Uncovering New Players and New Roles in Microbial Anoxic Carbon Transformations Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lindsey Marie Solden Graduate Program in Microbiology The Ohio State University 2018 Dissertation Committee: Professor Kelly Wrighton, Adviser Professor Jeffrey Firkins Professor Venkat Gopalan Professor Daniel Wozniak Copyrighted by Lindsey Marie Solden 2018 Abstract Organic carbon in anoxic ecosystems flows in a cascade from complex plant material to more labile sugars, and ultimately to short-chain fatty acids (SCFA) and gasses like carbon dioxide and methane. Microbial communities, groups of microorganisms that interact with one another, facilitate this process. Microbial anaerobic carbon degradation is exemplified in ruminants. These animals harness energy from plant material using the power of interacting microorganisms, which break down plant carbon into SCFA under largely anoxic conditions in the rumen. Because microbial SCFA can provide up to 80% of the animal’s energy, understanding microbial carbon degradation mechanisms in the rumen is important for many agricultural industries including the production of meat, milk, leather, and wool. Beyond domesticated ruminants, there are over 75 million wild ruminants that are fundamental members in ecosystems from Alaska to Australia. Furthermore, the microbial enzymes that break down plant material in the rumen have industrial applications for modifying enzymatic cocktails in biofuel production. The research presented here uses cultivation-independent and laboratory approaches to assign carbon degradation capabilities to specific members of the microbial community in the moose rumen. Moose, animals that naturally forage on woody biomass, were selected to provide access to natural rumen microbial communities that are especially adapted to a high lignocellulose diet. We sampled rumen fluid from moose in the spring, summer, and winter, along a seasonal gradient in lignocellulose. Rumen fluid was sampled via the rumen cannula, offering access into the active microbial interactions ii mediating complex carbon degradation. From these rumen fluid samples, we performed high-throughput shotgun metagenomics and metaproteomics, coupled to multiple methods for metabolite quantification (1H NMR, sequential fiber analyses, and carbohydrate microarray polymer profiling (CoMPP)). We binned hundreds of genomes, resulting in 77 unique (~>80% complete) genomes. A majority of these genomes (71%) belong to novel genera, families, and orders. Five of these genomes belong to an uncultivated, Bacteroidetes family, the BS11, which represent the first ever genomic representatives from this family. A newly resolved genus in this family was the most enriched member on a high lignocellulose diet and was found to ferment hemicellulose sugars. To uncover interactions between these microorganisms and determine their functional role, we mapped metaproteomics data to the unique genome data. This revealed most of the carbon degradation enzymes were encoded within polysaccharide utilization loci from uncultivated Bacteroidetes genomes. We then characterized the carbon metabolite chemistry focusing primarily on carbohydrate polymers and sugars of using CoMPP and 1H NMR. Linking our proteomes to metabolomics, we discovered that proteins from only seven Bacteroidetes genomes were processing all plant polymers detected, suggesting that a few generalist microorganisms are responsible for most of the carbon degradation in the rumen. One of these highly active Bacteroidetes genomes contained protospacer linkages to viral genomes, indicating that immunity against viral predation that may be required for some organisms to sustain carbon degradation in the rumen. iii Finally, winter rumen fluid with elevated condensed tannins was used to enrich for tannin-degrading microorganisms. From these reactors, we isolated a Streptococcus sp. that can degrade Sorghum condensed tannins (CT) in the presence of glucose. Label- free proteomics was performed to evaluate the dynamic proteome when the isolate was grown in the presence or absence of CT. CT lead to the enrichment of many proteins annotated as tannase enzyme, transcriptional regulators for phenolic metabolism, putative enzymes involved in phenolic metabolism, stress response proteins, and proteins originating from prophage. Cumulatively, this dissertation research examines the rumen on multiple scales, to identify microbial community and viral interactions, microorganism physiology, and the putative enzymes that facilitate how carbon flows through the rumen. iv