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Life in the Cold Biosphere: the Ecology of Psychrophile

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Life in the Cold Biosphere: the Ecology of Psychrophile Life in the cold biosphere: The ecology of psychrophile communities, genomes, and genes Jeff Shovlowsky Bowman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2014 Reading Committee: Jody W. Deming, Chair John A. Baross Virginia E. Armbrust Program Authorized to Offer Degree: School of Oceanography i © Copyright 2014 Jeff Shovlowsky Bowman ii Statement of Work This thesis includes previously published and submitted work (Chapters 2−4, Appendix 1). The concept for Chapter 3 and Appendix 1 came from a proposal by JWD to NSF PLR (0908724). The remaining chapters and appendices were conceived and designed by JSB. JSB performed the analysis and writing for all chapters with guidance and editing from JWD and co- authors as listed in the citation for each chapter (see individual chapters). iii Acknowledgements First and foremost I would like to thank Jody Deming for her patience and guidance through the many ups and downs of this dissertation, and all the opportunities for fieldwork and collaboration. The members of my committee, Drs. John Baross, Ginger Armbrust, Bob Morris, Seelye Martin, Julian Sachs, and Dale Winebrenner provided valuable additional guidance. The fieldwork described in Chapters 2, 3, and 4, and Appendices 1 and 2 would not have been possible without the help of dedicated guides and support staff. In particular I would like to thank Nok Asker and Lewis Brower for giving me a sample of their vast knowledge of sea ice and the polar environment, and the crew of the icebreaker Oden for a safe and fascinating voyage to the North Pole. None of this work would have been possible without the support of the NASA Astrobiology Institute, the University of Washington Astrobiology Program (via NSF IGERT), NSF Office of Polar Programs (now Division of Polar Programs), and the EPA STAR fellowship program. Programs and initiatives that directly supported individual chapters are listed in chapter acknowledgements. I would also like to thank the many co-authors and collaborators who worked directly with me on these projects, facilitated the fieldwork, or provided critical insight at some point. In particular, Hans-Werner Jacobi, Kevin Hand, and Søren Rysgaard graciously provided opportunities to go to the field, enabling much of the work described here. Matthias Wietz, Nikolas Blom, Shelly Carpenter, Jesse Colangelo-Lillis and many others made that fieldwork a lot more enjoyable; thanks to all of you for the assistance, entertainment, and collaboration! Eric Collins, Britney Schmidt, and a host of colleagues, co-authors, and friends, including the whole iv crew at the shaka house, provided, and continue to provide, critical insight and great discussion on this and related work. Last I would like to thank Sarah Purkey, my colleague and partner, for all the ideas, discussion, and help over the last six years. I can’t wait to see where life leads us next! v University of Washington Abstract Life in the cold biosphere: The ecology of psychrophile communities, genomes, and genes Jeff S. Bowman Chair of Supervisory Committee: Prof. Jody W. Deming The prevalence of low temperature habitats on Earth makes the ecology of organisms adapted to low temperature environments (psychrophiles) an important area of research. Studies of low temperature ecosystems including the deep sea, sea ice, glacial ice, permafrost, and snow have provided a wealth of knowledge on the resilience of psychrophilic microbial ecosystems in the face of anthropogenic and natural disturbance, the history of microbial life on Earth, and the potential distribution of life in extraterrestrial environments. Taking these three knowledge areas as motivation, this dissertation further explores psychrophile ecology. Chapter 1 introduces the history of research on psychrophiles, and the current state of knowledge regarding sea ice microbial communities, an important ecosystem dominated by psychrophiles. This chapter also explores one method for making the jump from descriptive ecological studies to process-based studies that have predictive power. Chapters 2 and 3 explore the diversity of Bacteria found in two understudied psychrophile habitats; multiyear sea ice and frost flowers. In the first quantitative analysis of sea ice community composition, Chapter 2 describes the multiyear sea ice microbial community as diverse, and in agreement with previous studies, dramatically different from the seawater microbial community. Chapter 3 describes an vi unusual community of Bacteria in frost flowers, dominated by members of the order Rhizobiales. The metabolic plasticity of the Rhizobiales could have important implications for