ABSTRACT COUNTS, JAMES AARON. Determinants for Hot Acid Microbial Lifestyles: Chemolithoautorophic Physiology of the Extremely Thermoacidophilic Archaeal Lineage Sulfolobales. (Under the direction of Dr. Robert M. Kelly). The Crenarchaeal order Sulfolobales consists exclusively of extremely thermoacidophilic organisms from terrestrial geothermal environments. This niche requires numerous adaptive features to tolerate harsh degrees of acidity (<4.5) and temperatures (>65˚C). Further, mixing with subsurface environments causes dramatic fluctuations in the above-mentioned stressors and provides capacious energy in the form of reduced inorganic sulfur compounds (RISCs) – primarily hydrogen sulfide, mineral sulfides, and zero-valent sulfur – and dissolved and mineral-bound transition metals (primarily iron). To better understand thermoacidophily, nine type-strains from culture repositories were sequenced, leveraging recent advances in third-generation sequencing technologies. This includes Acidianus ambivalens, Acidianus brierleyi, Acidianus infernus, Acidianus sulfidivorans, Metallosphaera hakonensis, Metallosphaera prunae, Stygiolobus azoricus, Sulfolobus metallicus, and Sulfurisphaera ohwakuensis. These organisms are largely chemolithotrophic, include several facultative anaerobes and the only obligate anaerobe from the Sulfolobales; they span 5 named genera, encapsulating a broad picture of the order. Analysis of these and 14 additional type-strain Sulfolobales genomes identifies a core genome of approximately 1,100 genes and an open pan genome. The addition of these genomes also provides resolution for poor branching in existing taxonomy compiled with earlier methods. Through the use of a Thermoprotei orthologous core of 97 proteins and average amino acid identity (AAI), a proposal is made for several taxnomical shifts within the order Sulfolobales. Proposed is the reclassification of Sulfolobus metallicus as Cocturalobus metallicus, including unnamed species Sulfolobus sp. JCM16833 and JCM16834, representing an extremely acidophilic (pH~2), thermophilic (65-70˚C) genus of obligate chemolitotrophic (coctura, ore smelting) aerobes. Sulfolobus islandicus spp. and the newly characterized Sulfolobus sp. A20 should be reclassified in the recently named genus Saccharolobus Finally, evidence suggests that the newly isolated, sequenced Sulfodiicoccus acidiphilus be reclassified in a new family: Sulfodiicoccaceae, which may represent a distantly branched group of sulfur-inhibited, chemoheterotrophs. Deeper Sulfolobales genome analysis suggests there are numerous well-conserved pathways for processing inorganic compounds. This includes the 3-hydroxypropionate/4- hydroxybutyrate (near complete excluding S. acidiphilus), the heterodisulfide reductase complex, thiosulfate:quinone oxidoreductase (except S. acidocaldarius) and sulfide:quinone oxidoreductase. Unsurprisingly, there are sulfur-related genes of relative rarity, including the sulfur oxygenase reductase (SOR), sulfur-reductase (Sre), and tetrathionate hydrolase (TetH), largely limited to facultative and obligate anaerobe(s), but also appearing in the most acidophilic Sulfolobales. Interesting are several genes closely linked with sulfur and acidophily, including an uncharacterized TetH paralogue, NiFe hydrogenase, and a novel (within the order) cytochrome bd-like oxidase. This last enzyme complex may represent a crucial element of pH homeostasis maintenance, given that these cytochromes usually have exceptionally high oxygen affinity and intransigence to strong reductants (e.g. hydrogen sulfide). Further, the putative cytochrome bd, as well as doxBCE (a universal Sulfolobales oxidase) are present even in the strict anaerobe Stygiolobus azoricus. Within this environment, iron-biooxidation is another prevalent phenotype. Metallosphaera sedula was the most proficient iron-biooxidizer with constitutively-expressed ferrous iron oxidation “fox” genes. Herein is demonstrated the activity of this complex (isolated from M. sedula) as well as identification of its constituents. Confirmation of its conservation among select members of the order cements its role in extremely thermoacidophilic iron biooxidation. Further, iron oxidation rates appear to relate to fox gene expression, particularly FoxABCDW, which likely constitute the active complex. Evolutionary analysis of the primary oxidase subunit FoxA suggests the speciation of cytochrome c-like oxidases follows the speciation of Crenarchaeal aerobes of the lineage Sulfolobales and the recently identified candidate phylum Marsarchaeota. Further, examining multiple genomes, a urease-like complex and a peroxiredoxin from the OsmC lineage suggest the importance of pH homeostasis and free-radical formation in