FLEXIBILITY IN THE MINERAL DEPENDENT METABOLISM OF A THERMOACIDOPHILIC CRENARCHAEOTE by Maximiliano Jose Amenabar Barriuso A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana August 2017 ©COPYRIGHT by Maximiliano Jose Amenabar Barriuso 2017 All Rights Reserved ii DEDICATION For my wife Ivonne who has been the pillar of support and love in my life and for my daughters Martina and Trinidad who are the ones that inspire my life every day. iii ACKNOWLEDGEMENTS I would like to extend my gratitude to Dr. Eric Boyd for all his time and patience in guiding me through all my Ph.D. It is hard to express my gratitude to him in just a few words, but I just could say that I have been really lucky to learn from him. I also would like to express my thanks to Dr. John Peters for giving me the opportunity to join his lab and making my life easier in applying and attending Montana State University. I am also thankful to everyone in the Boyd lab: Dan, John, Melody, Saroj, Eric D., Erik A., Libby and former lab member Matt Urschel for being such a fun and smart group of people to work with. Thanks also to the various co-authors who contributed to the chapters of these dissertation and to my committee members: Dr. Matthew Fields, Dr. Mark Skidmore and Dr. John Peters; all of you helped me grow as a scientist. I am also thankful to the Department of Microbiology and Immunology for believing in me and giving me the opportunity to join the Department to do my PhD, and also to all the former and current staff from the Department for helping me any time I need it. I am also thankful to the Chilean Government and its scholarship program “Becas Chile” for providing funding for 4 years of my doctoral program. I am also thankful to all my family and friends back in Chile who have supported me during these years. I would also like to thanks all my friends in the U.S.A. including those from Central Valley Fire District who have been like my second family. I am especially thankful to Anthony, Joanna, Carlos, John and Vicky Andre and his son Chris for helping me and my family when we needed them the most. Finally, but certainly not least, I would like to thanks my wife Ivonne for all her love and help. Without her support, none of this would have been possible. iv TABLE OF CONTENTS 1. GENERAL INTRODUCTION ........................................................................................1 Hydrothermal Ecosystems in YNP ..................................................................................4 Types of Thermal Features Present in YNP ....................................................................6 Geomicrobiology of ASC Hot Springs ............................................................................9 Sulfur Depositional Zone ...................................................................................11 Iron/Arsenic Depositional Zone .........................................................................13 Geomicrobiology of “Dragon Spring” ...........................................................................15 Acidianus sp. as a Model for the Study of Thermoacidophiles .....................................22 Metabolic Flexibility of Microbes Present in Terrestrial Hydrothermal Systems .........23 Prediction of Microbial Substrate Preference ................................................................24 References ......................................................................................................................27 2. MICROBIAL SUBSTRATE PREFERENCE DICTATED BY ENERGY DEMAND RATHER THAN SUPPLY ........................................................40 Contribution of Authors and Co-Authors ......................................................................40 Manuscript Information Page ........................................................................................41 Main text ........................................................................................................................43 Growth Yields and Substrate Transformation Kinetics. ....................................45 Preferential use of Redox Couples .....................................................................47 Energy Demand Dictates Substrate Preference .................................................49 Methods..........................................................................................................................53 Sample Collection and Enrichment of Acidianus Strain DS80 .........................53 Physiological Characterization ..........................................................................55 Preferential Substrate use Determinations .........................................................56 Evaluation of Growth and Activity ....................................................................56 Carbon Assimilation ..........................................................................................57 Energetics ...........................................................................................................58 Transmission Electron Microscopy ...................................................................60 Field Emission Scanning Electron Microscopy .................................................60 Data availability .............................................................................................................60 Acknowledgements ........................................................................................................61 Author information ........................................................................................................61 Competing Financial Interest .........................................................................................61 Tables .............................................................................................................................62 Figures............................................................................................................................67 References ......................................................................................................................76 v TABLE OF CONTENTS CONTINUED 3. EXPANDING ANAEROBIC HETEROTROPHIC METABOLISM WITH HYDROGEN ..........................................................................81 Contribution of Authors and Co-Authors ......................................................................81 Manuscript Information Page ........................................................................................82 Summary ........................................................................................................................84 Introduction ....................................................................................................................85 Material and Methods ....................................................................................................88 Culture Conditions .............................................................................................88 Isolation of Genomic DNA and Sequencing .....................................................89 Physiological Characterization ..........................................................................92 Carbon Assimilation and Mineralization ...........................................................93 Influence of Acetate on the Assimilation of Dissolved Inorganic Carbon (DIC) .....................................................................................94 Influence of DIC on the Assimilation and Mineralization of Acetate ...............94 Results ............................................................................................................................95 DS80 Genomic Properties and Phylogeny .........................................................95 Genomic Predictions of Electron Donor Usage .................................................96 Genomic Predictions of Electron Acceptor Usage ............................................99 Physiological Characterization of Acidianus strain DS80 ...............................100 Carbon Assimilation and Mineralization .........................................................101 Influence of Acetate on DIC Assimilation .......................................................103 Influence of DIC on Acetate Assimiliation/Mineralization .............................104 Discussion ....................................................................................................................104 Acknowledgments........................................................................................................113 Tables ...........................................................................................................................114 Figures..........................................................................................................................115 Supplementary File 1 ...................................................................................................127 Supplementary File 2 ...................................................................................................142 References ....................................................................................................................163 4. FLEXIBILITY IN MINERAL DEPENDENT ENERGY METABOLISM BROADENS THE ECOLOGICAL NICHE OF A THERMOACIDOPHILE .....................................................................170