Horizontal gene transfer in plant microbiomes: integrons as hotspots for cross-species gene exchange

Plant microbiomes play important roles in plant health and fitness. Bacterial horizontal gene transfer (HGT) can influence plant health outcomes, driving the spread of both plant growth-promoting and phytopathogenic traits. However, community dynamics, including the range of genetic elements and bacteria involved in this process are still poorly understood. Integrons are genetic elements recently shown to be abundant in plant microbiomes, and are associated with HGT across broad phylogenetic boundaries. They facilitate the spread of gene cassettes, small mobile elements that collectively confer a diverse suite of adaptive functions. Here, we analysed 5,565 plant-associated bacterial genomes to investigate the prevalence and functional diversity of integrons in this niche. We found that integrons are particularly abundant in the genomes of Pseudomonadales, Burkholderiales, and Xanthomonadales. In total, we detected nearly 9,000 gene cassettes, and found that many could be involved in plant growth promotion or phytopathogenicity, suggesting that integrons might play a role in bacterial mutualistic or pathogenic lifestyles. The rhizosphere was enriched in cassettes involved in the transport and metabolism of diverse substrates, suggesting that they may aid in adaptation to this environment, which is rich in root exudates. We also found that integrons facilitate cross-species HGT, which is particularly enhanced in the phyllosphere. This finding may provide an ideal opportunity to promote plant growth by fostering the spread of genes cassettes relevant to leaf health. Together, our findings suggest that integrons are important elements in plant microbiomes that drive HGT, and have the potential to facilitate plant host adaptation

Tn6603, a Carrier of Tn5053 Family Transposons, Occurs in the Chromosome and in a Genomic Island of Pseudomonas aeruginosa Clinical Strains

Transposons of the Pseudomonasaeruginosa accessory gene pool contribute to phenotype and to genome plasticity. We studied local P. aeruginosa strains to ascertain the encroachment of mer-type res site hunter transposons into clinical settings and their associations with other functional modules. Five different Tn5053 family transposons were detected, all chromosomal. Some were solitary elements; one was in res of Tn1013#, a relative of a reported carrier of int-type res site hunters (class 1 integrons), but most were in res of Tn6603, a new Tn501-related transposon of unknown phenotype. Most of the Tn6603::Tn elements, and some Tn6603 and Tn6603::Tn elements found in GenBank sequences, were at identical sites in an hypothetical gene of P. aeruginosa genomic island PAGI-5v. The island in clonally differing strains was at either of two tRNALys loci, suggesting lateral transfer to these sites. This observation is consistent with the membership of the prototype PAGI-5 island to the ICE family of mobile genetic elements. Additionally, the res site hunters in the nested transposons occupied different positions in the Tn6603 carrier. This suggested independent insertion events on five occasions at least. Tn5053 family members that were mer-/tni-defective were found in Tn6603- and Tn501-like carriers in GenBank sequences of non-clinical Pseudomonas spp. The transposition events in these cases presumably utilized tni functions in trans, as can occur with class 1 integrons. We suggest that in the clinical context, P. aeruginosa strains that carry Tn6603 alone or in PAGI-5v can serve to disseminate functional res site hunters; these in turn can provide the requisite trans-acting tni functions to assist in the dissemination of class 1 integrons, and hence of their associated antibiotic resistance determinants

The Natural History of Integrons

Integrons were first identified because of their central role in assembling and disseminating antibiotic resistance genes in commensal and pathogenic bacteria. However, these clinically relevant integrons represent only a small proportion of integron diversity. Integrons are now known to be ancient genetic elements that are hotspots for genomic diversity, helping to generate adaptive phenotypes. This perspective examines the diversity, functions, and activities of integrons within both natural and clinical environments. We show how the fundamental properties of integrons exquisitely pre-adapted them to respond to the selection pressures imposed by the human use of antimicrobial compounds. We then follow the extraordinary increase in abundance of one class of integrons (class 1) that has resulted from its acquisition by multiple mobile genetic elements, and subsequent colonisation of diverse bacterial species, and a wide range of animal hosts. Consequently, this class of integrons has become a significant pollutant in its own right, to the extent that it can now be detected in most ecosystems. As human activities continue to drive environmental instability, integrons will likely continue to play key roles in bacterial adaptation in both natural and clinical settings. Understanding the ecological and evolutionary dynamics of integrons can help us predict and shape these outcomes that have direct relevance to human and ecosystem health

Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR

Objectives: Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution.Methods: We applied a single-cell fusion PCR-based technique-emulsion, paired isolation and concatenation PCR (epicPCR)-to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host.Results: Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, namely the gain of a regulatory 5'-untranslated region (5'-UTR), the loss of attC recombination sites between adjacent gene cassettes, and the invasion of a 5'-UTR by an IS element. Notably, our experimental testing of a novel variant of the aadA11 gene cassette demonstrated that gaining the observed 5'-UTR contributed to a 3-fold increase in the MIC of streptomycin relative to the ancestral reference gene cassette in E. coli.Conclusions: Dissecting the observed signatures of molecular evolution of class 1 integrons allowed us to explain their effects on antibiotic resistance phenotypes, while identifying their bacterial hosts enabled us to make better inferences on the likely origins of novel gene cassettes and IS that invade known gene cassettes

Discovery of integrons in Archaea: platforms for cross-domain gene transfer

Horizontal gene transfer between different domains of life is increasingly being recognized as an important evolutionary driver, with the potential to increase the pace of biochemical innovation and environmental adaptation. However, the mechanisms underlying the recruitment of exogenous genes from foreign domains are mostly unknown. Integrons are a family of genetic elements that facilitate this process within Bacteria. However, they have not been reported outside Bacteria, and thus their potential role in cross-domain gene transfer has not been investigated. Here, we discover that integrons are also present in 75 archaeal metagenome-assembled genomes from nine phyla, and are particularly enriched among Asgard archaea. Furthermore, we provide experimental evidence that integrons can facilitate the recruitment of archaeal genes by bacteria. Our findings establish a previously unknown mechanism of cross-domain gene transfer whereby bacteria can incorporate archaeal genes from their surrounding environment via integron activity. These findings have important implications for prokaryotic ecology and evolution

Identification of integrons and gene cassette-associated recombination sites in bacteriophage genomes

Bacteriophages are versatile mobile genetic elements that play key roles in driving the evolution of their bacterial hosts through horizontal gene transfer. Phages co-evolve with their bacterial hosts and have plastic genomes with extensive mosaicism. In this study, we present bioinformatic and experimental evidence that temperate and virulent (lytic) phages carry integrons, including integron-integrase genes, attC/attI recombination sites and gene cassettes. Integrons are normally found in Bacteria, where they capture, express and re-arrange mobile gene cassettes via integron-integrase activity. We demonstrate experimentally that a panel of attC sites carried in virulent phage can be recognized by the bacterial class 1 integron-integrase (IntI1) and then integrated into the paradigmatic attI1 recombination site using an attC x attI recombination assay. With an increasing number of phage genomes projected to become available, more phage-associated integrons and their components will likely be identified in the future. The discovery of integron components in bacteriophages establishes a new route for lateral transfer of these elements and their cargo genes between bacterial host cells

Vegetable phylloplane microbiomes harbour class 1 integrons in novel bacterial hosts and drive the spread of chlorite resistance