these acidic, metal-laden environments. © Copyright 2020 by James Counts All Rights Reserved Determinants for Hot Acid Microbial Lifestyles: Chemolithoautorophic Physiology of the Extremely Thermoacidophilic Archaeal Lineage Sulfolobales by James Aaron Counts A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemical Engineering Raleigh, North Carolina 2020 APPROVED BY: Dr. Robert M. Kelly Dr. Balaji Rao Committee Chair Dr. Carlos C. Goller Dr. Amy Grunden BIOGRAPHY James Aaron Counts was born in Orlando, Florida in 1989 and spent his entire childhood in the western Orlando community. He attended West Orange High School, where he was a National Honor Society member, Percussionist, and long-time Oboist. Upon graduation, he attended the University of Florida studying Chemical Engineering, where he was a member of the engineering honor society Tau Beta Pi and took part both professionally and voluntarily in numerous K-12 STEM and academic outreach programs. While interning at a local pharmaceutical and contract research company in his final year, he decided to pursue post- secondary education, enrolling at North Carolina State University in chemical and biomolecular engineering. Since arriving at North Carolina State, James settled into the Hyperthermophiles lab of Robert M. Kelly, studying questions of extreme metabolic and physiological evolution, as well as origins of microbial life, working with novel thermoacidophilic enzymes and expanding our understanding of microbial life at the limits of the habitable universe. While studying at NC State, he completed a traineeship in the NIH Molecular Biotechnology Training Program and worked as an intern in process optimization at Novozymes NA, Inc. In addition to a love of science, he has a love of running, embarking on a secret career as an amateur long-distance runner, culminating in the completion this fall of his first road marathon in 3:00:15 and coaching middle school cross- country in Durham County Public Schools, where he resides. He hopes to continue research in the areas of environmental microbiology, evolution and ecology focusing on extreme/underexplored habitats, eventually moving into an academic lab at a research institution in the same field. ii ACKNOWLEDGMENTS It would be disingenuous to fail in acknowledging all of the friends that pushed me through this thesis and helped me stay motivated throughout my nearly six and a half years in this program. Many people have come and gone from my life but most of them had an important role in one way or another, intentional or unintentional, in keeping me on track. Many hikes and adventures, many long runs through the streets of Durham and the forests of the Carolinas, and many shared thoughts over local pints provided the fuel to keep persisting and challenging myself. Although, the single most important person for completing this process was my advisor, who not only kept the money flowing for me to continue my research but has never stopped mentoring (even despite his own circumstances). He also continuously tolerated the maturation process of a twenty-something trying to find their path in research and in life. For all of these people, I cannot help but feel thankful. iii TABLE OF CONTENTS LIST OF TABLES ..................................................................................................................... vii LIST OF FIGURES .................................................................................................................. viii CHAPTER 1: Physiological, MEtabolic, and BiotEchnological FEaturEs of ExtrEmEly Thermophilic Microorganisms ................................................................................................. 1 Abstract ............................................................................................................................... 2 Introduction ......................................................................................................................... 2 SulfoloblEs .......................................................................................................................... 3 Carbon Dioxide Fixation .................................................................................................. 4 Sulfur Utilization .............................................................................................................. 5 Metal Oxidation ............................................................................................................... 6 Pyrococcus furiosus ........................................................................................................... 8 Central Glycolytic Metabolism ........................................................................................