Determinants for Hot Acid Microbial Lifestyles: Chemolithoautorophic Physiology of the Extremely Thermoacidophilic Archaeal Lineage Sulfolobales.

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/4hydroxybutyrate (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