EXPERIMENTAL STUDIES ON FERMENTATIVE FIRMICUTES FROM ANOXIC ENVIRONMENTS: ISOLATION, EVOLUTION, AND THEIR GEOCHEMICAL IMPACTS By JESSICA KEE EUN CHOI A dissertation submitted to the School of Graduate Studies Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Microbial Biology Written under the direction of Nathan Yee And approved by _______________________________________________________ _______________________________________________________ _______________________________________________________ _______________________________________________________ New Brunswick, New Jersey October 2017 ABSTRACT OF THE DISSERTATION Experimental studies on fermentative Firmicutes from anoxic environments: isolation, evolution and their geochemical impacts by JESSICA KEE EUN CHOI Dissertation director: Nathan Yee Fermentative microorganisms from the bacterial phylum Firmicutes are quite ubiquitous in subsurface environments and play an important biogeochemical role. For instance, fermenters have the ability to take complex molecules and break them into simpler compounds that serve as growth substrates for other organisms. The research presented here focuses on two groups of fermentative Firmicutes, one from the genus Clostridium and the other from the class Negativicutes. Clostridium species are well-known fermenters. Laboratory studies done so far have also displayed the capability to reduce Fe(III), yet the mechanism of this activity has not been investigated further. We studied three clostridial organisms, Clostridium sp. FGH that was previously enriched from Oak Ridge, TN; Clostridium beijerinckii; and Clostridium acetobutylicum to determine how Fe(III) oxide reduction occurs. The results indicate that Fe(III) oxide reduction by fermentative clostridial species does not require direct cell-to-mineral contact, meaning that it is an extracellular process mediated by a soluble molecule. Fermentation oft leads to the accumulation of acidic by-products, ii which can act as Fe(III)-chelating ligands. But during glucose fermentation, soluble Fe(III) accumulation did not occur, nor did supplementation of organic acids to fermenting cultures stimulate either the rate or extent of Fe(III) reduction even though rapid Fe(III) solubilization was observed. This extracellular process may be mediated by flavins, and supplementation of flavin adenine dinucleotide (FAD), a flavin molecule that serves as an electron carrier intracellularly, stimulated Fe(III) oxide reduction. These results indicate the potential for Clostridium species to perform Fe(III) reduction in contaminated environments and may be relevant towards bioremediation. Clostridum sp. FGH was enriched as a co-culture with another spore-forming Firmicutes organism that we isolated and named Anaerosporomusa subterranea strain RU4. This organism is likewise an obligate fermenter but with a very narrow metabolic niche. Surprisingly, this organism in pure culture showed no ability to reduce Fe(III), despite the fact that it was isolated from an Fe(III)-reducing enrichment culture. Furthermore, the addition of typical terminal electron acceptors were not utilized by A. subterranea. However, this organism was interesting because it was a Gram-negative (or didermic, since they have two membranes in their cell wall structure) organism within a phylum that was previously thought to be only Gram- positive (or monodermic, since these microorganisms have only one membrane as part of their cell wall). We investigated how this organism could have evolved through comparative phylogenomics of outer membrane biosynthesis genes. We identified 46 potential protein sequences involved in lipopolysaccharide and outer membrane biosynthesis in the genome of A. subterranea. Specifically out of iii these proteins, we focused on the proteins LptACD that are responsible for transporting mature lipopolysaccharide from the inner membrane, across the periplasmic space, and to the outer membrane. This is because most Gram-negative or didermic bacteria require these proteins to synthesize an outer membrane. Our results demonstrated that A. subterranea shared approximately 50% sequence similarity for the LptACD proteins with other Negativicutes. When the alignment was expanded to other Gram-negative or didermic bacterial clades, on average 25% similarity was reported. This suggested significant homology within the Negativicutes and with other microorganisms, such that these genes in A. subterranea were ancestrally derived. Furthermore, phylogenetic comparisons using the LptACD protein sequences showed comparable tree topology to the widely-accepted 16S phylogenetic tree, illustrating vertical gene transfer and that recent horizontal gene transfer did not occur. In