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CATECHOL 2,3‐DIOXYGENASE‐ASSISTED CLEAVAGE OF AROMATICS BY “ANAEROBIC” TERMITE GUT SPIROCHETES AND GENOMIC EVIDENCE OF A COMPLETE META‐PATHWAY Thesis by Kaitlyn Shae Lucey In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 2014 (Defended June 13, 2013) i ii © 2014 Kaitlyn Shae Lucey All Rights Reserved iii ACKNOWLEDGMENTS As a young child I grew up in South Boston, or Southie. My family and I lived in a brick house that stood on the top of “Pill Hill” (named for the doctors and dentists who built homes and practiced there around the turn of the last century) at the center of the main street of the neighborhood, Broadway. Depending on a Westerly or Easterly direction, I could follow Broadway to the public housing projects or to Boston proper – an epicenter of cultural, artistic, and scholarly diversity. My house was a pivotal point from which my life began, and my early childhood in Southie left an indelible mark motivating me to continually challenge myself and strive for better while simultaneously giving back. Accordingly, first and foremost I would like to thank my mother, father, and younger brother, Gerald, who have believed in my potential, given me the right balance of guidance and freedom to choose my own path, and been genuinely invested in my success from the start. Members of my extended family have also been unparalleled role models and supporters, and I would like to thank my grandparents, William Spencer and Janet Van Dyke, my late grandmother, Betty Lucey, my Godfather, Michael Owens, and my Aunt Carole, Uncle Jim, Aunt Lynn, Uncle Greg, Uncle Dave, and Dave Roberts. Beginning at Wellesley College, I embarked upon a path in the sciences in hopes of both contributing solutions to the STEM‐related challenges our society and iv environment is facing, and inspiring others to engage in STEM also. My professors and mentors at Wellesley College, specifically Professor Dave Ellerby and Professor Mary Allen, were integral in helping me initiate my path in the sciences. I would also like to thank Wellesley College, in general, for focusing on undergraduate learning, including a laboratory component with nearly every science class, and providing undergraduates with original research opportunities and prominent responsibility in the lab. My undergraduate experience has played a significant role in where I am today and where I am going. At Caltech, former lab members Liz Ottesen and Eric Matson also helped kick‐star my research by sharing many molecular and physiological skill sets. I would also like to thank both Xinning Zhang and Adam Rosenthal who, in addition to sharing with me much knowledge, have been stellar mentors. And of course, I would like to thank Jared Leadbetter. I have learned so much about being a good scientist and a good teacher working in your lab and with you. TA‐ing Microbial Physiology, the Asilomar conference, and preparing my publication have been particularly significant learning experiences. I am grateful, too, to Victoria Oprhan, Sarkis Mazmanian, and Dianne Newman for serving on my thesis committee. It has been an absolute pleasure working with you and with students and staff in your labs, and I am thankful to have had these opportunities. I also have developed some amazing friendships at Caltech that I hope will extend far beyond our time here. First, I would like to thank Elitza Tocheva, Nathan v Dalleska, and Felica Hunt for being such great mentors and role models, in addition to friends. I would also like to thank Chinny Idigo, Meg Schwamb, Mandy Grantz, and Kate Schilling for their friendship since practically day one here at Caltech, as well as my newest friends Molly and Luna for their encouraging and supportive purrs. Lastly, I would like to thank Los Angeles. From the majestic San Gabriel Mountains, to the unpredictable Pacific Coast, I am very fortunate to have spent the past five years in such an inspirational setting. I am grateful for the top‐notch education and laboratory research opportunities I have had to date, and will continue to strive to move the field of environmental microbial forward as well as teach and mentor the next generation of microbial ecologists. At the very least, I hope to instill in others a technological literacy and appreciation for science even if they do not also pursue STEM careers. Those who have been with me along this journey have been excellent teachers, mentors, scientists, and role models, and I aspire to serve others like you all have inspired me. vi ABSTRACT The termite hindgut microbial ecosystem functions like a miniature lignocellulose‐ metabolizing natural bioreactor, has significant implications to nutrient cycling in the terrestrial environment, and represents an array of microbial