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Investigations Into How Bacteria Influence Nutrient Availability in Their Environment

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Investigations Into How Bacteria Influence Nutrient Availability in Their Environment INVESTIGATIONS INTO HOW BACTERIA INFLUENCE NUTRIENT AVAILABILITY IN THEIR ENVIRONMENT A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by David Robert Sannino December 2017 © 2017 David Robert Sannino INVESTIGATIONS INTO HOW BACTERIA INFLUENCE NUTRIENT AVAILABILITY IN THEIR ENVIRONMENT David Robert Sannino, Ph. D. Cornell University 2017 Bacteria shape many of their interactions with other organisms through the manipulation of nutrient availability, whether through cooperation by providing nutrients, or through competition for nutrients. Thiamin is an essential vitamin necessary for all life, however, how bacteria shape the ecological interactions between organisms for this nutrient is not well understood. We employed a Drosophila melanogaster-microbiota model utilizing a chemically defined diet to understand the interaction between how the host is influenced by the microbiota’s interaction with the dietary component thiamin. We found that the Drosophila melanogaster microbiota provisions thiamin to its host in a low thiamin environment. This provision rescued development of Drosophila on a no thiamin diet, as axenic flies were unable to develop on this diet. Our study was a clear demonstration supporting the long standing hypothesis that animal microbiotas function to provision thiamin and other vitamins to their host. A small subset of bacteria produce the enzyme thiaminase I, which degrades thiamin to its two moieties. The biological function of this enzyme is not understood. We used a genomic approach to investigate a potential function of this enzyme and found that it is located in a conserved operon in three thiaminase I producing Paenibacillus species, with other genes involved in thiamin salvage and production of the thiamin antimetabolite bacimethrin, suggesting it may play a role in thiamin salvage and competition for this nutrient. We further investigated the biological role of thiaminase I using Burkholderia thailandensis, where we generated thiamin auxotrophs. We found that the enzyme plays a role in thiamin salvage as it allows for auxotrophic strains to grow in media conditions when strains lacking thiaminase I cannot, as it recycles precursors from thiamin and certain analogs. Using a genomic approach, we also investigated the biosynthetic and metabolic potential of the unique, giant bacterial intestinal symbiont ‘Candidatus Epulopiscium viviparous’ of the surgeonfish Naso tonganus. We found that this bacterium’s genome is enriched for carbohydrate metabolism, as it has the potential to degrade a vast array of carbohydrates present in its host’s diet, allowing them to supply assimilable nutrients, vitamins, and protein to their host. BIOGRAPHICAL SKETCH Dave Sannino was born on October 24, 1987, and grew up in Millstone Township, NJ. He attended Rutgers University where he graduated Summa Cum Laude. While at Rutgers University, he worked as an undergraduate researcher in Dr. Elisabetta Bini’s laboratory studying the extremophilic organisms including the archaeon Sulfolobus solfataricus. In August 2011, he began his graduate career in the Department of Microbiology at Cornell University, ultimately finding himself in Dr. Esther Angert’s laboratory, where he began studying the biology and genomics of Epulopiscium spp. as well as the bacterial produced enzyme thiaminase I. Throughout his graduate career he was a teaching assistant for multiple microbiology courses and received the CALS Outstanding TA Award for microbiology in 2015, for his TAship in general microbiology. In his spare time, Dave enjoys playing hockey, drawing, and music. iv ACKNOWLEDGMENTS I am deeply grateful to my advisor Esther Angert for all of her guidance, assistance, hard work, and mentorship, as well as for giving me the freedom to explore multiple projects, systems, and techniques throughout my graduate career. Also for being very patient with me and not giving up on me particularly after my rough A-exam. I am grateful to my minor advisor Nicolas Buchon for taking me into your lab, giving me advice, expertise, and mentorship. I would not have been able to complete my PhD without you. I thankful to my lab-mate Francine Arroyo for all the assistance, the advice, hard work, genomic expertise, and willingness to always help me no matter what. I would not have been able to complete the Epulopiscium chapter without her and I am indebted to her for that. I would like to thank Cliff Kraft for all his insight, discussions, and conversations in regards to the thiaminase I project. I would like to thank Chuck Pepe-Ranney for his genomics wizardry, willingness to always