University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2018 Genomic and Physiological Characterization of Lignin-Derived Aromatic Catabolism Pathways in Roseobacters Michelle June Chua University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Recommended Citation Chua, Michelle June, "Genomic and Physiological Characterization of Lignin-Derived Aromatic Catabolism Pathways in Roseobacters. " PhD diss., University of Tennessee, 2018. https://trace.tennessee.edu/utk_graddiss/5295 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 Michelle June Chua entitled "Genomic and Physiological Characterization of Lignin-Derived Aromatic Catabolism Pathways in Roseobacters." 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. Alison Buchan, Major Professor We have read this dissertation and recommend its acceptance: Shawn R. Campagna, Elizabeth Fozo, Jill Mikucki, Erik Zinser 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.) Genomic and Physiological Characterization of Lignin-Derived Aromatic Catabolism Pathways in Roseobacters A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Michelle June Chua December 2018 Copyright © 2018 by Michelle Chua. All rights reserved. ii ACKNOWLEDGEMENTS I would like to first thank my parents and brother for supporting me and all my decisions. You have been the most reliable people throughout my life, and I am so lucky to have you as my family members. Secondly, I would like to acknowledge everyone in Knoxville I have formed (what I believe are meaningful) relationships with: former and current members of the Buchan lab, Eric O., Alicia P., Kevin S., Yu-Kai W., and Jia W., the relationships with whom are too long to praise within these acknowledgements. Elizabeth McPherson is included in this group. She has listened to me yammer on for countless hours (something I am obviously not known for) and for some reason, still enjoys seeing me. I am also happy to have joined the Dungeons & Dragons group spear-headed by Alex Grossman. It is not often I feel like I fit in in a group, but the people there make me feel welcome, despite my awkwardness. I also offer my gratitude to my committee members. Words cannot express how grateful I am that Drs. Alison Buchan, Shawn Campagna, Elizabeth Fozo, Jill Mikucki, and Erik Zinser joined and have stayed on my committee. I have said this before, I thank them all for their patience and helping me (what I hope) grow throughout my graduate school experience. A defining moment for me with Dr. Campagna was when he gave me a pep talk after I failed my first preliminary exam, a moment he may not remember, but is one I will remember for years to come. Thank you, Dr. Fozo, (during the irregular moments we have interacted) for being a kind and caring cheerleader for me. I thank Dr. Mikucki for supporting me (not only with emotional support, but food!) during my time as a graduate student with her and afterwards. I also thank Dr. Zinser for letting me rotate in his lab during my first semester at UT and for continuing to play a role in my dissertation. I very much enjoyed his Microbial Physiology class and non- standard Journal Clubs, which I will miss. iii Lastly, I want to acknowledge Dr. Buchan. I am extremely pleased that she let me join her lab. I have not only enjoyed the projects I have worked on, but also my interactions with Dr. Buchan. I appreciate her being patient with me when I am being difficult and also providing me with (emotional) support when I have needed it most. She is also hilarious, which makes my meetings with her and lab meetings quite enjoyable. Thank you, Alison. iv ABSTRACT Lignin provides vascular plants with structural rigidity and is the most abundant aromatic polymer on Earth. Lignin is rich in phenolic structures and its structural complexity contributes to its biological recalcitrance, yet it is susceptible to degradation via microbial enzymes. Biogeochemically relevant marine bacteria in the Roseobacter clade are capable of transforming lignin and mineralizing lignin-derived aromatic monomers. Roseobacters are prevalent in temperate and polar oceans, marine sediments, sea ice, and hydrothermal vents, and representatives are readily cultivated in the lab. Roseobacter abundances are frequently highest in coastal oceans, including the salt marshes of the Southeastern United States, where the dissolved organic carbon pool is enriched in aromatic moeities. This dissertation provides new insight into the aromatic compound catabolism potential by members of the Roseobacter lineage through both genomic analyses of publicly available genome sequences and genetic and physiological characterization of the Roseobacter strain Sagitulla stellata. Approximately 85% of the 311 Roseobacter genomes analyzed in this study have genes that encode for enzymes for at least one ring-cleaving pathway. These findings highlight the prevalence of this trait amongst lineage members, but also revealed little correlation between phylogeny, based on 89 conserved protein sequences, and genomic potential. Laboratory-based studies focused on the ligninolytic Roseobacter, S. stellata, with an emphasis on pathways mediating the degradation of the hydroxycinnamates (HCAs) ferulate and p-coumarate, which are important for cross-linking in lignin. Two genes annotated as feruloyl-CoA- synthases, enzymes that catalyze the first step of HCA degradation, were found to have overlapping specificities for the two HCAs in S. stellata, highlighting a functional redundancy not yet reported for other aromatic compound degrading bacteria. Additional studies were performed to examine the physiological response of S. stellata to mixtures of aromatic monomers. A series of studies performed with binary mixtures of aromatic compounds revealed a strong substrate preference for HCAs and their catabolic intermediates relative to v benzoate. Collectively, these data indicate S. stellata is adapted to growing on a mixture of lignin-derived aromatics, which would be advantageous in the aromatic compound-rich salt marshes in which it and its relatives are found. vi TABLE OF CONTENTS CHAPTER I: INTRODUCTION ............................................................................. 1 I. Aromatic compounds in the environment ................................................... 2 Sources ......................................................................................................... 2 Lignin ............................................................................................................. 2 II. Aerobic catabolism of aromatic compounds in bacteria ............................. 3 Aromatic catabolism: classic aerobic pathways ............................................. 3 Aromatic catabolism: aerobic “hybrid” pathways ............................................ 3 III. Significance of Roseobacter species ...................................................... 4 Roseobacter species in the environment ....................................................... 4 Aromatic catabolism by Roseobacter species ............................................... 4 Sagittula stellata E-37 .................................................................................... 5 IV. Objectives ............................................................................................... 6 V. References ................................................................................................. 8 VI. Appendix ............................................................................................... 12 CHAPTER II: CHARACTERIZATION OF RING-CLEAVING PATHWAYS IN ROSEOBACTER GENOMES ............................................................................. 14 I. Abstract .................................................................................................... 15 II. Introduction .............................................................................................. 17 III. Methods ................................................................................................ 20 IV. Roseobacter genomes.......................................................................... 21 V. Aerobic peripheral pathways .................................................................... 22 Protocatechuate, gentisate, and homogentisate pathways .......................... 23 Benzoate and phenylacetate pathways ....................................................... 25 VI. Evolutionary ecology of the marine Roseobacter clade ........................ 26 VII. Conclusions .......................................................................................... 28 VIII. References ........................................................................................... 30 IX. Appendix ..............................................................................................