metabolic diversity. Deciphering the intricacies of this microbial community to obtain as complete a picture as possible of how it functions as a whole, requires a combination of various traditional and cutting‐edge bioinformatic, molecular, physiological, and culturing approaches. Isolates from this ecosystem, including Treponema primitia str. ZAS‐1 and ZAS‐2 as well as T. azotonutricium str. ZAS‐9, have been significant resources for better understanding the termite system. While not all functions predicted by the genomes of these three isolates are demonstrated in vitro, these isolates do have the capacity for several metabolisms unique to spirochetes and critical to the termite system’s reliance upon lignocellulose. In this thesis, work culturing, enriching for, and isolating diverse microorganisms from the termite hindgut is discussed. Additionally, strategies of members of the termite hindgut microbial community to defend against O2‐stress and to generate acetate, the “biofuel” of the termite system, are proposed. In particular, catechol 2,3‐ dioxygenase and other meta‐cleavage catabolic pathway genes are described in the “anaerobic” termite hindgut spirochetes T. primitia str. ZAS‐1 and ZAS‐2, and the first evidence for aromatic ring cleavage in the phylum (division) Spirochetes is also presented. These results suggest that the potential for O2‐dependent, yet non‐ respiratory, metabolisms of plant‐derived aromatics should be re‐evaluated in termite hindgut communities. Potential future work is also illustrated. vii TABLE OF CONTENTS Acknowledgments ..…………………………………………………………………………….…………… iii Abstract …………………………………………………………………………………………………………... vi Table of Contents ……………………………………………………………………………………….…... vii List of Tables …..……………………………………………………………………………………………... viii List of Figures ………………………………………………………………………………………..……….... ix Chapter 1: Research with termite hindgut microorganisms & background ... 1‐1 Abstract ……………………………………………………………………………………………….. 1‐1 Introduction ……………………………………………………………………………………….... 1‐2 Materials and Methods ……………………………………………………………………….. 1‐16 Results …………………………………………………………………………………………….… 1‐24 Discussion ……………………………………………………………………………………..…… 1‐34 References …………………………………………………………………………………............ 1‐39 Appendix …………………………………………………………………………………………… 1‐44 Chapter 2: Exploring genomic evidence of acetogenic demethylation by Treponema azotonutricium str. ZAS­9 ……………………………………….…………………. 2‐1 Abstract ……………………………………………………………………………………………….. 2‐1 Introduction ……………………………………………………………………………………...…. 2‐2 Materials and Methods ……………………………………………………………………...….. 2‐9 Results …………………………....……………………………………………………….……...…. 2‐10 Discussion …………………………………………………………………………...…………….. 2‐17 References ……………………………………………………………………………………........ 2‐23 Appendix …………………………………………………………………………………………… 2‐26 Chapter 3: Catechol 2,3­dioxygenase and other meta­cleavage catabolic pathway genes in the “anaerobic” termite gut spirochete Treponema primitia …………………………………………………………………………………………………….…….… 3‐1 Abstract ……………………………………………………………………………………………….. 3‐1 Introduction ………………………………………………………………………...………………. 3‐2 Materials and Methods …………………………………………………………………………. 3‐4 Results …………………………....……………………………………………………….……….... 3‐10 Discussion ………………………………………………………………………………………….. 3‐38 References ……………………………………………………………………………………........ 3‐49 Appendix …………………………………………………………………………………………… 3‐57 Chapter 4: Conclusions …………………………………………………………………………………. 4‐1 References ………………………………………………………………………….………........... 4‐12 viii LIST OF TABLES Chapter 1 Table 1­1: Appearance of baker’s yeast and appearance and odor of corresponding yeast autolysate and autolysate supernatant. Table 1­2: Growth rates of Treponema primitia str. ZAS‐2 on 4YACo medium prepared from yeast autolysates of different brands and packaging types. Table 1­3: Treponema primitia str. ZAS‐2 enzymatic defenses against oxidative stress. Chapter 2 Table 2­1: Methylated compounds tested for growth on by Treponema azotonutricium str. ZAS‐9. Chapter 3 Table 3­1: Treponema primitia str. ZAS‐1 and ZAS‐2 meta‐cleavage pathway genes and pfam features. Table 3­2: Selection pressure analysis of Treponema primitia str. ZAS‐1 and ZAS‐2 meta‐cleavage pathway genes. Table 3­3: Monoaromatic compound cleavage by Treponema primitia str. ZAS‐1. Table 3­4: Average depth of O2 penetration in Treponema primitia str. ZAS‐1 and ZAS‐2 O2/aromatic gradient cultures. ix LIST OF FIGURES Chapter 1 Fig. 1­1: A phylogenetically “lower” termite, a dampwood termite Zootermopsis nevadensis worker, and a dissected intestinal tract of another Z. nevadensis worker specimen. Fig. 1­2: Phylogram of “higher” and “lower” termite families as well as household and wood roaches. Fig. 1­3: A phylogenetically

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