ASSESSING THE DIVERSITY OF FRESHWATER BACTERIA AND VIRUSES EMPLOYING METAGENOMICS TO CAPTURE THE DYNAMICS AND THE DIVERSITY OF FRESHWATER BACTERIA AND VIRUSES By MOHAMMAD MOHIUDDIN, M.Sc., Hons. B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Mohammad Mohiuddin, July 2019 DOCTOR OF PHILOSOPHY (2019) McMaster University Department of Biology Hamilton, Ontario, Canada TITLE: Employing Metagenomics to Capture the Dynamics and the Diversity of Freshwater Bacteria and Viruses AUTHOR: Mohammad Mohiuddin, M.Sc., Hons. B.Sc. (University of Dhaka, Bangladesh) SUPERVISOR: Professor Herb E. Schellhorn NUMBER OF PAGES: xx, 241 ii Lay Abstract Bacteria and viruses are abundant in freshwaters, yet our understanding of bacterial and viral communities in freshwaters remains inadequate. In addition, the changing patterns of waterborne diseases in recent years have warranted strong monitoring of recreational freshwaters across the globe. The goals of this thesis were to assess the dynamics and the diversity of freshwater bacterial and viral communities of the lower Great Lakes region and identify pathogenic bacterial and viral species. Employing a metagenomic approach, I have investigated the spatial and temporal distribution of bacterial and viral communities in recreational freshwaters. Using computational approaches, I have identified pathogenic bacteria and viruses and investigated the link between connected habitats. My research advances our understanding of bacterial and viral communities of the lower Great Lakes region and provides useful information to water quality decision makers for the optimization of municipal sampling programs currently employed in the Great Lakes area. iii Abstract Microbial water communities are a complex consortium of bacteria, viruses and protozoa. These complex microbial and viral communities may contain pathogens that cannot be detected by conventional methods. Next-generation sequencing (NGS) offers the potential to exhaustively characterize all microbial and viral components of a given water sample and facilitates the identification and quantification of pathogens of interest. The goals of this thesis were to assess the dynamics and the diversity of freshwater bacterial and viral communities of the lower Great Lakes region and identify pathogenic bacterial and viral species. We first assessed the diversity of viral communities in six different beaches of Lake Ontario and Lake Erie, two of the largest freshwater reservoirs in North America. We employed a robust and routinely applicable approach that can provide a comprehensive analysis of bacterial and viral community composition. Our analysis suggests that the viral communities of the lower Great Lakes region are dominated by bacteriophages but also contained viruses of plants and animals. Exhaustive characterization of bacterial communities indicates that the bacterial community composition is highly diverse, and the diversity differs between recreational waters and beach sands. In addition, we identified sequences of pathogens that are not currently included in traditional water monitoring schemes in both recreational water and beach sand. To investigate the impact of spatiotemporal and environmental factors on the distribution of bacterial species, we employed a computational approach and our analysis suggests that dissolved oxygen (DO) level is strongly associated with bacterial iv community diversity. Using a computational approach, we have also identified habitat- specific bacterial species and a possible link between inter-connected habitats. The findings of this thesis aid in our understanding of bacterial and viral community diversity in recreational waters and provide useful information to water quality decision makers. v Acknowledgements First and foremost, I would like to thank Dr. Herb Schellhorn for giving me the opportunity to work with him and for being an outstanding advisor and mentor. His patience and guidance have helped me grow intellectually, both as a researcher and as a person. I am truly grateful for my time learning from him. I am also thankful for the freedom and resources I have received from him to complete my graduate studies. I would also like to thank my supervisory committee members, Dr. Radhey Gupta and Dr. Brian Golding, for their guidance and support throughout my graduate studies. Thank you for meeting with me and for thoughtful discussions. Thanks are due to the collaborators, Dr. Tom Edge, Joshua Diamond, Glen Hudgin, for providing samples and resources throughout my graduate studies. I am grateful for the generous financial support I received from Norgen Biotek, Natural Sciences and Engineering Research Council (NSERC) of Canada, Niagara Peninsula Conservation Authority (NPCA), WaterSmart Niagara, MacWater and Niagara Region Public Health, for my research. Throughout my time in the Schellhorn lab, I have enjoyed the company of dynamic lab members. Thank you all for your support, for the creative ideas and for all the fun I had in and outside the lab. Special thanks go to Dr. Athanasios Paschos, Steven Botts and Yasser Salama for their input in my research. Finally, thank you to all my family, friends and loved ones, for their support and encouragement throughout my graduate studies. vi Table of Contents Lay Abstract ................................................................................................................. iii Abstract ......................................................................................................................... iv Acknowledgements ....................................................................................................... vi Table of Contents ......................................................................................................... vii List of Figures .............................................................................................................. xii List of Tables............................................................................................................. xviii Declaration of Academic Achievement ...................................................................... xix Chapter 1: Assessing the diversity of freshwater bacteria and viruses in the metagenomic era ............................................................................................................ 1 Abstract ....................................................................................................................... 2 1. Introduction .............................................................................................................. 3 2. Freshwater bacterial communities............................................................................. 5 2.1 Bacterial community structure ............................................................................ 5 2.2 Diversity of bacterial communities .................................................................... 13 3. Freshwater viruses .................................................................................................. 15 3.1 Viral community structure ................................................................................ 15 3.2 Diversity of viral communities .......................................................................... 20 4. Metagenomics- advantages and limitations ............................................................. 21 4.1 Factors that impact metagenomic studies .......................................................... 22 5. Conclusion ............................................................................................................. 37 References ................................................................................................................. 40 Chapter 2: Spatial and temporal dynamics of virus occurrence in two freshwater lakes captured through metagenomic analyses .......................................................... 59 Abstract ..................................................................................................................... 60 1. Introduction ............................................................................................................ 61 2. Materials and methods ............................................................................................ 64 2.1 Sampling sites and sample collection ................................................................ 64 2.2 Sample fractionation ......................................................................................... 64 2.3 Extraction of DNA from bacterial and VLP fraction ......................................... 65 vii 2.4 Preparation of DNA samples for sequencing ..................................................... 66 2.5 Bioinformatic analyses ...................................................................................... 66 3. Results ................................................................................................................... 68 3.1 Efficiency of the fractionation scheme .............................................................. 68 3.2 DNA in the environmental water samples ......................................................... 68 3.3 Abundance of bacterial and viral genome copies in freshwater .......................... 69 3.4 Overview of the viromes ..................................................................................