Interactions of Aquificae with Mercury And
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MICROBIALLY MEDIATED MERCURY DETOXIFICATION IN GEOTHERMAL ENVIRONMENTS: INTERACTIONS OF AQUIFICAE WITH MERCURY AND EVIDENCE FOR PHYLOGENETIC NICHE CONSERVATISM IN YELLOWSTONE NATIONAL PARK HOT SPRINGS By ZACHARY FREEDMAN A dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Ecology and Evolution Written under the direction of Professor Tamar Barkay And approved by __________________________________ __________________________________ __________________________________ __________________________________ __________________________________ __________________________________ New Brunswick, New Jersey January 2012 ABSTRACT OF THE DISSERTATION Microbially Mediated Mercury Detoxification in Geothermal Environments: Interactions of Aquificae with Mercury, and Evidence for Phylogenetic Niche Conservatism in Yellowstone National Park Hot Springs By ZACHARY FREEDMAN Dissertation Director: Dr. Tamar Barkay Geothermal features are generated by leaching of minerals and metals as superheated water flows through cracks and fissures in Earth’s crust. As this water reaches the surface, chemical, pH, and temperature gradients are created that drive life in these environments. Geothermal environments are often enriched with toxic metals, e.g. mercury (Hg), the focus of this dissertation. Resistance to toxic Hg(II) is controlled by the enzyme mercuric reductase (MR), which catalyzes Hg(II) reduction to Hg(0). The gene encoding MR, merA, is part of the mercury resistance (mer) operon, which at minimum includes genes encoding transport, enzymatic, and regulatory functions. The primary objective of my research was to achieve better understanding of biotic transformations that modulate Hg toxicity in geothermal environments. I characterized Hg-resistance in Aquificae, dominant primary producers in geothermal environments, and investigated the diversity and distribution of Hg-resistance genes in geochemically ii diverse hot springs in Yellowstone National Park (YNP), and 23 assemblages on a global scale. Two strains of Aquificae were obtained; Hydrogenobaculum sp. Y04AAS1 (AAS1) and Hydrogenivirga sp. 128-5-R1-1 (R1-1). Genome sequencing revealed homologous sequences to merA, and alignment of putative Hg-resistance genes, MerA, MerT (Hg(II) transporter) and MerP (periplasmic scavenger), reveal homology with the mer system of Tn501. Characterization of mer in AAS1 and R1-1 include growth at Hg concentrations >10 μM Hg(II), loss of Hg(II) from the growth medium, validation of Hg(0) production, and MR enzyme activity; mer induction was not observed, suggesting lack of regulatory function. Microbial mat biomass was collected from Bijah and Succession Springs, YNP, and environmental merA sequences were obtained from GenBank to determine the ecological controls on Hg-resistant communities in YNP hot springs, and on a global scale. merA assemblages exhibited grouping within each community, and total sequence pool, as indicated by positive net relatedness index and nearest taxon index values, respectively. Cluster analyses reveal different clustering patterns of 16S rRNA and merA gene assemblages from YNP, suggesting unique controls on 16S rRNA and merA gene community structure. Meta-analysis of merA communities from 23 assemblages encompassing 782 environmental sequences reveal clustering based on sample location, suggesting that geography structures Hg-resistant communities. iii Acknowledgements I would like to extend my deepest thank you first and foremost to my major professor, Dr. Tamar Barkay, who always made time to discuss ideas, give advice and criticism, all while motivating me with her support and encouragement. I am extremely thankful to the members of my dissertation committee: Dr. Tamar Barkay, Dr. Eric Boyd, Dr. John Dighton, Dr. Max Haggblom, Dr. Anna-Louise Reysenbach, and Dr. Costantino Vetriani for their guidance and support during the generation and completion of my dissertation work. I am very thankful to Drs. Theodore Chase, Max Haggblom, Anna- Louise Reysenbach, and Costantino Vetriani for their good advice and making their laboratory resources available to me, and to Drs. Eric Boyd and John Dighton for advice and guidance in forming my dissertation project. I am grateful to past and present members of the Barkay lab; Aspa, Riqing, Sharron, Melitza, Kim, Chu-Ching, Yanping, Heather, and Kritee for their help, direction, critiques, and lively discussions over the years. Thank you to the many undergraduate students who performed research in the Barkay lab, and contributed to research presented in this dissertation; Maribeth Armenio, Anthony DiBattista, Hunter Hao, and Tzeh Keong Foo. Also to Chengsheng