elemental cycling within young sea ice, particularly for nitrogen and sulfur species, an idea explored through an analysis of metabolic potential in Chapter 4. Chapter 5 describes a new method for evaluating genomic plasticity within any group of genomes, and applies this method to a comparative analysis of psychrophile and mesophile genomes, finding evidence for greater genome plasticity within psychrophile genomes. Application of a molecular clock to horizontal gene transfer events in these genomes suggests a link between cold periods in the Phanerozoic and the rate of retention of genes acquired by psychrophiles. Chapter 6 examines how psychrophilic enzymes are optimized for low temperatures through amino acid substitutions and introduces a model for further exploration of amino acid preferences. Experiments with this model suggest a role for serine, preferred in some psychrophile proteins, in adaptation to both low temperature and high salinity. Chapter 7 explores the potential for psychrophiles to degrade alkanes, a major component of crude oil, by the presence of genes coding for alkane hydroxylases. Several likely alkane hydroxylase genes in psychrophiles, not currently annotated as alkane hydroxylases, were identified. Analysis of the protein physical parameters coded by these genes suggests some optimization to low temperature. These findings have important implications for the in situ bioremediation of crude oil in cold environments, and for efforts to develop remediation technologies that function at low temperature. vii Table of Contents LIST OF FIGURES…………………………………………………………………………..…xvi LIST OF TABLES…………………………………………………………………………….xviii CHAPTER 1: AN INTRODUCTION TO SEA ICE MICROBIAL ECOLOGY……………1 1.1 A BRIEF HISTORY OF RESEARCH ON SEA ICE MICROBIAL COMMUNITIES……..2 1.2 THE PRESENT DAY UNDERSTANDING OF SEA ICE MICROBIAL ECOLOGY……...7 1.3 THE FUTURE OF SEA ICE MICROBIAL ECOLOGY……………………………….........9 REFERENCES…………………………………………………………………………………..14 FIGURES………………………………………………………………………………………...19 CHAPTER 2: MICROBIAL COMMUNITY STRUCTURE OF ARCTIC MULTIYEAR SEA ICE AND SURFACE SEAWATER BY 454 SEQUENCING OF THE 16S RNA GENE.…………………………………………………………………………………………...24 Bowman, J. S., Rasmussen, S., Blom, N., Deming, J. W., Rysgaard, S., & Sicheritz-Ponten, T. (2011). Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene. The ISME journal, 6(1), 11–20. ABSTRACT……………………………………………………………………………………...24 2.1 INTRODUCTION……………………………………………………………………….......25 2.2 METHODS…………………………………………………………………………………..27 2.2.1 SAMPLE COLLECTION AND PREPARATION………………………………..27 2.2.2 COMMUNITY STRUCTURE…………………………………………………….28 2.2.3 COMMUNITY COMPOSITION………………………………………………….29 2.2.4 COMMUNITY SIMILARITY…………………………………………………….29 2.2.4 16S rRNA GENE DIVERGENCE………………………………………………...30 2.3 RESULTS………………………………………………………………………………........31 2.3.1 ENVIRONMENTAL…………………………………………………………........31 viii 2.3.2 COMMUNITY STRUCTURE…………………………………………………….32 2.3.3 COMMUNITY COMPOSITION………………………………………………….33 2.3.4 16S rRNA GENE DIVERGENCE………………………………………………...34 2.3.5 COMMUNITY SIMILARITY…………………………………………………….35 2.4 DISCUSSION………………………………………………………………………………..36 2.5 SUPPLEMENTARY METHODS…………………………………………………………...39 2.5.1 SAMPLE COLLECTION………………………………………………………….39 2.5.2 ENVIRONMENTAL PARAMETERS……………………………………………40 2.5.3 DNA EXTRACTION, PURIFICATION, AND SEQUENCING…………………41 REFERENCES…………………………………………………………………………………..43 TABLES AND FIGURES……………………………………………………………………….48 CHAPTER 3: SELECTIVE OCCURRENCE OF RHIZOBIALES IN FROST FLOWERS ON THE SURFACE OF YOUNG SEA ICE NEAR BARROW, ALASKA AND DISTRIBUTION IN THE POLAR MARINE RARE BIOSPHERE…………………..........71 Bowman, J. S., Larose, C., Vogel, T. M., & Deming, J. W. (2013). Selective occurrence of Rhizobiales in frost flowers on the surface of young sea ice near Barrow, Alaska and distribution in the polar marine rare biosphere. Environmental microbiology reports, 5(4), 575–582. ABSTRACT……………………………………………………………………………………...71 3.1 INTRODUCTION……………………………………………………………………….......72 3.2 RESULTS AND DISCUSSION……………………………………………………………..75 3.3 CONCLUSIONS…………………………………………………………………………….80 3.4 SUPPLEMENTARY METHODS…………………………………………………………...80 3.4.1 SAMPLE COLLECTION AND DNA EXTRACTION…………………………...80 3.4.2 CLONE LIBRARY………………………………………………………………...82 3.4.3 MICROARRAY ANALYSIS……………………………………………………...83 3.4.4 T-RFLP…………………………………………………………………………….84 3.4.5 METAGENOMIC ANALYSIS………………………………………………........85 3.4.6 COMPARISON WITH OTHER DATA SETS………………………………........86 ix REFERENCES…………………………………………………………………………………..88 TABLES AND FIGURES……………………………………………………………………….94 CHAPTER 4: THE GENETIC POTENTIAL FOR KEY BIOGEOCHEMICAL PROCESSES IN ARCTIC FROST FLOWERS AND YOUNG SEA ICE REVEALED BY METAGENOMIC ANALYSIS................................................................................................101
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