On the taxonomy and functions of integrons
Creators: name: Ghaly, Timothy M.
Publisher: name: Macquarie University
Year: 2024
Subjects: classificationCode: 399999
subject: Other education not elsewhere classified

description: Integrons are genetic elements that helped precipitate the global antimicrobial resistance crisis. However, the role that integrons have played in the resistance crisis is just a specialised case of their more general role in microbial evolution. Their ability to capture a diverse range of gene cassettes means that they can drive rapid adaptation to diverse selection pressures. Integrons have played this role for perhaps hundreds of millions of years. They generate extensive genomic and phenotypic diversity through rearrangements of modular, mobile gene cassettes and the proteins that they encode.   
To date, most research has focused on mechanistic studies and their role in antimicrobial resistance. However, these studies have largely been based on integrons present in clinical settings. Large knowledge gaps on the taxonomic and functional diversity of environmental integrons remain. In this thesis, I employ a range of experimental and bio informatic approaches to establish novel methods and key findings regarding integron ecology, including:
<em>(i)</em> <em>Predicting sources of mobile gene cassettes</em>. I developed a novel approach to predict the taxonomic and environmental sources of individual gene cassettes. For this, I modelled the sequence and structural homology of gene cassette recombination sites (<em>attC</em>s) from the chromosomes of diverse taxa. I showed that these taxon-specific <em>attC </em>models could, with high accuracy (specificity ≥ 98%), predict their source taxa. I used this approach to predict the sources of known antibiotic resistance gene cassettes that are now prevalent among diverse bacterial species across the globe. Importantly, this work highlights key taxonomic and environmental hotspots for the emergence of resistance cassettes.  
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<em>(ii)</em> <em>Developing standardised methods</em>. Currently, a PCR-based approach represents the most efficient method of studying integrons within complex microbial communities. However, a consequence of this approach is the significant potential for non-genuine, off-target amplification. Until now, there has been no proposed method for dealing with this problem, which has serious implications for the reliability of any biological conclusions. In response to this, I developed reproducible experimental and computational methods for the PCR amplification, sequence processing, and importantly, the validation of gene cassette sequences from complex communities. These methods can aid future studies of integrons and can help foster reliable and standardised results.  
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<em>(iii)</em> <em>The discovery of integrons in Archaea</em>. Integrons, since their discovery more than 30 years ago, have been thought to only exist within Bacteria. However, in this thesis, I show for the first time that integrons are also prevalent among diverse Phyla within the domain Archaea. Importantly, integrons can mediate the recruitment of diverse archaeal gene cassettes by a bacterial host. Such a mechanism facilitates a previously unknown pathway of gene transfer between the two domains of prokaryotes, Archaea and Bacteria. 
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<em>(iv)</em> <em>Uncovering the functional diversity of gene cassettes</em>. Despite their broad taxonomic distribution, geographical prevalence, and evolutionary potential, the functions of gene cassettes remain poorly characterised. In this thesis, I examine the functional diversity of environmental gene cassettes. I find that a subset of functions that are largely involved with mediating biotic and abiotic interactions, is universally enriched relative to broader metagenomes. Beyond the dominant functions, most are rare and sample-specific, and are likely relevant to their microenvironment. Overall, the gene cassette pool is distinct from metagenomes, enriching niche functions of the prokaryotic pangenome. 
Together, work presented in this thesis provides significant advancements to the field of integron ecology. I present findings that expand our knowledge of the taxonomic distribution of integrons beyond Bacteria, highlight these elements as facilitators of cross-domain gene transfer, and investigate the functional repertoire of integron gene cassettes. Additionally, I develop experimental and bioinformatic methods for the analysis of integrons and their gene cassettes to aid future studies within the field.