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Browsing by Author "Santangelo, Thomas, advisor"

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    Archaeal isoprenoid biosynthesis
    (Colorado State University. Libraries, 2018) Liman, Lie Stefanus Geraldy, author; Santangelo, Thomas, advisor; Laybourn, Paul, committee member; Peebles, Christie, committee member
    Many high value natural products - including artemisinin, squalene, and farnesene – are isoprenoids. Efforts to commercially produce isoprenoids are often complicated by low concentrations of isoprenoid precursors and the toxicity of isoprenoids in common production platforms (i.e. bacteria and yeasts). Archaeal-based production platforms provide a potential solution to the precursor toxicity problems as archaea produce isoprenoids in large quantities to generate their unique membrane hydrocarbon chains. One roadblock to commercial archaeal isoprenoid production platforms is the uncharacterized pathway leading to isoprenoid precursor synthesis. This project details, genetically and biochemical, the first three steps in the proposed pathway of archaeal isoprenoid biosynthesis - from acetyl-CoA to mevalonate - in Thermococcus kodakarensis.
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    Archaeal transcription and replication: new insights into transcription-coupled DNA repair and origin-independent DNA replication
    (Colorado State University. Libraries, 2017) Gehring, Alexandra Marie, author; Santangelo, Thomas, advisor; Argueso, J. Lucas, committee member; Nyborg, Jennifer K., committee member; Peersen, Olve B., committee member
    The three Domains of extant life use similar mechanisms for information processing systems. Although many aspects of replication, transcription and translation are universally conserved, the evolutionary history of the enzymes involved is not always clear and domain-specific differences are known. The transcription apparatus, especially the multi-subunit RNA polymerase (RNAP), has a clear evolutionary conservation across all Domains. Elucidating the mechanisms of the transcription apparatus in Archaea will help further understanding of underlying transcription mechanisms and regulation of those mechanisms, not only in Archaea but also in Bacteria and Eukarya. Conversely, the DNA replication machinery, most notably the replicative DNA polymerases, are distinct for each Domain. Any demonstration of the activities of the replication proteins, and especially discovery of unique pathways and mechanisms underlying replication helps to improve the understanding of the larger evolutionary questions surrounding DNA replication. The compact nature of archaeal genomes necessitates timely termination of transcription to prevent continued transcription of neighboring genes while ensuring complete transcription of the gene of interest. Transcription elongation is processive, and the transcription elongation complex is exceptionally stable. The disruption of this transcription elongation process, transcription termination, is an essential step in the transcription cycle. The presence of DNA lesions causes early termination of transcription in Bacteria and Eukarya. The results of this dissertation demonstrate this is also true in Archaea. Archaeal RNAP arrests transcription at DNA lesions and likely initiates transcription-coupled DNA repair (TCR) as will be soon demonstrated using in vivo techniques developed during this dissertation work. DNA replication is a highly regulated cellular process, particularly initiation of DNA replication. The long-standing replicon hypothesis states a trans-acting replication initiation protein must recognize a cis-acting DNA element, the origin of replication. For the 50 years after the replicon hypothesis was first posited, the replication hypothesis was supported in phages, Bacteria, Archaea, and Eukarya. The work presented in this dissertation describes the non-essentiality of Cdc6 and the origin of replication, and further demonstrates that origin-independent DNA replication is the mechanism by which Thermococcus kodakarensis replicates its genome. The results of this study and others in the field brings forward questions about the evolutionary history of DNA replication in all three Domains of extant life.
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    Mapping the metabolic protein interactome that supports energy conservation at the limits of life
    (Colorado State University. Libraries, 2024) Williams, Seré Anne, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Pilon, Marinus, committee member; Anderson, G. Brooke, committee member; Snow, Christopher, committee member
    Distinct metabolic strategies yield energetic gains from a wide variety of substrates, yet only three overarching methods of energy conservation have been defined: substrate level phosphorylation, the generation of a charged membrane, and electron bifurcation. The dominant theme of known energy conservation mechanisms suggests that energy is conserved through the selective movement and management of electrons, thus essentially all life relies on redox (reduction and oxidation) reactions. Small molecule redox cofactors (such as NAD(P)+) and proteinaceous electron carriers (such as ferredoxins) are employed as electron carriers throughout the biosphere. Proteinaceous electron carriers offer the potential for selective protein-protein interactions to bridge reductive flow from catabolic reactions to the membrane, providing a "proteinaceous electron highway" for efficient electron shuttling. Specific redox protein partnerships have been shown to adapt to changing physiological conditions, suggesting that proteinaceous electron flux is tunable and provides a level of selectivity not possible with small molecule electron transport. While electron flux through a tunable and regulated system of protein interactions can offer exceptional energy conservation strategies, large gaps remain in our knowledge of how electron flux is regulated in vivo. Identification of bona fide in vivo protein assemblies – and how such assemblies dictate the totality of electron flow and thus cellular metabolism – is an important milestone to understand the regulation imposed on metabolism, energy-production, and energy conservation. Resolving the dynamic nature of nanoscale interactions in living systems is arguably the current frontier of molecular biology, and combinatorial methods – which layer multiple in vitro and in vivo techniques with large data analysis – have come to the forefront. This dissertation addresses energy conservation strategies of in vivo protein associations in a model, genetically accessible, hyperthermophilic archaeon (Thermococcus kodakarensis) by mapping the metabolic protein interactome using affinity purification mass spectrometry (AP-MS) and generating engineered strains where fusion proteins selectively redirect electron flux in vivo. Twenty-five proteins involved in distinct metabolic functions were tagged to reveal that each tagged-protein interacts with ~ thirty proteins on average. These interactions connected disparate functions suggesting catabolic and anabolic activities may occur in concert -- in temporal and spatial proximity in vivo. The AP-MS method also refined our understanding of previously determined stable complexes suggesting that protein complexes in vivo likely adapt to redox conditions. Engineered strains linking a proteinaceous electron donor to a proposed electron acceptor were viable and impacted electron flux in vivo. Fusion strains linking a ferredoxin to the hydrogen-generating respiratory system increased hydrogen gas output ~8% on average with one strain showing a ~45% increase over wild type. Fusion strains impacting lipid saturation were shown to inhibit saturation, and future studies aim to determine if electrons can be redirected from the vast reductant sink of lipids to the generation of hydrogen gas, a valuable biofuel.
