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Identification of the Essential Genome of Burkholderia Cenocepacia K56-2 to Uncover Novel

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Identification of the Essential Genome of Burkholderia Cenocepacia K56-2 to Uncover Novel Identification of the Essential Genome of Burkholderia cenocepacia K56-2 to Uncover Novel Antibacterial Targets by April S. Gislason A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology University of Manitoba Winnipeg, Manitoba, Canada Copyright © 2017 April S. Gislason ii Abstract Burkholderia cenocepacia K56-2 is a clinical isolate of the Burkholderia cepacia complex, a group of intrinsically antibiotic resistant opportunistic pathogens, for which few treatment options are available. The identification of essential genes is an important step for exploring new antibiotic targets. Previously, a conditional growth mutant library was created using transposon mutagenesis to insert a rhamnose-inducible promoter into the genome of B. cenocepacia. It was then demonstrated that conditional growth mutants under-expressing an antibiotic target were sensitized to sub-lethal concentrations of that antibiotic. This thesis describes the use of next generation sequencing to measure the enhanced sensitivity of conditional growth mutants after being grown together competitively. Evidence is provided to show that the sensitivity of a GyrB conditional growth mutant to its cognate antibiotic, novobiocin, was enhanced due to competitive growth. Screening eight antibiotics with known mechanisms of action against a pilot conditional growth mutant library revealed a previously uncharacterized two-component system involved antibiotic resistance in B. cenocepacia. With the goal of exploring new Burkholderia antibacterial targets, the essential genome of B. cenocepacia K56-2 was identified by sequencing a high density transposon mutant library. Comparison of the B. cenocepacia K56-2 essential gene set with the essential genomes predicted for B. thailandensis, B. pseudomallei, and B. cenocepacia J2315 revealed strain-specific essential genes and 158 essential genes that are common to species in the Burkholderia genus. Three genes were identified that are essential in Burkholderia but not in other bacterial essential genomes identified so far, suggesting that specific cell envelope functions are critical to survival of Burkholderia species. To increase the diversity of the conditional growth mutant library, a method to enrich conditional growth mutants from the high density mutant library was developed and used iii to isolate 830 conditional growth mutants. With the addition of these mutants, the conditional growth mutant library currently represents 23.2% of the essential operons in B. cenocepacia K56- 2 and 36.7% of the essential genes that are common to species in the Burkholderia genus. In summary, this thesis has contributed to create genomic tools available for antibiotic research and has identified novel targets for fighting Burkholderia related infections. iv Acknowledgements First and foremost, I would like to acknowledge my PhD supervisor, Dr. Silva Cardona, who inspired me to pursue my PhD studies. Throughout my time spent in her research laboratory, both as a technician and a student, she always gave me the opportunity to explore my interests and encouraged me to challenge myself. I will be forever grateful to her for her guidance and support. I would also like to thank my committee members, Dr. Brian Mark, Dr. Ivan Oresnik and Dr. Steve Whyard, I greatly appreciated their guidance and valuable suggestions. I would like to thank Dr. Xuan Li for his help with the statistical analysis. Thank you to Wubin Qu, Dr. Mike Domaratzki, Dr. Ruhi Bloodworth and Dr. Keith Turner for their help with bioinformatics analysis and Dr. Larry Gallagher for his help with the Tn-seq circle method. I would also like to acknowledge the support provided by Faculty of Science Graduate Scholarships and Graduate Students Association Travel Awards, during my PhD studies. I sincerely thank all the professors, graduate students and support staff in the Department of Microbiology, including the past and present members of the Cardona lab, for creating such a welcoming and friendly environment to work in. To Dr. Deb Court and Dr. Teri de Kievit, I sincerely appreciate your kindness and encouragement during the stressful times. I thank my wonderful lab family - Tasia Lightly, Dr. Silvina Stietz, Dr. Carrie Selin, Sol Haim, Tanya Pribytkova, Brijesh Kumar, Matthew Choy and Andrew Hogan for being so entertaining and for your friendship. Finally, to Mom, Dad, Amber, Sean, Kristjana, Gina and Mark - thank you for your love and support, and for always believing in me. v Contributions of authors Multiple authors contributed to the work described in Chapters 2, 3 and 4. For all the work outlined in these chapters, Dr. Silvia T. Cardona was involved in the experimental design and interpretation, as