The Pennsylvania State University The Graduate School Eberly College of Science COMPLEMENTARITY IN THE CORAL HOLOBIONT: A GENOMIC ANALYSIS OF BACTERIAL ISOLATES OF ORBICELLA FAVEOLATA AND SYMBIODINIUM SPP. A Thesis in Biology by Styles M. Smith ©2018 Styles M. Smith Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2018 The thesis of Styles M. Smith was reviewed and approved* by the following: Mónica Medina Associate Professor of Biology Thesis Advisor Steven W. Schaeffer Professor of Biology Head of Graduate Program Kevin L. Hockett Assistant Professor of Microbial Ecology *Signatures are on file in the Graduate School ii Abstract All holobionts, defined as a multicellular host and all of its associated microorganisms, rely on interactions between its members. Corals, which demonstrate a strong symbiosis with an algal partner, have a diverse holobiont that can be sequenced and analyzed that could reveal important roles of microbes that benefit its health. This microbial community has been predicted to be composed of nitrogen fixers, phototrophs, sulfur and phosphorus cyclers. However, the identity of these microbes responsible for these roles remain uncertain. In addition, there may be complementary roles that are unknown. Predicting these roles is challenging because…. A suggested method to overcome this problem is sequencing the genomes of the microbes found in the coral holobiont. I hypothesize that bacterial members of the holobiont play an important role in coral biology via complementary metabolisms in nutrient cycling and aiding the coral in stress response. The complete metabolic capabilities of ten bacteria isolated from the coral holobiont were examined via the sequencing and annotation of the whole genome, followed by pangenomic analysis with 31 whole genomes of closely related strains of bacteria previously sequenced. The metabolisms identified—including denitrification, dimethylsulfoniopropionate biosynthesis and degradation, and cobalamin biosynthesis—potentially complement the coral holobiont via nutrient cycling. These findings demonstrate the importance of genomic sequencing to reveal the interactions between members of the holobiont. iii Table of Contents List of Figures…………………………………………………………………………………………………………..………………..v List of Tables…………………………………………………………………………………………………………….…………..….vii Acknowledgments…………………………………………………………………………………………………………………..viii Chapter 1 Introduction: Defining the Coral Holobiont 1 Bibliography……………………………………………………………………………………………………………………4 Chapter 2 Metabolic Pathways Identified in Bacterial Members of the Coral Holobiont 6 Abstract………………………………………………………………………………………………………..……………….6 Introduction…………………………………………………………………………………………………………….…….7 Materials and Methods…………………………………………………………………..……………………………..9 Results………………………………………………………………………………………………………………………….16 Discussion…………………………………………………………………………………………………………………….30 Conclusion……………………………………………………………………………………………………………………37 References……………………………………………………………………………………………………………………39 iv List of Figures Figure 2-1 |F24,565 gene clusters were found across 41 isolate genomes. The isolates sequenced in this study are found in red boxes (A 44A11, B130DD11, B 452B1, L 44C2F, M 452T16, N 064newd, R 44A6S, R 452T9, S B7newh, and V 130DD58). Gene clusters group together based on co-occurrence in genomes. The genomes are organized by shared gene clusters, calculated with Euclidean distance and Ward linkage. A darker shade represents occurrence of a gene cluster in that genome. Genomes are colored based on genus. A heatmap of relative gene distribution in RAST subsystem categories is shown in the top right for each genome (white=0, dark blue=max)………………………………..…………..……………………………………………19 Figure 2-2 | RAST subsystem category gene distribution of the pangenome Relative gene distribution into RAST subsystem category is shown for each of the 41 isolate genomes (A). Genome names are first letter of genus followed by strain number, and genera are grouped based by color.……………………………………………………………………………..……..................20 Figure 2-3 | Pangenome gene distribution in specific subsystems Specific subsystems gene distribution for sulfur metabolism (A), nitrogen metabolism (B), and cofactors, vitamins and pigments (C) show gene count sum for each genus.…...