, and the generation of altered or novel functions. Indeed, we see multiple families of gene cassettes that share high sequence homology spanning the whole ORF and attC site [25,26]. This diversity may be, at least in some part, a product of gene duplication events. The dynamics of cassette insertion and excision may help explain the diversification of integron cassette arrays, and the gene cassettes themselves. The Remarkable Success of Antibiotic-Resistance Gene Cassettes Given the huge diversity of the cassette metagenome, it is important that we try to predict which cassette-encoded traits might be selected in current and future clinical settings. Before we examine future traits, however, we should first ask: Why have antibiotic-resistance cassettes been so successful in disseminating across the globe? More specifically, why is there a disproportionately large number of resistance cassettes carried by ‘mobile integrons’? At least five classes of integrons have become embedded within mobile DNA elements, and these events appear to have occurred in the recent past [8,27,28]. Their mobility allows rapid movement across broad phylogenetic boundaries, and consequently they gain access to a significant portion of the cassette metagenome. Indeed, cassettes associated with mobile integrons appear to have been collected from multiple genomic backgrounds. This can be inferred by the inconsistent codon usage in their cassette ORFs, and the considerable sequence diversity in their attC sites [11]. By contrast, chromosomal integrons often have cassette arrays with highly similar attC sites [16,29–31]. These homologous attC sites are species specific and cluster according to host topology [29,31], indicating that chromosomal integrons generally recruit gene cassettes of intraspecific origin, while mobile integrons disperse between species and can sample cassettes with diverse origins. Despite the heterogenous origins of cassettes associated with mobile integrons, they exhibit remarkably homogenous functionality, with the majority of cassettes conferring resistance to antibiotics [6,25]. Even outside clinical settings, the cassette arrays of mobile integrons regularly consist of antibiotic-resistance genes [10,32–34]. Resistance genes are under strong positive selection imposed by the heavy use of antibiotics in medicine and agriculture [35]. However, an important further explanation for their overrepresentation is that resistance cassettes are particularly good examples of single-gene/single-phenotype entities, which do not have to be integrated into metabolic networks and therefore have minimal disturbance to the existing genome [36]. In general, the successful integration of heterologous genes into a new host depends on their interaction with specific components of gene regulatory networks and host physiology [37,38]. In mobile integrons, heterologous cassettes are much more likely to be disruptive to host fitness. 4
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This may explain why the majority of cassettes associated with mobile integrons encode antibioticmodifying enzymes that function without significant cellular interactions [25]. It is therefore clear that resistance cassettes of mobile integrons do not proportionately represent all mechanisms of resistance. Rather, these cassettes largely encode β-lactamases, acetyltransferases, and nucleotidyl transferases [25]. By contrast, genes conferring antibiotic resistance through regulatory mechanisms are rarely observed within mobile integrons [25]. This should become a key factor when considering which traits mobile integrons might disseminate under future selection regimes. Predicting Future Traits of Clinical Relevance Predicting cassette-encoded traits that may be harmful to human health is a formidable task, given that more than 80% of sequenced gene cassettes encode proteins of unknown functions [13,39,40]. However, since these diverse gene cassettes can be targeted by PCR [13,41], they can be cloned into a surrogate host. Despite their relatively low abundance in environmental DNA, their functions could be assessed via a functional metagenomic approach [42,43]. In particular, environmental compartments could be systematically screened for cassettes that encode clinically relevant functions, including novel antimicrobial-resistance mechanisms, virulence, and pathogenicity. Particular focus should be given to traits that promote colonisation and invasion of the human host [7]. These may comprise a broad range of functional traits such as biofilm formation, signalling, cell surface adhesion, secretion, stress responses, detoxification, or nutrient acquisition. Importantly, these traits could be screened by first targeting environments known to be reservoirs of antibiotic-resistance genes, such as hospital effluents and pharmaceutical waste [44]. These environments likely represent the same selection pressures applied in clinical settings, and are thus ideal sites to detect novel cassette-encoded functions that might affect human health. In addition to screening cassettes based on function, we can also screen cassettes based on sequence homology [45]. For this, we should begin by examining the more prevalent genes in the cassette metagenome. There appear to be a subset of