Characterization of the Structure, Regulation, and Function of CsgD- mediated Escherichia coli Biofilms By William Henry DePas A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Microbiology and Immunology) in the University of Michigan 2014 Doctoral Committee Associate Professor Matthew Chapman, Chair Assistant Professor Eric Martens Assistant Professor Lyle Simmons Professor Michele Swanson To my parents, Dennis and Mary, and my brother Wade ii Acknowledgements First of all, I would like to thank Matt Chapman for his guidance and support throughout my graduate career. Matt loves life, loves science, and is great at both of them. His curiosity and enthusiasm are infectious, and I knew from the first time I interviewed with him that he would be a fantastic boss to work for. Matt sets the tone of the lab as a fun, open, and productive place to do work. I cannot imagine a better environment to have performed my thesis work in or a better PI to have worked for. My fellow Chapman lab members have not only been fantastic co-workers, they have been some of my best friends while at the University of Michigan. Specifically Yizhou, Maggie, and Dave have made my time here extraordinarily enjoyable. Yizhou is an amazing scientist who offered patient help no matter how busy she was. She was a constant smiling presence in the lab. Maggie joined the lab at the same time I did, and has been a fantastic friend and colleague for the past five years. Maggie’s ideas and opinions have been invaluable, and she has developed her project into one of the coolest stories I have heard of. The trip to Sweden I took with Matt, Maggie, and Yizhou has been one of the highlights of my life. As soon as Dave started rotating, it was obvious that he fit into the lab well, and he has since become a great friend. Dave shares my passion for wrinkled colonies, and seeing him develop into an insightful, independent scientist has been a great experience. I could not imagine a better trio to share five years with. I am confident that Neha will carry on the proud Chapman lab traditions, she is already fitting in very well. The Chapman lab old guard also deserves a large share of thanks. Neal Hammer trained me well in lab and starcraft techniques, both of which proved useful. Dan Smith’s encyclopedic knowledge of all things curli was a valuable resource for me in my early graduate career. Former post-docs Luz Blanco and Matt Badtke were great iii friends and taught me a lot of useful techniques. The knowledge base created by all the other former lab members has been a fantastic tool. I have been fortunate to share lab space with Bob Bender, Blaise Boles, and their respective labs. I cannot think of anything about E. coli metabolism that Bob does not know. He is a fun and knowledgeable mentor. Bob’s last graduate student, Ryan Frisch, was also a great colleague. Even though I was a young roton in a different lab, Ryan went out of his way to teach me techniques and science that I still use to this day. Seeing Blaise start a faculty job and succeed immediately has been fun and inspirational. He has been a great resource. The Boles lab has offered up an energetic, intelligent group of people to do science with. Kelly, Adnan, Dave, Matt, Matt, and Rachel have been nothing but supportive and helpful during my time here. They have also been very good friends. I’ve had the opportunity to work most with Adnan due to his love of making agar plates out of various substances and his appreciation for all things ecological. Collaborating with him on chapter 4 was a great experience. My undergraduate and masters research assistants, David Warshaw, John Lee, and Vinay Saggar, have been very helpful, and working with them has been a lot of fun. Without their hard work and willingness to take on new projects, I would have a much narrower and less exciting dissertation. My committee has been amazingly supportive throughout my graduate career. They were constantly offering suggestions, reagents, and time, all of which helped me along. Being in the Microbiology and Immunology department while residing in an MCDB lab has often presented logistical problems, but both department staffs have been amazing and infinitely patient while dealing with me. Heidi Thompson and Mary Carr specifically have been awesome. Gregg Sobocinski has been a huge help with the imaging aspects of my project. I would also like to thank the Rackham graduate school and the Genetics Training Grant for providing me with funding. Most importantly, I would like to thank my family for their constant support and encouragement. iv Table of Contents Dedication……………………………………………………………………………………..…ii Acknowledgements……………………………………………..………………………………iii List of Figures…….……………………………………………………………..