……………………..21 Figure 2-4 | DMSP biosynthesis, cleavage, and demethylation by isolates. There are two sources of DMSP in the coral holobiont, Symbiodinium spp. and Labrenzia spp. Both Ruegeria isolates are able to demethylate DMSP. The cleavage of DMS by R. arenilitoris 452T9 and L. aggregata 44C2F leads to sulfur aerosols in the atmosphere which can act as cloud condensing nuclei (CCN)…………………………………………………………………………………………………………………............23 Figure 2-5 | Nitrogen cycling pathways in bacterial isolates genomes, host, coral, and Symbiodinium. The metabolic pathways, nitrate reduction to ammonia, and urea catabolism recycle nitrogen within the holobiont. Denitrification results in loss of nitrogen from the holobiont. The gene(s) necessary for a pathway are next to the arrow. Symbols indicate the genome of origin for each gene. Orange symbols indicate bacteria isolated from O. faveolata and green symbols indicate bacteria isolated from Symbiodinium spp…………………....................26 Figure 2-6| Aerobic Anoxygenic Photosynthesis Pathway of Isolate Ruegeria arenilitoris 452T9. The isolate’s AAnP pathway has complete gene sets to synthesize the necessary enzymes and proteins for bacteriochlorophyll (light blue), carotenoids (orange), photosystem II- like reaction center (purple), and bacterial light harvesting complex (pink). Letters correspond to genes within the operon of that subsystem. The isolate is compared to the α- proteobacterium Rhodobacter sphaeroides, showing that the genetic repertoire and order of genes are similar.…………………………………………………………………………………………………………………….28 v Figure 2-7 | Phylogeny of marker gene pufM in anoxygenic photosynthesizing α- proteobacteria. The neighboring joining tree (1000 BS) of 197 sequences of the pufM gene. Bootstraps values on branches. Evolutionary distance computed using Maximum composite likelihood method. Isolate R. arenilitoris 452T9 clusters (orange box) within Rhodobacterales (green).………………………………………………………………………………………………………………………..…………29 vi List of Tables Table 2-1 | Isolates collected from Orbicella faveolata colonies……………………………………………..10 Table 2-2 | Isolates collected from Symbiodinium cultures……………………………………………………..11 Table 2-3 | A-5 MiSeq assembly stats of the bacterial isolates from O. faveolata……………………13 Table 2-4 | A-5 MiSeq assembly stats of the bacterial isolates from Symbiodinium…………………13 Table 2-5 | The 31 complete genomes from NCBI used for the pangenomic analysis………………14 Table 2-6 | 16S rRNA classification of bacterial isolates ………………………………………………………….16 Table 2-7 | Basic stats of Pangenome for each genus. Singletons are shown as number of genes with known COG function within total number of singleton genes found. Isolates listed are those sequenced for this study, while genomes include other genomes that were used for comparative purposes……………………………………………………………………………………………………………..18 vii Acknowledgements There are several people I would like to extend my gratitude to for their help, guidance, advice, assistance, mentoring, support, and friendship. I profoundly appreciate the support, patience, and kindness of my advisor Dr. Mónica Medina. She has giving me countless lessons for my own development. She made me immediately feel at home with a family despite being thousands of miles away from any relatives. I would also like to recognize a great many of faculty and staff of Penn State for their professional and personal guidance, including Dr. Stephen Schaeffer, Dr. Tracy Langkilde, Dr. Chuck Fisher, Dr. Todd LaJeunesse, Dr. Roberto Iglesias-Prieto, and Dr. Iliana Baums. Special thanks to Dr. Fisher for inviting me on the Nautilus research vessel before I even accepted the offer from Penn State and Dr. Schaeffer for being an invaluable resource for my professional development. I want to also give a ginormous thank you to Allison Lewis, Zach Fuller, Cam Venable, Catherine Herzog, and Sam Vohsen for so many things. I knew Allison, Zach, and Cam would become my long-term friends after all the time they gave me when I was stranded at PSU during recruitment weekend. Since that day they have given me fantastic support personally and contributed to my work. Sam, my roommate of two years has been a wunderkind of a friend and colleague. I would not have made it this far with him. Also, thank you to all the students in the Biology department and any students that helped create a stronger community via the BDGSA. Final thanks are dedicated to my parents, Enola and Darren Smith, and my brothers Jacob and Chad. You guys are simply the best family a goof like me could ever ask for. I acknowledge the following grants for support of my research NSF (OCE 1442206) and NSF (INCLUDES 1649184). viii Chapter 1 Introduction A holobiont is characterized by a multicellular host interdependently associated with bacteria, archaea, fungi, and a variety of other eukaryotic microorganisms1. These symbionts can complement the holobiont through metabolic interactions that potentially benefit the host or other holobiont members. There are several well-established symbioses between animals and microorganisms, including nitrogen-fixing bacteria and cellulolytic protozoa within the gut of termites 2. Corals exhibit a mutualism with single cell algae from the