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    Post-initiation activities of the archaeal RNA polymerase in a chromatin landscape
    (Colorado State University. Libraries, 2020) Sanders, Travis James, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Peersen, Olve B., committee member; Ben-Hur, Asa, committee member
    The machineries that control transcription initiation and elongation in Archaea and Eukarya are highly homologous. These similarities support the prevailing evolutionary theory of Archaea being the progenitor of Eukarya. Due to the retention of a core transcription apparatus, while lacking complexities of the eukaryotic counterpart, archaeal systems offer the unique potential to study and characterize the basic protein components necessary for transcription. Transcription termination was less well understood in both Archaea and Eukarya. Shared homology of the initiation and elongation phases argued for a homologous method of termination in Archaea and Eukarya. Additionally, both the archaeal and eukaryotic transcription apparatuses are frequently impeded by histone proteins bound to DNA. Like the transcription complex, archaeal histones are a simplified mirror to eukaryotic histones, permitting evaluation of all steps in the transcription cycle in the context of a chromatin landscape. This thesis summarizes the core molecular machineries involved in the regulation of archaeal transcription during elongation and termination in the greater context of archaeal histone-based chromatin. Thus, the discoveries made have contributed to both the transcription and chromatin fields by providing mechanistic details of the core, conserved transcription apparatus in the framework of evolution.
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    Post-initiation regulatory mechanisms of transcription in the Archaea
    (Colorado State University. Libraries, 2023) Wenck, Breanna Renée, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Osborne Nishimura, Erin, committee member; Wilusz, Carol, committee member
    Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. Archaeal systems contain simplified, basal regulatory transcription components and mechanisms homologous to their eukaryotic counterparts, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. Many archaeal genomes are organized with histone proteins that resemble the core eukaryotic histone fold, which permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that impacts transcription regulation and gene expression. Despite such semblance between the eukaryotic and archaeal core histone folds, archaeal genomes lack the canonical N and C terminal extensions that are abundantly modified to regulate transcription in eukaryotic genomes. Much of what is known regarding factor-mediated transcription regulation in the Archaea is limited; however combined and continued efforts across the field provide tidbits of information, but many pieces are still missing. This thesis aims to i) delineate the role key residues within the histone-DNA complex and archaeal histone-based architecture and key residues within the histone-DNA complex have on the progression of the transcription apparatus, characterize factor-mediated transcription termination, and explore chromatin- and TFS-mediated regulatory effects on transcription via global RNA polymerase (RNAP) positions.
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    The role of Ferredoxin 3 in hydrogen metabolism in the hyperthermophilic archaeon Thermococcus kodakarensis
    (Colorado State University. Libraries, 2022) Stettler, Meghan, author; Santangelo, Thomas, advisor; Hansen, Jeffrey, committee member; Peers, Graham, committee member
    Life faces innumerable challenges to cellular maintenance and reproduction, including access to sufficient energy. As such, all domains of life ubiquitously utilize energetically conservative mechanisms to maximize energy gains from the environment. Use of proteinaceous electron carriers, like ferredoxins, allows cells to harness energy from catabolic reactions that would otherwise be lost to the system as entropy or enthalpy. The hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis is of particular interest as a target for bioengineering to maximize total energy gains, as it natively produces hydrogen gas resulting from terminal electron transport through a Membrane Bound Hydrogenase. T. kodakarensis encodes for three physiologically distinct ferredoxins. Prior to this thesis, only the sequence and molecular weight of the T. kodakarensis ferredoxins were known. Efforts in this thesis laid the groundwork for the biophysical characterization of each ferredoxin isoform via protein-film voltammetry and x-ray crystallography by the development of a reliable recombinant expression and purification scheme. Preliminary biophysical assay trials resulted in a Ferredoxin 1 crystal capable of diffracting to 1.1 Ångstroms, and midpoint reduction potentials for Ferredoxin 1 and Ferredoxin 3 confirming the predicted redox center geometry, demonstrating the efficacy of the developed protein expression and purification scheme for producing high-quality samples. Further investigation into the activity of the ferredoxins resulted in the generation of T. kodakarensis strains encoding for a tether protein between Ferredoxin 3 and its presumed sole electron acceptor Membrane Bound Hydrogenase at two respective locations. The parent strain includes a deletion of Ferredoxin 3, resulting in a deficient phenotype during sulfur-independent growth. The tethered strains of T. kodakarensis demonstrates a full recovery of sulfur-independent growth. Additionally, western-blotting revealed retention of the tethered protein in-vivo, and headspace measurements demonstrated restoration of hydrogen gas production compared to the parent deletion strain, and a reduction in total hydrogen gas output per cell compared to the lab parent strain. These findings implicate the importance of Ferredoxin 3 in hydrogen metabolism in T. kodakarensis and indicate Ferredoxin 3 as a potential target for bioengineering. Furthermore, this thesis is the foundation for further characterization of the T. kodakarensis ferredoxins as proteinaceous electron carriers with potential applications outside of this model organism.