University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2017 Thermophilic Endospore-Forming Bacteria as Models for Exploring Microbial Dispersal in Time and Space Cramm, Margaret Cramm, M. (2017). Thermophilic Endospore-Forming Bacteria as Models for Exploring Microbial Dispersal in Time and Space (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28742 http://hdl.handle.net/11023/4275 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Thermophilic Endospore-Forming Bacteria as Models for Exploring Microbial Dispersal in Time and Space by Margaret Anne Cramm A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA DECEMBER, 2017 © Margaret Anne Cramm 2017 Abstract Thermophilic endospore-forming bacteria – “ thermospores ” – are particularly useful model organisms for exploring microbial biogeography because they remain viable for long geologic time periods owing to a dormant state that confers resistant to extreme conditions. Using high temperature incubation experiments and 16S rRNA gene amplicon sequencing, geographic and temporal thermospore dispersal in marine sediments was explored. Thermospores detected in surface sediments across the North Atlantic are likely to originate from multiple warm temperature habitats, and are viable in sediments buried ~15 000 years ago. These approaches also revealed thermospore viability following the extreme stress of prolonged -80°C exposure. Thermospore viability and dispersal on a scale of millions of years was explored in a 1.2 km long sediment core. Uneven thermospore germination posed a challenge for thermospore detection but their capacity for use as models of biological dispersal remains valid. ii Preface This thesis is the original, unpublished, independent work by the author, Margaret Cramm iii Acknowledgements I would like to thank the funding agencies NSERC, Genome Canada, MEOPAR, ArcticNet and, the Province of Alberta for supporting my research. I would like to thank Anirban Chakraborty for teaching me everything I know about microbiological labwork. Anirban’s patience and dedicaton to teaching me was central to the establishment of my research foundation and the completion of this thesis. Addionally, I would also like to thank Carmen Li for her patience and assistance with the molecular work required for this research. Carmen was integral in the collection and interpretation of DNA sequencing data that was central to this research. Emil Ruff is a wizard of microbial community analysis. I am grateful for Emil’s dedication to teaching me that went beyond simply teaching me analysis techniques but also ensured that I understood the underlying concepts. Finally, I would like to thank Casey Hubert, my supervisor, for mentoring and championing me throughout my graduate student research career. I am particularly grateful to Casey for allowing me to steer the direction of my research based on my curiosity, increasing the quality of my scientific writing, and supporting me in participating in unique domestic and international research opportunities that elevated my research skill set. My time as a graduate student has offered me diverse learning experiences that have advanced me as a researcher and evolved me personally. Much of what I have gained during this time is a direct result of my sense of adventure meeting Casey’s open-minded approach to graduate student learning and for this reason I know that of all the supervisors in the world Casey is the best one for me. iv Dedication I would like to dedicate this work to Albert Stephen Bishop Cramm and Ann Ida (Appelt) Cramm whose support made my education and participation in this research possible. v Table of Contents Abstract .......................................................................................................................... ii Preface........................................................................................................................... iii Acknowledgements ...................................................................................................... iv Dedication ...................................................................................................................... v Table of Contents ......................................................................................................... vi List of Tables ................................................................................................................ ix List of Figures ............................................................................................................... xi List of Supplementary Tables ..................................................................................... xv List of Supplementary Figures .................................................................................. xvi List of Abbreviations ................................................................................................... xx Chapter 1: Introduction ................................................................................................. 1 1.1 Bacterial endospores ............................................................................................. 1 1.2 Endospore strategies for survival and longevity ..................................................... 3 1.3 Thermophilic endospore-forming bacteria .............................................................. 5 1.4 Microbial biogeography and thermospores ............................................................ 5 1.5 Thermospores in anoxic environments .................................................................. 8 1.6 Panspermia and the last universal common ancestor .......................................... 10 1.7 Thesis overview ................................................................................................... 14 Chapter 2: Materials and Methods ............................................................................. 15 2.1 Sediment sample collection ................................................................................. 15 2.2 High temperature incubation ................................................................................ 17 2.3 Sulfate and organic acid measurement ................................................................ 17 2.4 16S rRNA gene amplicon sequencing and analyses ........................................... 18 2.4.1 DNA extraction .............................................................................................. 18 2.4.2 16S rRNA gene amplification and sequencing .............................................. 18 2.4.3 16S rRNA gene amplicon sequence analysis ................................................ 19 Chapter 3: Freezing tolerance of thermophilic endospores in Arctic marine sediment ....................................................................................................................... 21 3.1 Abstract .............................................................................................................. 21 3.2 Introduction ........................................................................................................ 23 3.3 Materials and Methods ...................................................................................... 25 3.3.1 Freezing pretreatment ................................................................................... 25 3.3.2 High temperature incubation ......................................................................... 25 3.3.3 Sulfate and organic acid measurement ......................................................... 26 3.3.4 DNA extraction and 16S rRNA gene amplicon sequencing ........................... 26 3.3.5 Gas composition measurement after freezing pretreatment .......................... 27 3.3.6 Repeat freezing pretreatment and incubation to observe the effect of O2 contamination on bacterial community structure .................................................... 27 vi 3.4 Results ................................................................................................................ 29 3.4.1 Sulfate reduction during 50°C incubations ..................................................... 29 3.4.2 Organic acid depletion ................................................................................... 30 3.4.3 16S rRNA gene amplicon library analysis and thermospore OTU identification ............................................................................................................................... 31 3.4.4 Gas analysis after freezing pretreatment and the gas permeability of rubber stoppers after freezing ............................................................................................ 40 3.4.5 Bacterial community structure after repeat pre-freezing and incubation ........ 40 3.5 Discussion .......................................................................................................... 47