ENTEROBACTIN EXPORT IN ESCHERICHIA COLI VIA P43 (ENTS) AND ASSOCIATED COMPONENTS A Dissertation Presented to the Faculty of the Graduate School University of Missouri-Columbia In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy by JASON L. FURRER Dr. Mark A. McIntosh, Dissertation Supervisor DECEMBER 2006 The undersigned, appointed by the dean of the Graduate School of the University of Missouri- Columbia, have examined the dissertation entitled: ENTEROBACTIN EXPORT IN ESCHERICHIA COLI VIA P43 (ENTS) AND ASSOCIATED COMPONENTS presented by Jason Lawrence Furrer, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Dr. Mark McIntosh, Dissertation Supervisor Dr. Sandra Abell Dr. Dongsheng Duan Dr. David Lee Dr. Mark Milanick Dr. Judy Wall ii DEDICATION I dedicate this work to my family, Jennifer & my “chubble-hootmonkey-werewolf” son Caleb. They have put up with my random schedules and working late, and kept me encouraged when I was frustrated. Also, this is dedicated to my mom and dad, Carol & Larry, as well as my grandparents, Al & Betty, for supporting me with constant encouragement, guidance, and financial means all throughout school and life. Finally, to all the friends I have met and kept in graduate school: I’ve loved being part of the MMI family and look forward to working with all of you in the future as we progress into our science. iii ACKNOWLEDGEMENTS First and foremost, I have to thank my advisor, Dr. Mark McIntosh. His science, suggestions, mentoring, and genuine interest in students make him a wonderful example for how I should conduct myself as a future scientist. I don’t know if he fully understands in what high regard I hold him for having an active research lab in addition to being able to perform his duties as departmental chair, steer core facilities, have a family, and still be willing to give a pestering graduate student time out of his day. I was given a lot of freedom in lab to design and conduct my own experiments, which always provided a learning experience despite success or failure. Mark was always there to offer suggestions (“What if you made 10 L of radiolabeled enterobactin?”). The lab atmosphere was always one of doing good science and being treated as a colleague rather than a peon. Plus, the man plays golf. What more does one need in an advisor? It’s scary looking back, as I almost didn’t even rotate in Dr. McIntosh’s lab if it wasn’t for someone canceling at the last minute. But, Mark took me in and I have been nothing but happy with my choice. Next, I would like to thank my other committee members, Dr. Sandra Abell, Dr. Dongsheng Duan, Dr. David Lee, Dr. Mark Milanick, and Dr. Judy Wall. Their advice and suggestions have influenced both my experiments and my graduate training. Dr. Milanick deserves special recognition for his technical expertise and suggestions related to the inverted membrane vesicle work. Because of my committee members’ flexibility, I was allowed to pursue interests outside of the laboratory such as teaching and earning a Graduate Minor in College Teaching. In that regard, I have to especially thank Dr. Sandra Abell, who started me down a teaching path I never originally considered and that I now walk confidently. Outside of my committee, I have to thank Dr. Louise Thai and Dr. Michael Misfeldt. Dr. Thai has become a friend and fellow educator, as I have been involved in her class, Micro 3200. Dr. Misfeldt is another teacher I have the utmost respect for and who has additionally provided me with iv excellent advice, conversations, and guidance as a “surrogate boss” during times when my mentor is occupied as departmental chair. As anyone in MMI knows, you acquire a new set of mothers when you join the department. The office staff of Jana Clark, Shelly Crawford, Karen Ehlert, and Cathy LaMere watches over everyone and are always happy to help the graduate students with everything from making copies to figuring out why we didn’t get paid for a month! I shudder to think where the department would be without these fantastic people. If you’ve been here long enough, you also got to know Julie Mitchell, who acted as our graduate coordinator until diagnosed with cancer. I really owe my graduate career to Julie, who fought to get me into the program in the first place and with whom I developed a real bond. I would like to extend my best wishes and fondest memories to her family for all her help in my graduate career. Finally, if that wasn’t enough, I have a day-to-day mother as well in lab: Dr Manijeh Heidari. Manijeh does more than just ordering and daily activities in lab. She is a friend and confidant, even if she and I don’t always see eye-to-eye about the “usefulness” of things in lab. I could not imagine a more caring and selfless person than Manijeh and I wish her (and her daughter Peri, whom I harass on the phone everytime she calls) all the best in the world. I know that one day, Manijeh will win her battle with “the man” (even if she still never gets the joke that we laugh about every time she says it…). I must thank other lab members, including India Hook-Barnard, Jennifer Lavrrar, Wendell French, and Travis Barnard. We had some good times, both in and out of lab, that are too numerous to mention here, although “Cinco de Mayo” and crickets come to mind. I’ll leave that to the reader’s imagination. I have been lucky enough to have two amazing undergraduate workers in Lauren Roberts and Matt Hibbard. Thanks for all the countless cleanings, media preps, and monotonous techniques we pawned off on you. I enjoyed working with you and hope you know I’ve always appreciated having you here and trusted you as I do myself when it came to doing experiments. I know that you both have wonderful careers ahead of you in whatever you choose. I offer a huge “thank you” to all of the v graduates (Arka Ghosh, Bethany Kent-Hall, Alexa Turner-Dickson, Victoria Blaho-Coady, Miranda Hvinden, Risa Benwell) and countless undergraduates that rotated or volunteered with us and helped out on all my ten-thousand side projects from epitope tagging, Brucella, and cranberry juice. I never could have finished as much as I did without people and friends like you. Last, but certainly not least, are those people in the “other lab” we share space with: Dr. Becky Jorgensen and Maggie Lange. Even though you both have strayed from the path and became eukaryote-loving immunologists… I love you to death as we have gone through this whole experience together. Thanks for always sharing sage advice and a good laugh, or consolation and suggestions when things aren’t so great. I wish you the best of everything in your careers and lives. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................ iv LIST OF FIGURES................................................................................................... x ABSTRACT.............................................................................................................. xi CHAPTER 1. INTRODUCTION Iron and microbial cells......................................................................... 1 Siderophores ........................................................................................ 2 The enterobactin operon and synthesis................................................ 3 The regulation of enterobactin in E.coli................................................. 3 The transport of enterobactin................................................................ 4 E.coli P43 (ybdA) in the enterobactin operon ....................................... 4 Hypothesis and research objectives ..................................................... 6 Additional goals and results.................................................................. 7 Chapter 1 figures.................................................................................. 8 2. CHARACTERIZATION OF P43 INVOLVEMENT IN ENTEROBACTIN TRANSPORT Introduction........................................................................................... 13 Materials and methods ......................................................................... 15 Results.................................................................................................. 18 Discussion and conclusion ................................................................... 22 Chapter 2 figures.................................................................................. 26 vii 3. P43-MEDIATED TRANSPORT OF ENTEROBACTIN AND ENERGY REQUIREMENTS IN INVERTED MEMBRANE VESICLES Introduction........................................................................................... 32 Materials and methods ......................................................................... 34 Results.................................................................................................. 36 Discussion and conclusion ................................................................... 39 Chapter 3 figures.................................................................................. 44 4. IDENTIFICATION OF ADDITIONAL COMPONENTS INVOLVED IN ENTEROBACTIN RELEASE Introduction........................................................................................... 48 Materials and methods ......................................................................... 52 Results.................................................................................................