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University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2019 Investigating the microbial “dark matter” of the human oral microbiome using cultivation-dependent and –independent approaches Karissa Cross University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Cross, Karissa, "Investigating the microbial “dark matter” of the human oral microbiome using cultivation- dependent and –independent approaches. " PhD diss., University of Tennessee, 2019. https://trace.tennessee.edu/utk_graddiss/5637 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Karissa Cross entitled "Investigating the microbial “dark matter” of the human oral microbiome using cultivation-dependent and –independent approaches." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Mircea Podar, Major Professor We have read this dissertation and recommend its acceptance: Heidi Goodrich-Blair, Erik Zinser, Frank Loeffler, Stephen Kania Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Investigating the microbial “dark matter” of the human oral microbiome using cultivation-dependent and –independent approaches A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Karissa Lynn Cross August 2019 Copyright © 2019 by Karissa Lynn Cross All rights reserved. ii ACKNOWLEDGEMENTS This acknowledgements section is no doubt one of the most difficult sections for me to write because there have been so many people along my journey who have supported me and have helped make this possible. There are not enough words to address them all but for all of them I am beyond thankful. First, I would like to thank my advisor Dr. Mircea Podar for his support and vision to encourage me to push the limits of what a PhD degree could mean. I thank all of the current and past Podar Lab members: Dr. Snehal Joshi, Shafer Belisle, Zamin Yang, Dr. Manasi Balachandran, Stephen Lebreux, Dr. Jim Campbell, Dr. Alisha Campbell, and Dr. Louie Wurch. I would also like to thank my committee Dr. Heidi Goodrich-Blair, Dr. Erik Zinser, Dr. Frank Loeffler, Dr. Stephen Kania, and formerly Dr. Igor Zhulin who have stood by my side year after year and offered their insight and support. Thank you to my collaborators both near and far throughout the years who have taught me how to listen and learn and have afforded me the opportunities to push my science beyond my comfort zone. Thank you to my friends both at ORNL and UTK whose companionship and love have no doubt made this possible. A special thank you goes to William Ellis Baltz who I met my first year of graduate school and has continued to be by my side through not just the good days but the bad ones as well. I told him if he could make it through law school and I through graduate school at the same time, there is no limit to what we could accomplish together, and he continues to support my dreams steadfast. Also, I am thankful for his family Liana, Billy, and Sarah (and many more) who have made me feel welcomed and loved. Lastly, and at the top of my list, I thank my family. My grandparents, Nancy and Lowell, raised my sister and I and have supported us through all of our ventures. With their support I have had the confidence to pursue my goals where no one in our family has yet gone. They have served as strong role models demonstrating where hard work and perseverance can take you, and about never giving up or backing down. To my sister Ashley, thank you for the many phone calls and advice as we transverse this life together. With every step you have given me the assurance and love to keep moving forward when at times it felt impossible and I look forward to where this life continues to take us. iii ABSTRACT The advancement of sequencing technology has provided a renewed appreciation for the diversity of life on earth with most environments being dominated by yet-uncultured microorganisms. Analysis of 16S rRNA gene sequencing libraries of the original Human Microbiome Project dataset revealed that approximately 70% of sequences belonged to bacteria that were uncultured in the laboratory and current metagenomic analysis of the human microbiome has proposed 77% of taxa lacked sequenced genomes. As the human microbiome is important for healthy host physiology, these findings emphasize just how little we currently know about the microorganisms that reside in the human body. Understanding the microorganisms that make up the normal human microbiome is the first logical step in determining, during dysbiosis and disease, how the community changes as a whole so as to permit investigation into preventative and/or treatment measures during disease. For example, many disease-associated lineages of bacteria in periodontal disease remain uncultured in the laboratory and therefore limited insight is available as to whether or not these organisms play a role in disease etiology or their presence is a secondary effect of the disease state. Therefore, cultivating more microorganisms from the human microbiome is critical as it enables hypothesis-driven experimentation into the roles of those organisms in human health and disease. The work presented here represents a two-fold approach for cultivation of bacteria from the human oral microbiome by both classical means and by the development of a novel, “reverse- genomics” approach. First, I identified a specific interaction between Desulfobulbus oralis and Fusobacterium nucleatum that resulted in the isolation of the first Desulfobulbus genus member from the human oral cavity and I characterized this bacterium both physiologically and genomically. Next, I investigated what causes the recalcitrance of some oral bacteria to cultivation and what contributes to their dependence on other bacteria, such as F. nucleatum. Lastly, we leveraged existing public sequence data to target specific TM7 bacteria from within the microbial community for both single-cell genomic characterization and cultivation. Collectively this work demonstrates how cultivation-dependent and cultivation-independent methodology can be combined to investigate the “dark microbial matter” in the human oral cavity. iv TABLE OF CONTENTS CHAPTER ONE - Introduction ......................................................................................................... 1 Publication Note ................................................................................................................................................ 2 The Human Microbiome and Uncultured Bacteria ............................................................................. 2 The Oral Microbiome and Oral Diseases ................................................................................................ 3 Cultivation-independent Studies ............................................................................................................... 5 Cultivation-dependent Studies ................................................................................................................... 7 Sulfate Reducing Bacteria ............................................................................................................................ 9 Candidate Phylum TM7 Bacteria ............................................................................................................. 10 Non-bacterial Diversity of the Human Oral Cavity .......................................................................... 11 Research Objectives ..................................................................................................................................... 12 CHAPTER TWO - Insights into the evolution of host association through the isolation and characterization of a novel human periodontal pathobiont, Desulfobulbus oralis ......................................................................................................................... 15 Publication Note ............................................................................................................................................. 16 Abstract .............................................................................................................................................................. 16 Importance ........................................................................................................................................................ 17 Introduction ...................................................................................................................................................... 17 Results and Discussion .............................................................................................................................. 19 Materials and Methods................................................................................................................................
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