cassettes that are ubiquitous across the globe [13]. Cassettes sequenced from different soils across Australia and Antarctica revealed almost 600 cassettes that were universally present [13]. Furthermore, some of these cassettes have also been recovered in independent studies from various locations across Australia, Canada, and France [14,19,40,46]. Remarkably, the presence of these cassettes appears to be independent of environment type, having been recovered from a variety of soils, freshwater sediment, and tar pond sludge. The ubiquity of these cassettes suggests that their functions warrant further investigation, particularly because they occur in diverse environments, and their prevalence suggests that they would be the most accessible cassettes for mobile integrons to capture and express. Among this set of ubiquitous cassettes are a suite which encodes a diverse range of putative virulence proteins [13]. These include permeases, dihydrolipoyl dehydrogenase, peptidase, nucleotidyl transferase, glycosyl transferase, N-acetyltransferases, iron-sulphur proteins, a VWA-containing protein, and an XRE family transcriptional regulator. In addition, there are several cassette-encoded proteins potentially involved in phage resistance, including endonucleases, methylases, and a CRISPR–Cas3 protein [13]. This raises some concerns over the future use of phage therapy as an alternative to antibiotic treatment, since integrons appear to have ready access to phage-resistance genes. In addition, there have been a number of previously identified gene cassettes that encode virulence proteins, such as lipocalins [47], surface polysaccharides [48], enterotoxins [49], isochorismatases [39], lipases [14,29], and methionine sulfoxide reductases [10]. Together, these findings highlight the role that integron gene cassettes may play in the acquisition of novel phenotypes of significance for future clinical settings.
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We have seen how proficiently integrons, particularly class 1 integrons, can disseminate resistance genes across the globe. The formation and subsequent spread of this class of integron is largely due to its association with diverse mobile elements, coupled with sustained anthropogenic selection pressures [28,50,51]. Indeed, millions to billions of copies of class 1 integrons now exist in every gram of faeces from humans and agricultural livestock, with up to 1023 copies being shed into the environment every day [52]. This remarkable abundance places class 1 integrons in an ideal position to sample the diversity of the cassette metagenome at any location on the planet. Given the presence of clinically relevant genes in the worldwide pool of gene cassettes, any selection that favours the acquisition of such genes is likely to rapidly promote the fixation of such gene transfers. These traits highlight the peril of integron activity and should be considered when developing novel antimicrobials or alternate infection-treatment strategies.
The Promise The promise of integrons lies in their potential applications in industry, medicine, biotechnology, and synthetic biology. Here we highlight two promising avenues of research. The first is screening the cassette metagenome for industrially relevant enzymes. The second is controlling integron activity and gene cassette formation for the purpose of genome manipulation. Gene Cassettes as a Resource for Enzyme Discovery Enzymes are crucial for the development of environmentally sustainable industrial processes, and the vast majority of enzymes used in industry are of microbial origin [53]. Therefore, prospecting for microbial enzymes is a key step in the advance of biotechnology and industry. Here, the greatest benefit is likely to be had by focussing on novel proteins that are encoded by rare genes, thereby tapping into the hitherto unsampled repertoire of microbial functions. The overrepresentation of novel proteins and protein folds within the cassette metagenome [13,54] highlights the potential of cassette screening as an efficient method for discovery of novel functions. Furthermore, the high local richness and rapid spatial turnover of gene cassettes [13] suggest that repeated functional screening projects would substantially increase the discovery of diverse enzymatic activities. As mentioned previously, amplified gene cassette libraries can be cloned in a surrogate host, allowing high-throughput screening based on function. The vast repertoire of cassetteencoded functions can therefore be examined. Gene cassettes are an important component of bacterial adaptation [6]. In particular, they help bacteria adapt to specific niches and help generate ecotypes [55–57]. This suggests that the cassette metagenome collectively encodes a vast number of proteins that could have industrially useful activities. In polluted sediments, for example, cassettes are known to encode diverse enzymes related to the biodegradation of xenobiotic compounds [40,55]. Functional screening of gene cassettes in areas exposed to intense selection pressures or from harsh environmental conditions could lead to the discovery of novel enzymes that can be used for industrial purposes. We might also be able to impose artificial selection pressures to prospect for genes that encode functions of interest [58]. We have already seen this occur naturally, exemplified by the global prevalence of antibiotic-resistance gene cassettes. Resistance cassettes only constitute a tiny proportion of the cassette metagenome in environmental compartments [13,40,56]. However, intense antibiotic selection for resistance cassettes dramatically increased their distribution and abundance, highlighting the potential for directed evolution to recover genes of interest. Artificial selection could expose a vast number of functionally desirable genes that otherwise occur at vanishingly low abundance and thus remain undetected. Given the size and diversity of the cassette metagenome, an untold number of functions await discovery. 6
