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<I>AUREOCOCCUS ANOPHAGEFFERENS</I> University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2016 MOLECULAR AND ECOLOGICAL ASPECTS OF THE INTERACTIONS BETWEEN AUREOCOCCUS ANOPHAGEFFERENS AND ITS GIANT VIRUS Mohammad Moniruzzaman University of Tennessee, Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Environmental Microbiology and Microbial Ecology Commons Recommended Citation Moniruzzaman, Mohammad, "MOLECULAR AND ECOLOGICAL ASPECTS OF THE INTERACTIONS BETWEEN AUREOCOCCUS ANOPHAGEFFERENS AND ITS GIANT VIRUS. " PhD diss., University of Tennessee, 2016. https://trace.tennessee.edu/utk_graddiss/4152 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 Mohammad Moniruzzaman entitled "MOLECULAR AND ECOLOGICAL ASPECTS OF THE INTERACTIONS BETWEEN AUREOCOCCUS ANOPHAGEFFERENS AND ITS GIANT VIRUS." 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 requirements for the degree of Doctor of Philosophy, with a major in Microbiology. Steven W. Wilhelm, Major Professor We have read this dissertation and recommend its acceptance: Tim E. Sparer, Mark R. Radosevich, Jill A. Mikucki, Willie Wilson Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) MOLECULAR AND ECOLOGICAL ASPECTS OF THE INTERACTIONS BETWEEN AUREOCOCCUS ANOPHAGEFFERENS AND ITS GIANT VIRUS A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Mohammad Moniruzzaman December 2016 Copyright © 2016 by Mohammad Moniruzzaman All rights reserved ii To Ma, Abba, and Boro Mama iii Acknowledgements The body of work presented in this dissertation reflects the dedication of an incredible team of researchers who assisted me in every way from the beginning. My sincere thanks to my supervisor, Professor Steven W. Wilhelm, for his unflagging enthusiasm through the entire course of my research. His deep passion about research is contagious and has always inspired me to keep exploring, no matter how complex the scientific problem was. Choosing Dr. Wilhelm as my PhD supervisor was one of the best decisions I ever made. I also must thank Dr. Gary LeCleir, who supported me from the very beginning by teaching me new skills and providing valuable suggestions on the projects that I undertook. Thank you, Big Poppa! Without my collaborators, it would be impossible to accomplish a number of projects. My sincere thanks to Dr. Christopher Gobler, Dr. Christopher M. Brown, Dr. Sonya Dyhrman, Dr. Harriet Alexander and Dr. Louie Wurch, who provided me with environmental samples and access to datasets. My sincere thanks to my committee members who gave valuable suggestions and have been patient with my throughout these years. They provided specific guidelines on the directions I took, saving me a lot of time. Specifically, I have to mention Dr. Willie Wilson from Sir Alister Hardy Foundation for Ocean Sciences who flew to Knoxville every year and attended my committee meetings. It has been an incredible journey of friendship and scientific camaraderie. Over the years, I have interacted with an amazing team of graduate students in the Wilhelm Lab. Morgan Wurch constantly inspired me with her hard work, dedication and great discussion on science and life’s goals. Jackson, Lauren, Robbie, Sam and Josh – I am grateful to you for the wonderful time and discussion we had together in and outside the lab. I learned a lot as I mentored Eric Gann and Mark McDonald, who received training as undergraduates in Wilhelm lab. Thank you, for always being there during the long experiments and whenever I needed to vent out my frustration or crazy ideas! Over the years, I have had the opportunity to travel to different parts of the USA because of my incredible photographer friends living all over the country. I am grateful to you for your hospitality and the memorable trips we took together. iv Abstract Viruses are increasingly being recognized as an important biotic component of all ecosystems including agents that control the rapid ecological events that are harmful algal blooms (HABS). Aureococcus anophagefferens is a pelagophyte which causes recurrent ecosystem devastating brown tide blooms along the east coast of the USA and has recently spread to China and South Africa. It has been suggested that a large virus (AaV) is possibly an important agent for demise of brown tide blooms. This observation is consistent with the recognition of a number of other giant viruses modulating algal blooms in marine systems. In this dissertation, we investigated both the