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(Title of the Thesis)* ICE ASSOCIATION IN MICROBES by Sandra Louise Wilson A thesis submitted to the Department of Biology In conformity with the requirements for the degree of Doctor of Philosophy Queen’s University Kingston, Ontario, Canada (September, 2010) Copyright © Sandra Louise Wilson, 2010 Abstract Microbes have a remarkable ability to adapt to a host of environmental stressors, including low temperature, high pressure and osmotic stresses. The adaptations of resistant microbes to low temperatures are varied, and may include the accumulation of solutes to maintain osmotic balance, the production of antifreeze proteins (AFPs) or ice nucleation proteins (INPs) to manipulate ice growth or formation. AFPs depress the freezing point, inhibit ice recrystallization, and have been reported to inhibit or delay the growth of gas hydrates. Conversely, INPs precipitate ice formation at relatively high subzero temperatures. Collectively, these activities can be described as ‘ice-association’ activities. Here, ice-affinity and/or freeze-thaw cycling were used to either select for isolates with ice association properties or to assess the low temperature resistance of microbial consortia derived from various environments. Ice-affinity successfully selected psychrotolerant microbes from cultured temperate and boreal soils, some of which had been previously reported in glaciers and Arctic/Antarctic sites. Many of the recovered microbes demonstrated ice-association activities. Freeze-thaw selection also greatly decreased the abundance and diversity of consortia from distinct sites, and allowed the recovery of individual isolates, many of which demonstrated ice-association. Freeze- thaw selection was also used to assess the role of cross-tolerance between osmotic and freeze-thaw stresses, based on the common challenge of desiccation. Microbial consortia from lakes with varying degrees of salinity were subjected to freeze-thaw stress, and the consortia from more saline lakes tended to show greater low temperature resistance. While few of the recovered microbes demonstrated ice-association activities, those from ii the more saline lakes tended to contain a higher intracellular solute concentration and were more likely to form biofilms. This underscores the diversity of resistance strategies and supports the notion of cross-tolerance. To determine if these selective regimes would have applications for hydrate growth inhibition, microbes derived from an oil well sample were subjected to freeze-thaw stress. Selection reduced microbial abundance, shifted the diversity, and resulted in the recovery of microbes with some ice-association activity. Taken together, this thesis demonstrates that the application of low temperature stress can be used to successfully investigate stress resistance mechanisms within microbial communities from distinct environments. iii Co-Authorship The following researchers contributed to the success of this thesis, and are therefore greatly appreciated and are acknowledged with co-authorship. Chapter 2 Deborah Kelley assisted with the ice-finger experiments, identification and screening of the recovered microbes for ice-association activity as part of her undergraduate honours thesis. Chapter 3 Dr. Paul Grogan kindly provided the Daring Lake soil samples as well as lab space. iv Acknowledgements First and foremost, huge thanks to Virginia, who is a wonderful supervisor and mentor! Without her constant support, guidance, encouragement and ‘outside of the box’ thinking, this thesis would not exist. I look forward to working as your peer in the future. Of course, enormous thanks are happily due to my family. Mom and dad, thanks for fostering my love of science, encouraging and supporting me through this process. Jen and Ryan, thanks for all your help, support and the constant adventures – you’re wonderful siblings, and I love you all. Thanks to Bev for your friendly advice and constant thoughts, you’re a fabulous cousin. And dearest Gramps, where would I be without all the love and good times while you were with us? To my closest friends, Sarah Bean, Jennifer May-Focht and Sabrina Mueller, thanks for the constant smiles and laughter, which kept me going. And to my favourite roomie and fantastic friend, Chantelle Peter, your support was more helpful then you could realize – thanks for the walks and talks. Lastly, to those at FBCK who supported me, thanks. Finally, thanks to the bug-groupers: J. Affleck, A. Brown, M. Chalifoux, J. Choi, H. Esposito, R. Gordienko, E. Huva, N. Kumar, K. Lauerson, S. Nowickyj, W. Qin, T. Vanderveer, S. Wu, and the AFP-groupers, past and present for the suggestions and advice in lab meetings, and the good times in the lab. v Table of Contents Abstract ...................................................................................................................................... ii Co-Authorship ........................................................................................................................... iv Acknowledgements ..................................................................................................................... v Table of Contents ....................................................................................................................... vi List of Figures............................................................................................................................ ix List of Table.....................................................................................................................................xi List of Abbreviations: ................................................................................................................ xi Chapter 1 : General Introduction and Literature Review .............................................................. 1 Literature Cited: ...................................................................................................................... 3 Chapter 1.1: Selection of Low Temperature Resistance in Bacteria and Potential Applications .... 4 Abstract: ................................................................................................................................. 4 Introduction: ........................................................................................................................... 6 Challenges of Low Temperature and Osmotic Stresses: ....................................................... 6 Adaptations to Hyperosmotic Stress: ................................................................................... 8 Adaptations to Low Temperature Stress: ............................................................................. 9 Antifreeze Proteins: ........................................................................................................... 10 Ice Nucleation Proteins: .................................................................................................... 12 Freeze-Thaw and Ice-Affinity Selective Methods and Ice Assays: ......................................... 14 Equipment for Selection: ................................................................................................... 14 Microbe Ice-Association Activities: ................................................................................... 16 Collection Sites and Culture Conditions: ........................................................................... 17 Isolate Identification: ......................................................................................................... 20 Experimental Selection and Resistance: ................................................................................. 21 Soil Communities and Isolates:.......................................................................................... 21 Lake Communities and Isolates: ........................................................................................ 30 Biotechnological Implications: .............................................................................................. 33 Acknowledgements: .............................................................................................................. 35 Literature Cited: .................................................................................................................... 36 Chapter 1.2: Gas Hydrates and Microbes ................................................................................... 45 Literature Cited: .................................................................................................................... 49 Chapter 1.3: Research Objectives .............................................................................................. 52 vi Chapter 2 : Ice-Active Characteristics of Soil Bacteria Selected by Ice-Affinity......................... 55 Abstract: ............................................................................................................................... 55 Introduction: ......................................................................................................................... 56 Materials and Methods: ......................................................................................................... 58 Results: ................................................................................................................................. 63 Discussion: ........................................................................................................................... 73 Acknowledgements: .............................................................................................................
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