conclusion, our data suggests that the ancestor to the Firmicutes phylum was originally didermic and that A. subterranea may represent an ancestral lineage. iv ACKNOWLEDGEMENTS AND DEDICATION I would like to start by expressing my gratitude towards my advisor, Dr. Nathan Yee. I respect him in being able to guide me through this whole process because it has been a tough 5 years. His guidance and counsel over time has developed me as a scientific researcher, forced me to adopt new thought processes, and changed the way I think and behave as a microbiologist and scientist. Additionally, I would like to extend this gratitude towards my thesis committee members Dr. Lily Young, Dr. Costatino Vetriani, and Dr. Katherine Dawson for their time and commitment. This research was also made possible through various sources of funding. I would like to thank the Department of Biochemistry and Microbiology here at Rutgers for funding me for multiple years as a Teaching Assistant and for the Woodruff Fellowship I received in my first year. Lastly, I’d like to thank the U.S. Department of Energy Office of Science (Grant Nos. DE-FG02-08ER64544 and DE-764 SC0007051) for funding my research. During my time here at Rutgers, I’ve met and received help from many people. Of them, the most important person would be Dr. Madhavi Shah who helped me to get acquainted with and started in the lab as well as with my research. Even after she graduated, she continued to give me advice and help when I needed, and I cannot thank her enough. Additionally, she helped tremendously with the preparation of Chapters 2 and 3. My time in the lab was also enjoyable thanks to the friends I made in the lab, specifically Dr. Sarah Janssen, Cindy Wang, and Thomas Wang. I had the pleasure of working with numerous undergraduate students who helped me with v various aspects of my project, so I would like to acknowledge Jennifer Marin, Amanda Steitz, Chioma Ekedede, Beverly Chiu, and in particular Yelyzaveta Orlovetska for her contribution to the research outlined in Chapter 4. Dr. Ines Rauschenbach and Dr. Ramaydalis Keddis gave me a lot of direction and advice during my time as a teaching assistant here at Rutgers, and helped me to improve myself as a mentor and instructor of higher education. Lastly, I would like to acknowledge the staff of the Department of Biochemistry and Microbiology and of the Department of Environmental Sciences for their help in dealing with a plethora of technicalities and logistics. Specifically, I’d like to thank Ms. Maria Rivera because she was such a huge help with numerous things and became such a wonderful friend and support system; thank you so much for everything. I would like to thank my friends Ananya Agarwal, Ashley Grosche, and Sushmita Patwardhan, the girls whom I’ve had the pleasure of sharing this entire experience with since day 1 of my PhD. We’ve gone through the good, the bad, and the ugly altogether, and I couldn’t have asked for a better support group. Lastly, I would like to thank my absolutely fantastic parents, Dr. Sung Hee Choi and Sungsook Choi for their unconditional support and love. I don’t know how they did it, but they were ultimately the ones responsible for bringing me up to this point and I wouldn’t be the person I am today with them. They are my rock and my world. Lastly, I would like to dedicate this work to two people. First, my grandfather, Dr. Sangup Choi, the most intelligent man I’ve known. For reasons that I still am confounded by, I have thought of him very frequently during my PhD years and miss him dearly. He left the world a much better place for me, and I didn’t really realize that vi until after his passing. Therefore, through this dedication I would like to show him my utmost gratitude, appreciation, and love. Thank you Grandpa. I would also like to dedicate my work to my nephew, Kane Ko, in hopes that I have made and will continue to make this world a better place for him, much like what my grandfather did for me. I also hope through this dedication that he grows up and chooses to pursue science for his future career! vii PREFACE Chapter 2 on clostridial Fe(III) oxide reduction is under preparation for publication as Jessica K. Choi, Annette R. Rowe, Lori Zacharoff, Nathan Yee. 2017. Jessica K. Choi participated in writing the manuscript and performed experiments involving growth of the Clostridium species with ferrihydrite, Fe-beads, and supplementation using either citrate or FAD. Chapter 3 has been published as Jessica K. Choi, Madhavi Shah, Nathan Yee. 2016. Anaerosporomusa subterranea gen. nov., sp. nov., a spore-forming anaerobe belonging to the class Negativicutes isolated from saprolite. IJSEM 66:3848-3854. DOI 10.1099/ijsem.0.001275.