molecular underpinnings of Aureococcus-AaV interactions and the dynamics of AaV and the associated viral community in situ. We determined the genome sequence and phylogenetic history of AaV using high throughput sequencing approach and revealed it’s intertwined evolutionary history with the host and other organisms. Building upon the available genome of AaV and its host, we took an RNA-seq approach to provide insights on the physiological state of the AaV-infected Aureococcus ‘virocell’ that is geared towards virus production. In situ activity of AaV was detected by targeted amplicon and high throughput community RNA sequencing (metatranscriptomics) from Quantuck Bay, NY, a site with recurrent brown tide blooms. AaV and associated giant algal viruses in the Mimiviridae clade were found to respond to environmental changes, indicating that this newly recognized phylogenetic group is an important contributor to the eukaryotic phytoplankton dynamics. Analyzing time series metatranscriptomics from two distinct coastal sites recovered diverse viruses infecting microeukaryotes (including AaV) as part of interacting networks of viruses and microeukaryotes. Results from these studies testify AaV as an important factor for brown tide bloom demise, reveals the molecular underpinnings of AaV-host interactions and establishes the ecological relevance of Mimivirus-like algal viruses. We also provide foundation for using metatranscriptomics as an important tool in marine virus ecology – capable of recovering associations among coexisting marine microeukaryotes and viruses. v Table of Contents Chapter I: Literature review 1 Objectives 19 List of references 21 Chapter II: Genome of Brown Tide virus (AaV), the little giant of the Megaviridae, 32 elucidates NCLDV genome expansion and host-virus coevolution Publication note 33 Abstract 34 Introduction 35 Materials and Methods 36 Results and discussion 40 Conclusion 53 List of references 55 Chapter II Appendix 64 Chapter III: The transcriptomic landscape of Aureococcus virocell 104 Publication note 105 Abstract 106 Introduction 107 Materials and Methods 108 Results 110 Discussion 132 Conclusion 138 List of references 140 Chapter III Appendix 148 Chapter IV: Diversity and dynamics of algal Megaviridae members during a harmful 155 brown tide caused by the pelagophyte, Aureococcus anophagefferens Publication note 156 vi Abstract 157 Introduction 158 Materials and Methods 160 Results 163 Discussion 168 List of references 176 Chapter IV Appendix 182 Chapter V: Virus-host infection dynamics for marine microeukaryotes resolved from 185 metatranscriptomics Publication note 186 Abstract 187 Introduction 188 Materials and Methods 189 Results and discussion 194 Conclusion 215 List of references 217 Chapter VI: Conclusion 223 List of references 227 Vita 228 vii List of Tables Table 2.1: Mimivirus cluster of orthologous groups (MimiCOGs) present in AaV. The universal 78 NCLDV core genes present in AaV are also listed. Table 2.2: CDS name, location, length, functional annotation and probable phylogenetic origin 80 of putative AaV coding sequences. Table 2.3: Paralogous genes in AaV clustered in groups. 100 Table 2.4: tRNAs in the AaV genome. 101 Table 2.5: Genes uniquely present in AaV among the nucleocytoplasmic large DNA viruses 102 (NCLDVs) Table 2.6: Putative AaV CDSs having homologs only in the NCLDVs (NCLDV specific 103 ORFans). Table 3.1: Over and underexpressed genes encoding components of photosystem I and II across 127 the time course compared to uninfected control. Table 3.2: AaV genes with COG/NCVOG assignments and time point when their expressions 148 were first observed. Genes with no COG/NCVOG assignments are not shown. Table 4.1: Spearman’s correlation co-efficient (ρ) indicating significant relationship between cell 184 counts vs. environmental variables. (PC_Cyano: Phycocyanin rich cyanobacteria.). Table 5.1: RNA–seq library sizes after quality trimming. The Quantuck Bay samples were sequenced as single reads, while paired-end sequencing was carried out for the Narragansett Bay 192 samples (denoted by X2). viii List of Figures Figure 1.1: Maximum likelihood phylogenetic reconstruction of NCLDV major capsid protein sequences from environmental metatranscriptomic datasets. 10 Figure 1.2: A number of ultrastructure analyses of Aureococcus and associated virus from different studies. 14 Figure 2.1: Best BLASTp hits of AaV proteome against NCLDVs
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