Building the Biofilm Matrix: Gene Regulation and Cell Organization by Janet Elizabeth Price A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and Developmental Biology) in The University of Michigan 2020 Doctoral Committee: Professor Matthew R. Chapman, Chair Professor Robert A. Bender Professor Philip C. Hanna Professor Ursula Jakob Professor Lyle A. Simmons Janet E. Price [email protected] ORCID iD: 0000-0002-4385-9283 DEDICATION For Kyle, Pierce, and Elliot, who always were there to remind me there is life outside of lab. For my family. For Mika, my loudest and proudest cheerleader. ii ACKNOWLEDGEMENTS Progress in a PhD would not be possible without a dedicated community of friends and supporters. I have been fortunate to always have an ear to listen to all my ideas and a shoulder to cry on when experiments go awry. Through their actions I am able to complete this dissertation. To all that have been there for me over the years, a heartfelt thank you. To a few individuals instrumental in my success, I wish to acknowledge them individually. I could not have imagined a better mentor than Sir Dr. Professor Matt Chapman. Matt has an enthusiasm for science that can be felt after spending any amount of time with him. He welcomed me into his lab and pushed me to be a better scientist every day. Knowing that I didn’t plan to stay in academia, Matt went above and beyond to encourage me to seek out opportunities to grow in business and communication. While his impressive dance moves never rubbed off on me, I owe him everything for the scientist I am today. The Chapman Empire has been my home away from home. I want to thank Dave Hufnagel, Maggie Evans, and Neha Jain for showing me the ropes and setting a high bar to aspire to. For a while, Neha and I were the Chapman Lab. We spent many long days in lab together and our research bay always echoed with her loving yells. Elizabeth Gichana (eLiz) is one of the most generous people I have ever met. She has looked out for me and helped me get out of my comfort zone by being my wing person at all the intimidating biotech mixers. The whole Chapman Lab has been made full of laughs, inside jokes, poorly drawn turtles, and amazingly supportive friends. For that I thank Sujeet Bhoite, Anthony Balistreri, Kanna Nagamatsu, Hema Swasthi, Nana Britwum, and Mark Gomulinski. I also want to thank the undergraduates I have worked with: Hakam Nouri, Matt Tyl, and Serge Albarian. They were tirelessly dedicated to the lab and their own curated projects. Our lab is surrounded by brilliant scientists. The Vecchiarelli and Miller Labs have been the ideal neighbors and partners in awesome costumes. Anne Hakim is a tour de force. She was my partner in commiseration and a model of how to have a full family life while taking no quarter in lab. Banter between Joe Basalla, Lisa Tran, and Rees Rillema is sharp and mirthful, but they are always willing to bounce ideas and lend a hand. My favorite time of year is when the Freddolino lab and I travel to Madison, WI for the Phage conference. I cherish times on the patio geeking out over the recent talks with the whole gang. One of the finest minds I met at U of M belongs to Taylor Nye. Her drive and confidence inspired me to always work harder. I will sorely miss our philosophizing over beverages. Shyama Nandakumar was with me through the ups and downs of grad school. I do not know where she finds the energy to do everything she does, but she is kind enough to keep me in the loop. We started together and are now finishing together. I want to thank the Department of MCDB for all the support. Mary Carr is an invaluable resource and always ready with kind words and advice. She made sure all the i’s were dotted iii and t’s were crossed for everyone in her charge. I also want to thank Gregg Sobocinski for all the help he gave me with imaging. Even without a window, the microscope basement is a cheery place. And Gregg keeps it running like a swiss watch. I want to thank my committee for always pushing me. Their candid discussions have led to many interesting scientific rabbit holes to explore. A few of which made it into these pages. I am be endlessly grateful for the time and effort they spent shaping me into the scientist I am today. Finally, I want to thank my family for believing in me. iv TABLE OF CONTENTS DEDICATION ..................................................................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................................... iii LIST OF FIGURES ............................................................................................................................ viii LIST OF TABLES ................................................................................................................................. x ABSTRACT ........................................................................................................................................ xi CHAPTER I........................................................................................................................................ 1 Introduction ................................................................................................................................ 1 Life in a Microbial World ............................................................................................................ 1 Biofilm Model Systems ............................................................................................................... 2 Master Biofilm Regulator, CsgD .................................................................................................. 3 Components of the Extracellular Matrix ................................................................................... 4 Identifying Cell Processes Which Effect Biofilm Formation ...................................................... 5 Conclusion .................................................................................................................................. 6 CHAPTER II....................................................................................................................................... 8 The Assembly of Curli Amyloids Fibers is Deeply Integrated into the Biology of Escherichia coli ..................................................................................................................................................... 8 Abstract ....................................................................................................................................... 8 Introduction ................................................................................................................................ 9 Results and Discussion .............................................................................................................. 10 Materials and Methods ............................................................................................................. 23 Conclusions ............................................................................................................................... 28 Figures and Tables .................................................................................................................... 29 CHAPTER III.................................................................................................................................... 70 Establishment of Biofilm Subpopulation and Structure Requires Anaerobic Respiration ....... 70 v Abstract ..................................................................................................................................... 70 Introduction .............................................................................................................................. 71 Methods .................................................................................................................................... 73 Results ....................................................................................................................................... 79 Discussion.................................................................................................................................. 84 Figures and Tables .................................................................................................................... 88 CHAPTER IV ................................................................................................................................. 112 Thiol Starvation Induces Redox‐Mediated Dysregulation of Escherichia coli Biofilm Components ............................................................................................................................ 112 Abstract ................................................................................................................................... 112 Importance .............................................................................................................................. 114 Introduction ............................................................................................................................ 114 Results ..................................................................................................................................... 116 Discussion...............................................................................................................................