Bacterial hosts in vegetable phylloplanes carry mobile genetic elements, such as plasmids and transposons that are associated with integrons. These mobile genetic elements and their cargo genes can enter human microbiomes via consumption of fresh agricultural produce, including uncooked vegetables. This presents a risk of acquiring antimicrobial resistance genes from uncooked vegetables. To better understand horizontal gene transfer of class 1 integrons in these compartments, we applied epicPCR, a single-cell fusion-PCR surveillance technique, to link the class 1 integron integrase (intI1) gene with phylogenetic markers of their bacterial hosts. Ready-to-eat salads carried class 1 integrons from the phyla Bacteroidota and Pseudomonadota, including four novel genera that were previously not known to be associated with intI1. We whole-genome sequenced Pseudomonas and Erwinia hosts of pre-clinical class 1 integrons that are embedded in Tn402-like transposons. The proximal gene cassette in these integrons was identified as a chlorite dismutase gene cassette, which we showed experimentally to confer chlorite resistance. Chlorine-derived compounds such as acidified sodium chlorite and chloride dioxide are used to disinfectant raw vegetables in food processing facilities, suggesting selection for chlorite resistance in phylloplane integrons. The spread of integrons conferring chlorite resistance has the potential to exacerbate integron-mediated antimicrobial resistance (AMR) via co-selection of chlorite resistance and AMR, thus highlighting the importance of monitoring chlorite residues in agricultural produce. These results demonstrate the strength of combining epicPCR and culture-based isolation approaches for identifying hosts and dissecting the molecular ecology of class 1 integrons

The Natural History of Integrons

Integrons were first identified because of their central role in assembling and disseminating antibiotic resistance genes in commensal and pathogenic bacteria. However, these clinically relevant integrons represent only a small proportion of integron diversity. Integrons are now known to be ancient genetic elements that are hotspots for genomic diversity, helping to generate adaptive phenotypes. This perspective examines the diversity, functions, and activities of integrons within both natural and clinical environments. We show how the fundamental properties of integrons exquisitely pre-adapted them to respond to the selection pressures imposed by the human use of antimicrobial compounds. We then follow the extraordinary increase in abundance of one class of integrons (class 1) that has resulted from its acquisition by multiple mobile genetic elements, and subsequent colonisation of diverse bacterial species, and a wide range of animal hosts. Consequently, this class of integrons has become a significant pollutant in its own right, to the extent that it can now be detected in most ecosystems. As human activities continue to drive environmental instability, integrons will likely continue to play key roles in bacterial adaptation in both natural and clinical settings. Understanding the ecological and evolutionary dynamics of integrons can help us predict and shape these outcomes that have direct relevance to human and ecosystem health

Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities

Integrons are microbial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the performance of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class one integrons are collecting and concentrating resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample

Exploring the role of mobile genetic elements in shaping plant–bacterial interactions for sustainable agriculture and ecosystem health

Societal Impact Statement Plants and bacteria interact in complex ways that are crucial to the health and productivity of native vegetation and croplands. While the range of characterised plant‐beneficial bacterial traits continues to grow, key questions remain regarding the distribution and mobility of genes associated with these traits. This work explores the diversity of mobile genetic elements carried by bacteria associated with plant root surfaces, assessing their capacity to help shape plant–bacterial interactions. The significance of this work lies in the potential to contribute to new strategies for enhancing plant health, promoting sustainable agriculture and managing plant diseases in an era when we must respond to environmental change. Summary Integrons are gene capture and expression systems that contribute to bacterial adaptation. Integron research has mainly focused on the role that these elements play in spreading antimicrobial resistance. However, their contribution to niche adaptation is potentially much broader because integrons can sample the vast repertoire of diverse functions encoded by integron gene cassettes. Integrons and gene cassettes have been identified in many bacterial lineages residing in soil and water across varied ecosystems, but there has been little investigation of integrons in plant‐associated bacteria. Bacteria and plants have complex, dynamic relationships that influence plant health and productivity. To investigate whether integrons contribute to adaptative processes in plant microbiomes, we examined gene cassette and microbial taxonomic profiles in rhizoplanes of four important crop species grown under controlled glasshouse conditions. We identified 38,546 unique gene cassettes, including elements carrying genes associated with antibiotic resistance, type II toxin–antitoxin systems and genes with putative functions associated with plant growth promotion, along with a larger set encoding genes of unknown functions. Rhizoplane microbiomes of different plant species showed more similarity in their community composition profiles than in their gene cassette profiles, with complex and distinct suites of gene cassettes associated with each plant species, suggesting that gene cassettes might have a role in specific plant–bacterial interactions. We show that rhizoplane microbiomes carry diverse integron gene cassettes that could play a role in establishing and maintaining rhizoplane communities