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Investigation Into the Evolution of Heterogeneity Within Secondary Replicons and Their Maintenance in Genus Variovorax

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Investigation Into the Evolution of Heterogeneity Within Secondary Replicons and Their Maintenance in Genus Variovorax California State University, San Bernardino CSUSB ScholarWorks Electronic Theses, Projects, and Dissertations Office of aduateGr Studies 8-2021 INVESTIGATION INTO THE EVOLUTION OF HETEROGENEITY WITHIN SECONDARY REPLICONS AND THEIR MAINTENANCE IN GENUS VARIOVORAX Christopher Ne Ville California State University - San Bernardino Follow this and additional works at: https://scholarworks.lib.csusb.edu/etd Part of the Bioinformatics Commons, Biology Commons, Biotechnology Commons, and the Evolution Commons Recommended Citation Ne Ville, Christopher, "INVESTIGATION INTO THE EVOLUTION OF HETEROGENEITY WITHIN SECONDARY REPLICONS AND THEIR MAINTENANCE IN GENUS VARIOVORAX" (2021). Electronic Theses, Projects, and Dissertations. 1309. https://scholarworks.lib.csusb.edu/etd/1309 This Thesis is brought to you for free and open access by the Office of aduateGr Studies at CSUSB ScholarWorks. It has been accepted for inclusion in Electronic Theses, Projects, and Dissertations by an authorized administrator of CSUSB ScholarWorks. For more information, please contact [email protected]. INVESTIGATION INTO THE EVOLUTION OF HETEROGENEITY WITHIN SECONDARY REPLICONS AND THEIR MAINTENANCE IN GENUS VARIOVORAX A Thesis Presented to the Faculty of California State University, San Bernardino In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology by Christopher Ne Ville August 2021 INVESTIGATION INTO THE EVOLUTION OF HETEROGENEITY WITHIN SECONDARY REPLICONS AND THEIR MAINTENANCE IN GENUS VARIOVORAX A Thesis Presented to the Faculty of California State University, San Bernardino by Christopher Ne Ville August 2021 Approved by: Dr. Paul Orwin, Committee Chair, Biology Dr. Jeremy Dodsworth, Committee Member Dr. Daniel Nickerson, Committee Member © 2021 Christopher Ne Ville ABSTRACT Approximately 10% of all bacterial genomes sequenced thus far contain a secondary replicon. This considerable genetic reservoir contains many potentially mobilizable elements, allowing for the formation of many unique secondary replicons. This property of bacterial populations vastly increases the genomic diversity available to species that effectively take up and maintain these replicons. Members of the genus Variovorax have extensive heterogeneity in genome architecture, including sequenced isolates containing plasmids, megaplasmids, and chromids. Using available Illumina data on the NCBI database, we have completed these assemblies using 3rd generation sequencing methods on 17 members of this genus. We have sequenced, assembled, and evaluated these now complete Variovorax genomes to examine the diversity of elements. From these assemblies 9 chromids, 7 megaplasmids, 2 plasmids, and an integrated megaplasmid were identified using genomic frequency characteristics. We observed in the genomic characteristics data that there is a pattern of evolution where a plasmid is picked up by the organism and over evolutionary time, there is expansion of the replicon in size through acquisition of mobile elements and interreplicon transfer. As this process goes on, the secondary replicons’ genomic frequency characteristics regress toward the chromosome. Given the evolutionary pattern seen, we were interested to find out if the high levels of heterogeneity present in Variovorax are due to factors controlling secondary replicon maintenance present within the primary iii chromosome. Using two strains of Variovorax paradoxus strains, VAI-C (multipartite genome) and EPS (single chromosome) and two plasmids pRU1105 and pBBR5pemIKpBAD (which contains a Toxin-Antitoxin system), were used to evaluate the maintenance of plasmids in the absence of selection. Surprisingly, the Toxin-Antitoxin system had a deleterious effect and showed no additional phenotype in either organism. iv ACKNOWLEDGEMENTS We would like to thank Cal State University, San Bernardino’s Office of Student Research and the office of Associated Students Incorporated their funding of this project. We also thank the Vital and Expanded Technologies Initiative at CSU San Bernardino for funding of classroom-based sequencing. Without their support much of the sequencing done in this project would not have been possible. We would also like to thank our wonderful collaborators Dr. Jeff Dangl at UNC-Chapel Hill, Dr. Jared Ledbetter at Cal Tech, Dr. Kostas Konstantinidis at Georgia Institute of Technology and the Noble research group for providing the Variovorax strains and Illumina data used in this project. Starting out on this project we were a bit unsure of ourselves if we were actually going to be able to make this project work, without your support early on it would have