Browsing by Author "Kirk, Ashlyn Michelle"

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    Exploration of the antimicrobial capabilities of Pantoea: Insights into a metabolically diverse genus
    (Faculty of Graduate Studies and Research, University of Regina, 2024-06) Kirk, Ashlyn Michelle; Stavrinides, John; Russell, Jennifer
    The rise of antimicrobial resistance has once again renewed interest in the development of novel antimicrobials for the treatment of resistant pathogens. The genus of bacteria Pantoea, a member of the Erwiniaceae, has been shown to produce several unique antimicrobials with diverse targets. Chapter one gives a review of the known antimicrobials produced by Pantoea including their discovery, genetic origins, biosynthetic pathways, structures, mechanisms of action and resistance, distributions, and insights into their evolutionary histories. Chapter two describes a replica-plating technique that was developed to quickly and easily screen transposon mutant libraries to discover the biosynthetic gene clusters responsible for antimicrobial biosynthesis in bacteria, using Pantoea as a model. This method uses filter paper to transfer up to 200 mutants simultaneously onto soft agar overlays or spread plates containing a target microbe to screen for loss of antibiotic production. The efficacy of this method was demonstrated by re-screening for antibiotic biosynthetic gene clusters for three previously identified Pantoea antibiotics: Pantoea natural product (PNP) 1, 2, and 3. This method was then used to identify the biosynthetic gene cluster that was producing an unknown antibiotic in the clinical isolate P. agglomerans 20KB447973, which we named PNP-5. The recovered biosynthetic gene cluster for PNP-5 shows similarity to the broadspectrum dithiolopyrrolone antibiotic, holomycin. PNP-5 shows broad-spectrum activity against members of the Enterobacteriaceae, Erwiniaceae, and Streptococcaceae, including clinically relevant pathogens such as Klebsiella spp. and Escherichia coli. We also identified a second antibiotic produced by P. agglomerans 20KB447973: pantocin A. Chapter three aims to uncover the distribution and provide insight into the potential evolutionary history of known Pantoea antimicrobial biosynthetic gene clusters. A survey of 12 known clusters against public databases and our genome collection show that some clusters are found strictly in Pantoea, while others are more broadly distributed in distantly related genera within Vibrionaceae, Pectobacteriaceae, Yersiniaceae, Morganellaceae, and Hafniaceae. We compared the evolutionary history of these gene clusters to a cpn60-based species tree, considering the flanking regions of each cluster to provide some context for potential horizontal transfer events. Our analyses identified potential occurrences of horizontal gene transfer in most clusters, supporting relatively frequent exchanges of these metabolite biosynthetic genes. Chapter four provides conclusions and future directions for this work. Overall, this work provides insight into the antimicrobial versatility of Pantoea and its potential for providing novel antimicrobial scaffolds that may be useful for therapeutic development. Keywords: Pantoea, antimicrobial resistance, bacterial natural products, antimicrobial biosynthetic gene clusters