Acknowledgments This body of work would not have been possible without the help, guidance, and support from so many people. First and foremost, I would like to thank my PhD advisor Dr. Kelly Wrighton. It has been an honor to be one of her first graduate students. Kelly has provided every possible opportunity to learn, grow, and challenge myself. She has taught me both consciously and unconsciously how to navigate being a female academic scientist. Her passion and intense enthusiasm for her research is both motivational and inspiring and I hope to emulate her in the future. I appreciate all of her time spent teaching me how to design experiments, manage scientific collaborations, and communicate findings in talks and writing. I am indebted for her patience, generosity, mentorship, and most importantly friendship. I would not be the scientist or person I am today without her. I truly cannot thank her enough. I also would like to thank my graduate committee, Dr. Jeffrey Firkins, Dr. Daniel Wozniak and Dr. Venkat Gopalan, for making themselves available for helpful discussions, critical comments, and for sharing the most precious of all resources, time. I would also like to thank Dr. Barbara Wolfe for providing support, career development opportunities, and mentorship. Finally, I am especially indebted to my family, friends, and lab members who have provided me with kind words, friendship, and love. All of v you are essential in my amazing support system, and I wouldn’t be where I am today without you. I am incredibly lucky. vi Vita 2009………………………………………..Mentor High School 2013………………………………………..B.S. Microbiology, The Ohio State University 2013 to present …………………… ............ Graduate Teaching/Research Associate, Department of Microbiology, The Ohio State University Publications Naas AE, Solden LM, Norbeck AD, Brewer H, Hagem LH, Heggenes IM, McHardy AC, Mackie RI, Pasa-Tolic L, Arntzen MO, Eijsink VGH, Koropatkin NM, Hess M, Wrighton KC, Pope PB. (2018) “Candidatus Paraporphyromonas polyenzymogenes” encodes multi-modular cellulases linked to the type IX secretion system. Microbiome 6 (44). Solden LM, Wrighton KC. (2017) Finding life’s missing pieces. Nature Microbiology 2, 1458-1459. Angle JA, Morin T, Solden LM, Narrowe A, Smith G, Borton MA, Rey-Sanchez C, Daly RA, Mirfenderesgi G, Hoyt DW, Riley W, Miller CS, Bohrer G, Wrighton KC. (2017) Methanogenesis in oxygenated soils is a substantial fraction of wetland methane emissions. Nature Comm 8: 1567. Solden LM, Hoyt DE, Collins WB, Plank JE, Daly RA, Hildebrand E, Beavers TJ, Wolfe R, Nicora CD, Purvine SO, Carstensen M, Lipton MA, Spalinger DE, Firkins JL, Wolfe BA, Wrighton KC. (2017) New roles in hemicellulose degradation for the uncultivated Bacteroidetes family BS11. The ISME J 11(3): 691. Borton MA, Sabag-Daigle A, Wu J, Solden LM, O’Banion BS, Daly RA, Wolfe R, Gonzalez JF, Wysocki VH, Ahmer BMM, Wrighton KC. (2017) Chemical and pathogen induced inflammation disrupt the murine intestinal microbiome. Microbiome 5.47. vii Faulkner MJ, Wenner BA, Solden LM, Weiss WP (2017). Source of supplemental dietary copper, zinc, and manganese affects fecal microbial relative abundance in lactating dairy cows. J Dairy Science 100 (2): 1037-1044. Solden LM, Lloyd K, Wrighton KC. (2016). The Bright Side of Microbial Dark Matter: Lessons learned from the uncultivated majority. Current Opinions in Microbiology, 31: 217-226. Fields of Study Major Field: Microbiology viii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments ............................................................................................................... v Vita .................................................................................................................................... vii List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1: Introduction ....................................................................................................... 1 1.1 Rumen microbial membership and community genomics ................................................ 1 1.2 Rumen viruses are currently undersampled ...................................................................... 6 1.3 The first ecosystems perspective on the rumen microbiome ............................................ 8 Chapter 2: New roles in rumen hemicellulosic sugar fermentation for the uncultivated Bacteroidetes family BS11 ................................................................................................. 9 2.1 Introduction ........................................................................................................................