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Diverse Environmental Pseudomonas Encode Unique Secondary Metabolites That Inhibit Human Pathogens

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Diverse Environmental Pseudomonas Encode Unique Secondary Metabolites That Inhibit Human Pathogens DIVERSE ENVIRONMENTAL PSEUDOMONAS ENCODE UNIQUE SECONDARY METABOLITES THAT INHIBIT HUMAN PATHOGENS Elizabeth Davis A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2017 Committee: Hans Wildschutte, Advisor Ray Larsen Jill Zeilstra-Ryalls © 2017 Elizabeth Davis All Rights Reserved iii ABSTRACT Hans Wildschutte, Advisor Antibiotic resistance has become a crisis of global proportions. People all over the world are dying from multidrug resistant infections, and it is predicted that bacterial infections will once again become the leading cause of death. One human opportunistic pathogen of great concern is Pseudomonas aeruginosa. P. aeruginosa is the most abundant pathogen in cystic fibrosis (CF) patients’ lungs over time and is resistant to most currently used antibiotics. Chronic infection of the CF lung is the main cause of morbidity and mortality in CF patients. With the rise of multidrug resistant bacteria and lack of novel antibiotics, treatment for CF patients will become more problematic. Escalating the problem is a lack of research from pharmaceutical companies due to low profitability, resulting in a large void in the discovery and development of antibiotics. Thus, research labs within academia have played an important role in the discovery of novel compounds. Environmental bacteria are known to naturally produce secondary metabolites, some of which outcompete surrounding bacteria for resources. We hypothesized that environmental Pseudomonas from diverse soil and water habitats produce secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. To address this hypothesis, we used a population based study in tandem with transposon mutagenesis and bioinformatics to identify eight biosynthetic gene clusters (BGCs) from four different environmental Pseudomonas strains, S4G9, LE6C9, LE5C2 and S3E10. Of the eight BGCs identified, seven had putative products of non-ribosomal peptide synthetases and one had a putative product of a phenazine. All compounds appeared to be diverse and potentially novel, but further biochemical research must be done to verify these findings. Overall, we were able to identify genes that encode secondary iv metabolites capable of inhibiting the growth of CF derived P. aeruginosa as well as other human pathogens. This research has created ground work for the possibility of extracting, characterizing and developing new antibiotics. v For Mikayla vi ACKNOWLEDGMENTS I would like to acknowledge Bowling Green State University and the Biological Sciences department for this wonderful opportunity to learn and grow. I would like to acknowledge my committee members for all their help and support. Dr. Ray Larsen for always being right across the hall, and allowing me to stop in at any time with questions. Also for being such a big supporter of myself and my research. Dr. Jill Zeilstra-Ryalls for all the wonderful advice and the great walks to class twice a week. Working with her has been a great pleasure and I am very thankful for her support during this process. I want to acknowledge Dr. Hans Wildschutte for being an amazing advisor and leading by example through hard work and dedication. I also cannot thank him enough for the feedback and help at any time of the day, even if he was super busy, he always stopped and helped with anything. I would like to acknowledge my lab mates because without them my time in the lab would have been much more difficult. Thank you, Payel Chatterjee, for always helping me. Thank you for teaching me techniques and life skills that will be so beneficial in the future. Thank you, Joe Basalla, for always giving amazing insight on my research and being able to think outside the box. Also thanks to both Payel and Joe for being my coffee break buddies. Finally, I must acknowledge, Britney Eggly, Mahnur Khan and Emily Vervrugge for all the help with mutant hunts and any other task done in the lab. Finally, I would like to acknowledge my family for love and support throughout this process and my friends for always letting me vent to them. I want to give the biggest thank you to Matthew Burgess. He was such an important part of this process, supporting me and believing in me when I did not believe in myself. vii TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION ........................................................................................... 1 1.1 The antibiotic resistance crisis ............................................................................. 1 1.2 Cystic fibrosis and Pseudomonas aeruginosa ..................................................... 4 1.3 Pseudomonas as a model organism ..................................................................... 5 1.4 Antagonistic activity of environmental Pseudomonas ........................................ 8 1.5 Objectives ............................................................................................................ 11 CHAPTER II. MATERIALS AND METHODS .................................................................. 12 2.1 Selecting isolates .................................................................................................. 12 2.2 Growth conditions ................................................................................................ 12 2.3 Transposon (Tn) mutagenesis .............................................................................. 13 2.3.1 Tri-parental mating filter method .......................................................... 14 2.3.2 Tri-parental mating spotting method .................................................... 14 2.4 Optimization of transposon (Tn) mutagenesis ..................................................... 15 2.5 Replica plating ..................................................................................................... 16 2.6 Optimization of mutant screening ........................................................................ 16 2.7 Scaled up mutant hunt and verification of mutants ............................................. 18 2.8 Antagonistic assay using human pathogens ......................................................... 19 2.9 Mutant DNA extraction and PCR to identify transposon insertion ..................... 19 2.9.1 Linker-mediated (LM) PCR .................................................................. 19 2.9.2 Arbitrary PCR ....................................................................................... 20 2.10 Wildtype DNA extraction and genome sequencing ........................................... 21 viii TABLE OF CONTENTS Page CHAPTER III. RESULTS ..................................................................................................... 22 3.1 Strains tested for the ability to undergo Tn mutagenesis ..................................... 22 3.2 Optimization of Tn mutagenesis .......................................................................... 23 3.3 Optimization of mutant screen ............................................................................. 25 3.4 Optimization and mutant hunt results .................................................................. 26 3.4.1 Strain S4G9 ........................................................................................... 26 3.4.2 Strain LE6G11 ...................................................................................... 27 3.4.3 Strain LE6C9 ........................................................................................ 27 3.4.4 Strain LE5C2 ........................................................................................ 28 3.4.5 Strain S3E10 ......................................................................................... 28 3.5 Antagonistic assay against humans pathogens .................................................... 29 3.6 PCR and sequencing results ................................................................................. 29 3.7 Analysis of genomes and biosynthetic gene clusters (BGCs) ............................. 31 3.7.1 Alignment of mutants to wildtype genome ........................................... 31 3.7.2 Wildtype whole genome annotation results .......................................... 32 3.7.3 Identification and characterization of biosynthetic gene clusters (BGCs) ..................................................................................... 32 3.7.4 S4G9 gene clusters ................................................................................ 34 3.7.5 LE6C9 gene clusters ............................................................................. 34 3.7.6 LE5C2 gene clusters ............................................................................. 35 3.7.7 S3E10 gene clusters .............................................................................. 36 ix TABLE OF CONTENTS Page 3.7.8 Comparison of gene clusters ................................................................. 37 3.7.9 Average nucleotide identity (ANI) ....................................................... 37 CHAPTER IV. DISCUSSION............................................................................................... 39 4.1 Population-level diversity of Pseudomonas......................................................... 39 4.2 Transposon mutagenesis and bioinformatics ....................................................... 41 4.3 Secondary metabolites of Pseudomonas .............................................................. 43 4.4
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