……………...vii List of Tables……..……………………………..………………………………………………ix Chapter 1: General Introduction..…………………………...…………………………………1 Escherichia coli………………………….………………………………………………1 Biofilms……..…………………………………….………………………………………3 CsgD-mediated Biofilms.…………………….…………………………………………4 Curli and Cellulose in vivo..…………………………..………………………………10 Curli and Cellulose in vitro..……………..……………………………………………12 Rugose Biofilms……..…………………………………………………………………13 Figures……..………………………………...…………………………………………16 Notes and Acknowledgements…………….…………………………………………21 References……………………………………..………………………………………21 Chapter 2: Iron Induces Bimodal Population Development by Escherichia coli...……...38 Abstract………………..………………………………………………………..………38 Introduction………………………………………………………………………..……38 Results.……………………..……………………………………………..……………40 Discussion…………………….....……………………………..………………………46 Materials/Methods.……………………...……………………..………………………49 Figures and Tables……………………..………………………..……………………54 Notes and Acknowledgements..……………..………………………………………71 References…………………………………..…………………………………………71 Chapter 3: ArcAB Modulates Escherichia coli Biofilm Formation………………………...77 Abstract…..……………………………………………………………………..………77 v Introduction………………………………………………………………………..……78 Results.……………………………………………..……………………..……………80 Discussion…………..………………………………………….………………………85 Materials/Methods.…………………………...………………..………………………88 Figures and Tables………………………………………………..…………………..91 Notes and Acknowledgements..……………………………………………………105 References……………………………………………………………………………105 Chapter 4: Biofilm Formation Protects Escherichia coli Against Predation...….………111 Abstract…..………………………….………………………………………..………111 Introduction…………………………………….………………………………..……111 Results.………………………………………....………………………..……………113 Discussion…………..………………………….……………..………………………120 Materials/Methods.……………………….…………………..………………………124 Figures.…………………………………………………………..……………………129 Notes and Acknowledgements..……………………………………………………143 References……………………………………………………………………………143 Chapter 5: Discussion, Perspectives, and Future Directions……………...…………….149 Dynamics of bimodal population development…………..………………..………149 Iron and rugose biofilm formation……………………………………………..……152 Iron and CsgA polymerization…………………...…………………………….……154 Rugose biofilm function………………………….…………..………………………156 Figures.………………………………………………....………..……………………159 Materials/Methods…………………………………………...……………………….167 Notes and Acknowledgements..……………………………………………………168 References……………………………………………………………………………168 vi List of Figures Figure 1.1 Genetics of E. coli biofilm formation……………………………………………16 Figure 1.2 Nucleation-precipitation curli fiber assembly…………………………………..17 Figure 1.3 General schematic for amyloid polymerization………………………………..18 Figure 1.4 UTI89 biofilm models…………………………………………………………….19 Figure 1.5 Iron induces UTI89 rugose biofilm formation………………………………….20 Figure 2.1 UTI89 forms CsgD-dependent Rugose Biofilms ………..……………………54 Figure 2.2 Iron induces UTI89 rugose biofilm formation……………………………….…55 Figure 2.3 Both ferrous and ferric iron can trigger UTI89 rugose biofilms ……..………56 Figure 2.4 Two separable populations, matrix producing and non-matrix-producing, are present in a rugose biofilm…………………………………..………………………..………57 Figure 2.5 Washout bacteria do not produce curli or cellulose…………………………..58 Figure 2.6 Confocal microscopy reveals bimodal rugose biofilm architecture…….……59 Figure 2.7 Rugose biofilm structure in low and high iron conditions…………………….60 Figure 2.8 A fur mutant wrinkles in low iron conditions……………………………...……61 Figure 2.9 Iron and superoxide stress drives rugose biofilm formation…………………62 Figure 2.10 Gallium and aluminum can also trigger rugose biofilm formation……….…63 Figure 2.11 Rugose biofilm formation coincides with H2O2 resistance………….………64 Figure 2.12 RpoS-dependent KatE activity is increased in the matrix fraction…………65 Figure 2.13 Iron-dependent rugose biofilm formation in other enterics…………………66 Figure 3.1 Screening redox-regulators for rugose biofilm formation…….……...……….91 Figure 3.2 OxyR affects colony spreading…………………………………………..……..92 Figure 3.3 Single arc mutants and complementation on low iron plates……………..…93 Figure 3.4 ArcB78-778 represses rugose biofilm formation on iron replete plates….....…94 Figure 3.5 rpoS expression is required for rugose biofilm formation but is not sufficient to induce rugose biofilm formation………………………………………………………...…95 vii Figure 3.6 ArcZ increases biofilm wrinkling……………………….………………..………96 Figure 3.7 Rugose biofilm formation in an arcAB mutant is dependent on csgD…..….97 Figure 3.8 ArcAB represses CsgD in low iron conditions…………………………………98 Figure 3.9 arcAB mutant has a decreased washout/matrix ratio……………………...…99 Figure 3.10 Effect of ArcAB on bimodal population development………………...……100 Figure 4.1 Biofilm formation protects