help and answer questions, and great insight. Without him, I would still be lost trying to figure out how to assemble the type B genome. I would like to thank Katie Edwards for all of her expertise on thiamin, for supplying me with multiple analogs, for all her hard work and late nights performing the thiamin assays, and for always taking the time to thoughtfully answer every question I have about thiamin. I thank my other committee member John Helmann for his insight and helpful discussions. I would like to thank Adam Dobson for his mentorship, statistical help, fly insight, friendship, and all the good nights at the Chapter House, Statler, and Westy. I am thankful to the past Angert lab members; Betsy Hutchison and Dave Miller for getting me acquainted with epulos, Jen Fownes for all her help with thiaminase I, and Verena Carvalho-Salman, Bobby Loren, Abe Francis, Jenny Zhang, and Zanah Francis. I would like to thank the Buchon lab, in particular Alessandro Bonfini, Phil Houtz, Yoni Revah, Xi Liu, and Xiaoli Bing for their help with fly work. I thank Anthony Hay and Steve Winans for the 2 great semesters TAing 2900, as well as Steve Zinder, Barb Eaglesham, and Sue Merkel for all their mentorship with teaching. I would like to thank Joe Peters and Ahmed Gaballa for their insight, as well as Pete Newell for his insight and mentorship in my early stages of my grad career. I would like to thank Francoise Vermeylen at the Cornell Statistical Consulting Unit and Qi Sun v from the Cornell Institute of Biotechnology for all their assistance. I would like to thank my parents, Robert and Irene Sannino for their love and support throughout these 6+ years, as well as my brother Doug and my sister Liane. I would not have been able to last the 6+ years without you. I would like to thank my cohort, Olya Lastovetsky, Chantal Koechli, and Evgeniya Nazarova for the fun and friendship. I am thankful to Jared, Schwenke, Ginny, Marc, Bryan, Sara, Calum, Sӧren, Daniel, Dane, and everyone else for the friendship and good times. I am thankful to Margarita Kazakova and Rasa Žalakevičiūtė as well for their love and support when we were together. I would like to thank James Orcutt for helping to grow the hockey community in Ithaca, all the hockey people (except Pure Romance), a lot of my best times in Ithaca were on Cass Park and the Rink ice. Last but certainly not least, I have to thank Andrew, Barb, Bondar, Ceante, Leland, Mike, Pete, and Tom, for all the support and everything else. vi TABLE OF CONTENTS BIOGRAPHICAL SKETCH .................................................................................................................... iv ACKNOWLEDGEMENTS ....................................................................................................................... v CHAPTER 1. INTRODUCTION .............................................................................................................. 1 CHAPTER 2. THE DROSOPHILA GUT MICROBIOTA PROVISIONS THIAMIN TO ITS HOST .................................................................................................................................................................... 22 CHAPTER 2.1 ABSTRACT ...................................................................................................................... 22 CHAPTER 2.2 IMPORTANCE ................................................................................................................. 23 CHAPTER 2.3 INTRODUCTION ............................................................................................................. 23 CHAPTER 2.4 RESULTS .......................................................................................................................... 27 CHAPTER 2.5 DISCUSSION .................................................................................................................... 37 CHAPTER 2.6 MATERIALS AND METHODS ....................................................................................... 42 CHAPTER 2.7 ACKNOWLEDGMENTS ................................................................................................. 48 CHAPTER 2.8 REFERENCES .................................................................................................................. 49 CHAPTER 2.9 SUPPLEMENTARY MATERIAL .................................................................................... 52 CHAPTER 3. GENOMIC INSIGHTS INTO THE THIAMIN METABOLISM OF PAENIBACILLUS THIAMINOLYTICUS NRRL B-4156 and P. APIARIUS NRRL B-23460 ........... 62 CHAPTER 3.1 ABSTRACT ...................................................................................................................... 62 CHAPTER 3.2 ABBREVIATIONS ........................................................................................................... 62 CHAPTER 3.3 INTRODUCTION
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