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Integron Manipulation To control integron activity is to control gene acquisition, rearrangement, and expression within an in vivo system. Such attempts have already been made. First, integrons have been used to recruit chromosomal gene cassettes onto plasmid vectors for downstream applications [59]. Second, an integron-mediated cloning technique has been developed. Here, synthetic and natural gene cassettes were introduced into cells, resulting in their acquisition by pre-existing integrons [60]. This allows an alternate method for molecular cloning that does not rely on a vector or antibiotic selection. Finally, the natural integron-integrase activity can be manipulated to select optimal arrangement and content of genes within biological pathways [61]. Proof-of-concept has been demonstrated via the construction and optimisation of a tryptophan biosynthesis pathway [62]. Here, Bikard et al. [62] delivered tryptophan biosynthesis genes, along with regulatory elements, as individual gene cassettes in a synthetic integron. Upon induction of integronmediated recombination, thousands of gene cassette combinations were generated overnight. A subset of these gene arrangements were found to enhance tryptophan biosynthesis by an order of magnitude relative to their natural gene arrangement [62]. Such applications have huge potential for optimising known biochemical pathways and refining synthetic organisms with engineered genomes [63]. The genes involved in these pathways, however, must already be identified, limiting the usefulness of this strategy to construct entirely novel pathways. As will be discussed later, we might take better advantage of the integron system by controlling the de novo formation of gene cassettes (Box 1). Controlling this process in vivo could allow the sampling of unknown genes and, ultimately, the creation of entirely new biochemical pathways. Potential Future Applications of the Integron System Fully realising the promise of integrons involves controlling integrase activity and manipulating the de novo creation of gene cassettes. The former is regulated by the bacterial SOS response [64] and can be artificially induced. However, the mechanism for the latter still awaits discovery (Box 1). Control of this system would be invaluable for synthetic biology. Presumably, the cassette metagenome is populated by genes recruited from the bacterial pangenome. Determining this process and understanding what genes can be ‘sampled’ by this mechanism would allow better control of their transformation into functional gene cassettes. Box 1. Gene Cassette Genesis How do gene cassettes arise? This is widely regarded as the single most important unanswered question relating to integron biology [6]. Answering this question is the key to fully realising the promise of integron activity. The leading hypothesis for this process involves reverse transcription of an mRNA ancestor molecule [65,66]. This suggestion is based on the fact that gene cassettes generally lack a promoter, and are composed of a single ORF with minimal non-coding DNA. The hypothesised mechanism is catalysed by group IIC–attC introns, which are ribonucleoprotein complexes that possess both self-splicing and reverse-transcription activity. They have an affinity for inserting adjacent to stem–loop motifs, such as transcriptional terminators or attC sites [67–70]. The proposed model involves two independent intron insertion events. One intron inserting at a solitary attC site, and the other at the transcriptional terminator of a gene of interest. Homologous recombination between the two intron copies would create a gene–intron–attC intermediate. Once this intermediate is transcribed, the intron would self-splice and reverse transcribe the gene–attC RNA template to form a DNA gene cassette. This model, however, lacks experimental validation, and has been disputed [6]. The primary criticism is that this model does not account for the creation of cassettes that have promoters, such as toxin–antitoxin cassettes, as the promoter region cannot be present in the mRNA transcript. Group IIC–attC introns have been shown, at least in vitro, to also possess DNA-dependent polymerase activity [65], which could potentially explain the presence of promoters in such cassettes. However, an exact model for how this might occur has not been presented, nor has any explanation of why this would occur so infrequently. Uncovering the mechanism of gene cassette genesis is of considerable importance.