never got off the ground. We thank Jose de la Torre for generously providing the MiSeq short read sequencing of V. paradoxus CSUSB. I personally would like to thank Dr. Paul M. Orwin, for taking me under his wing as an undergraduate and through my graduate studies. Without his unwavering support and dedication, I would not be here today. I spend much of my first year in his office (I am fairly certain he thought I was a pain in his rear) because I felt so overwhelmed, and behind in my studies. It is through his guidance and relentless challenging of me that I was able to catch up in my studies and graduate with both my degrees. I personally would also like to thank Dr. Dan P. Nickerson, that first year in molecular biology I was a slacker full of v self-doubt that had failed too many times. It was your early belief and assistance that allowed me to finally start clawing my way back into my scholastic studies. Thank you for guidance throughout my experience at CSUSB. Lastly, I would like to thank Dr. Jeremy Dodsworth it was his passion for genomics in my final year of my undergraduate studies which helped alter the course of my studies. It was from those two years I got to TA with you that I learned a lot about scientific education and working with students. Not to mention our many random hallway chats that helped me develop much of the DNA analysis in my thesis turning it into what it became. I would like to thank all 3 of these professors without their ceaseless dedication I was able to grow as a scientist, a scholar and grow as a person. I would like to thank my partner Monique Quinn for her amazing support throughout my graduate degree, there were many nights where I was tired and on the verge of a total mental collapse. Without your support during this time, I do not know what I would have done or what would have happened. It was your support during the CoViD-19 crisis that helped me get through it and focus on my thesis rather than the world falling apart around us. I also thank my cat Rex for being the Fluffiest lambkin kitty and helping to relieve stress with the going got rough. Finally, I would like to thank my family, it was because of your support that I was able to make this far. To my grandparents thank you for your moral support and the many hours you spent listening to me, as well as your financial support during my education it is due to your assistance, I was able to afford to go to vi university. To my loving parents I would like to thank you for their moral encouragement, financial assistance as well their spiritual support in my college education. Without whom I would have never been able to enjoy the many opportunities I have been given. It is unfortunate that my father passed in my 2nd year of my master’s thesis and never got to see me finish but I am certain he would be proud. Thank you to my mother, I know that last couple years have been rough, but you have always supported and been there for me. You have always been there for me and there are no words to thank you for all that you have done for me. vii TABLE OF CONTENTS ABSTRACT .......................................................................................................... iii ACKNOWLEDGEMENTS ..................................................................................... v LIST OF TABLES ................................................................................................. xi LIST OF FIGURES .............................................................................................. xii CHAPTER ONE: INTRODUCTION ...................................................................... 1 Types of Replicons .................................................................................... 2 How Secondary Replicons Arise ................................................................ 3 Genomic Signatures and What They Tell Us ........................................... 11 Mobilization of Large Secondary Replicons and Purpose ........................ 16 Toxin Antitoxin Systems ........................................................................... 21 Project Focus ........................................................................................... 23 Thesis Statement ..................................................................................... 30 Specific Aims ........................................................................................... 32 Aim 1 ............................................................................................. 32 Aim 2. ............................................................................................ 33 CHAPTER TWO: MATERIALS AND METHODS ............................................... 35 Specific Aim 1 .......................................................................................... 35 Genomic DNA Isolation ................................................................
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