well as manuscript preparation. Dr. Xuan Li provided extensive help with the statistical analysis outlined in section 2.2.4. Wubin Qu designed the multiplex primer set outlined in 2.2.3. Matthew Choy carried out the experiments described in sections 2.2.8, 2.2.9 and 2.2.10. Dr. Silvina Stietz carried out the microscopy described in section 2.2.12. Dr. Keith Turner developed and performed the bioinformatics analysis for essential genes outlined in section 3. Dr. Ruhi Bloodworth and Dr. Mike Domaratzki wrote code and provided bioinformatic support for the analysis of the CG mutant library outlined in Chapters 3 and 4. Carmicheal Mabilangan was involved in generating and screening the conditional growth mutants described in Chapter 4. vi List of copyrighted material for which permission was obtained Chapter 1: Critical Reviews in Microbiology 2014 March; 41(4):465-72, Cardona ST, Selin C, Gislason AS. Genomic tools to profile antibiotic mode of action. Copyright © 2014 Informa Healthcare USA, Inc. http://dx.doi.org/10.3109/1040841X.2013.866073. Excerpt reproduced with permission. MicrobiologyOpen. 2013 April; 2(2):243-258. doi: 10.1002/mbo3.71. Burkholderia cenocepacia conditional growth mutant library created by random promoter replacement of essential genes. (Figure 1.2) Published under the Creative Commons Attribution License, allowing reproduction with attribution. Chapter 2: Antimicrobial Agents and Chemotherapy 2016 Dec; 27;61(1), Gislason AS, Choy M, Bloodworth RA, Qu W, Stietz MS, Li X, Zhang C, Cardona. Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia. Copyright © 2017, American Society for Microbiology, authors retain the right to reuse the full article in his/her thesis. Chapter 3: MBio. 2011 Jan 18;2(1):e00315-10. Gallagher LA, Shendure J, Manoil C. Genome- scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. Copyright © 2011 Gallagher et al. Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. vii Table of contents Abstract ii Acknowledgements iv Contributions of authors v List of copyrighted material for which permission was obtained vi List of tables x List of figures xi List of abbreviations xiii Chapter 1: Literature review 1 1.1 The Burkholderia cepacia complex 2 1.1.1 Ecology 2 1.1.2 Infections with Bcc 4 1.1.3 Immunocompromised individuals 4 1.1.4 Chronic granulomatous disease 5 1.1.5 Cystic fibrosis 6 1.2 B. cenocepacia 8 1.2.1 B. cenocepacia genome 8 1.2.2 Genomic islands 9 1.2.3 B. cenocepacia antibiotic resistance 11 1.2.4 Modified lipopolysaccharide 11 1.2.5 Biofilms 12 1.2.6 Efflux pumps 13 1.2.7 Porins 13 1.3 Antibiotic development 14 1.3.1 In vitro assays 15 1.3.2 Whole cell screens 16 1.4 Essential genes as antibiotic targets 17 1.4.1 Essential genes 17 1.4.2 Identification of essential genes 18 1.4.3 High density transposon mutagenesis 18 1.4.4 Analysis for determining the essential genome 19 1.4.5 Whole cell chemogenetic screens 21 1.4.6 Essential genes in chemogenetic screens 23 1.5 Thesis objectives 29 Chapter 2: Competitive growth enhances conditional growth mutant sensitivity to antibiotics and exposes a two-component system as an emerging antibacterial target in Burkholderia cenocepacia 30 2.1 Introduction 31 viii 2.2 Materials and methods 33 2.2.1 Bacterial strains and growth conditions 33 2.2.2 Growth inhibitors 38 2.2.3 Multiplex PCR optimization, amplicon library preparation and Illumina sequencing of amplicons 38 2.2.4 Data analysis 44 2.2.5 Artificial depletion of CG mutants 45 2.2.6 Competitive enhanced sensitivity assay 45 2.2.7 Comparison of pooled and clonal growth 48 2.2.8 Construction of the unmarked esaS deletion mutant, MKC4 (ΔesaS) 50 2.2.9 Site directed mutagenesis to construct the esaR conditional growth mutant MKC2 (CGesaR). 54 2.2.10 MIC ratios 54 2.2.11 Ala-Nap uptake assay 55 2.2.12 Microscopy analysis 55 2.3 Results 58 2.3.1 Quantification of CG mutant relative abundance by Illumina sequencing of multiplex- PCR amplified transposon junctions 58 2.3.2 A competitive enhanced sensitivity assay enhanced the specific depletion of a CG mutant to its corresponding antibiotic 64 2.3.3 The competitive enhanced sensitivity assay reveals a two-component system (TCS) involved in regulation of antibiotic resistance 68 2.3.4 Mutational analysis of the esaSR locus suggests that esaR is an essential gene 71 2.3.5 The esaSR locus is involved in drug efflux activity and cell membrane integrity 75 2.4 Discussion 81 Chapter 3: Identification and comparative analysis of the essential genome of Burkholderia cenocepacia K56-2 provides evidence for conservation of genus-specific essential genes 87 3.1 Introduction 88 3.2 Materials and methods 90 3.2.1 Bacterial strains and growth conditions 90 3.2.2 Production of the 1 million high-density transposon mutant library 92 3.2.3 Molecular biology techniques 93 3.2.4 Genomic DNA isolation 93 3.2.5 Tn-seq circle 94 3.2.6 Data analysis to determine gene essentiality 99 3.2.7 Bioinformatics 100 3.3.
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