Zhu for his work with Bayesian analysis of MerA presented in chapter 2. I want to thank and Yitai Liu and Gilbert Flores in the Reysenbach lab for guiding me on how to properly culture the strains of Aquificae included in this dissertation. I would like to extend my gratitude to Ileana Perez-Rodriguez for invaluable advice on media preparation, and a good friend and colleague who always kept the bar high. iv I would have been lost without the guidance of Marsha Morin, Eileen Glick, Arleen Nebel, Kathy Maguire, and Jesse Maguire, whose far-reaching institutional knowledge and assistance made my time at Rutgers far more enjoyable. My most sincere thank you to other faculty members, in Lipman Hall, ENR and elsewhere who have provided much help and assistance; Drs. Bill Belden, Jeff Boyd, Joan Ehrenfeld, Doug Eveleigh, Karl Kjer, Julie Lockwood, Rebecca Jordan, Peter Morin, John Reinfelder, Peter Smouse, Gavin Swiatek, and Nathan Yee. Also a big thank you to my friends which made the roller-coaster of graduate school an enriching and fun time; Aabir, Allie, A. J., Andrea, Ben, Blake, Brandon, Brian, Charlie, Curtis, David, Dom, Elena, Holly, Faye, Isabel, James, Jess, Josie, Norah, Orion, Robbie, Tiff, Sean, and Wes. I want to acknowledge funding sources that made this dissertation possible; the Yellowstone National Park Research Coordination Network, National Science Foundation GK-12 teaching fellowship, Robert A. and Eileen S. Robison Award, Department of Ecology and Evolution Small Grants, and the Graduate School for travel assistance. Last, but most definitely not least, I want to extend my deepest and most sincere gratitude to my family and loved ones that have provided a great amount of unconditional support throughout my time in graduate school. To my parents, Stephen and Eileen who have taught me what it means to not only work hard, but to do it with dedication and enthusiasm. To my brother Noah, who will never call me Dr., and whose time in Los Angeles, Chicago, and road trips to Vegas have provided great retreats over the last 6 years. And to Kate, whose never-ending caring, patience, and understanding has kept me strong and made life far more enjoyable for the past year and a half. v Dedication I want to dedicate this thesis to my parents, Eileen and Stephen, who always believed I could succeed if I put my mind to it, even despite what they may have been told at parent/teacher conferences. To my brother Noah, who always seems to have the right words at the right time, and to my girlfriend Kate, for all her support, love, and patience. vi Table of Contents Pages Abstract ...................................................................................................................... ii Acknowledgments ..................................................................................................... iv Dedication .................................................................................................................. vi List of tables .............................................................................................................. viii List of figures ............................................................................................................. ix . Chapter 1 - Introduction .......................................................................................... 1 - 15 Chapter 2 – Characterization of mer-mediated mercury resistance in Hydrogenobaculum sp. Y04AAS1 and Hydrogenivirga sp. 128-5-R1-1................ 16 - 55 Chapter 3 – Diversity and distribution of merA in geothermal environments and on a global scale: novel insights into the ecological structure of mercury resistant communities............................................................................................... 56 - 93 Chapter 4 – Summary and Conclusions ................................................................ 94 - 108 References .............................................................................................................109 - 112 vii List of Tables Page Table 2.1: Disassociation constants used in MINEQL+ modeling. Adapted from Crespo-Medina et al. (32)………………………………………………………...20 Table 2.2: Primers sets used in qPCR of merA and gyrA in strains AAS1 and R1-1.....26 Table 2.3 Comparison of physiological traits of merA+ Aquificae cultures and merA- control Physiological Data from the Aquificales Data Warehouse (http://alrlab.research.pdx.edu/Aquificales)…………………………………………....35 Table 2.4 Results of MINEQL+ modeling of Hg speciation in Aquificae culture 2- media with thiosulfate (S2O3 ) and hydrogen (H2) as sole energy sources…………….36 Table 2.5: Reduction of Hg(II) to Hg(0) by Aquificae cultures……………………….42 Table 3.1: Geochemical parameters