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The diversity of the cassette metagenome [13] suggests that, at the least, a large number of bacterial genes can naturally act as templates for cassette formation. By manipulating this system, we might be able to expand the range of genes that can be turned into cassettes and increase the rate at which this occurs. Further, by controlling integron-integrase activity, newly formed cassettes could then be acquired, rearranged, and expressed within a synthetic integron platform (Figure 3). By carefully targeting an environment of interest, or by applying artificial selection, we could increase the likelihood of capturing novel genes with desirable functions. Such a strategy might allow sampling of a significant proportion of the protein universe and could ultimately be used to build entirely new biochemical pathways for bioremediation or biosynthesis of industrial or therapeutic molecules. The strength of such an application lies in its capacity to construct industrially valuable operons that encode completely novel protein folds without any prior knowledge of their functions.
Concluding Remarks Integrons have played a significant role in the spread of antibiotic resistance across the globe [25]. This phenomenon has been driven by the sustained selection pressures imposed by human antimicrobial use in agriculture and medicine [7]. As pathogens become increasingly antibiotic resistant, we must consider which bacterial traits will arise from the next wave of anthropogenic selection. In particular, these might include increased virulence or pathogenicity, as well as resistance to novel antimicrobial strategies. It is almost certain that integrons will play a significant role in the acquisition of such traits. Pre-emptive efforts to identify the candidate genes that are likely to be involved would allow us to better avoid further perils of integron-mediated bacterial evolution.
Outstanding Questions How are gene cassettes formed de novo? What conditions influence the rate of cassette genesis? Do cassettes encode traits, other than resistance, that are relevant in clinical settings? Are these traits likely to be mobilisable by integron platforms? How can we screen cassettes for useful activities? How can we efficiently sample environmental gene cassettes for novel pathway construction?
Exploring the functional diversity of the cassette metagenome will also give us access to a huge genetic resource that we can exploit for enzyme discovery. Furthermore, understanding integron activity will allow us to take better advantage of potential benefits of these elements as genetic ‘pathway construction’ tools. In particular, knowledge of the formation of gene cassettes and
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Figure 3. The Use of Integrons as Platforms for Enzyme Discovery and as a Tool for Construction of Genetic Pathways. Integron gene cassettes could be created from available genes in the bacterial pangenome. By controlling this mechanism, we could increase the rate of cassette formation. We can exploit the vast genetic diversity held within the cassette metagenome to prospect for enzymes of industrial relevance. This can be achieved by a functional metagenomic approach, or by manipulating integron-integrase activity to construct novel biochemical pathways. By applying appropriate selection regimes, we could screen for traits of interest conferred by single genes, or by novel cassette operons.
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the molecular machinery involved might enable us to harness this process as a powerful molecular tool. This technology would have important applications for bioremediation, industry, agriculture, and medicine (see Outstanding Questions). Acknowledgements T.M.G. thanks Mary Ghaly for loving support, critical discussions, and for reviewing earlier drafts of the manuscript.
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