Theses and Dissertations at Montana State University (MSU)
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Item Investigating the metalloproteome of bacteria and archaea(Montana State University - Bozeman, College of Letters & Science, 2024) Larson, James Daniel; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Metalloproteins are proteins that rely on a bound metal for activity and comprise 30-50% of all proteins which are responsible for catalyzing imperative biological functions. Understanding the interplay between essential and toxic metals in the environment and the metalloproteins from an organism (metalloproteome) is important for a fundamental understanding of biology. A challenge in studying the metalloproteome is that standard proteomic methods disrupt protein-metal interactions, therefore losing information about protein- metal bonds required for metalloprotein function. One of the focuses of my work has been to develop a non-denaturing chromatographic technique that maintains these non-covalent interactions. My approach for investigating the native metalloproteome together with leading- edge mass spectrometry methods was used to characterize microbial responses to evolutionarily relevant environmental perturbations. Arsenic is a pervasive environmental carcinogen in which microorganisms have naturally evolved detoxification mechanisms. Using Escherichia coli strains containing or lacking the arsRBC arsenic detoxification locus, my research demonstrated that exposure to arsenic causes dramatic changes to the distribution of iron, copper, and magnesium. In addition, the native arsRBC operon regulates metal distribution beyond arsenic. Two specific stress responses are described. The first relies on ArsR and leads to differential regulation of TCA-cycle metalloenzymes. The second response is triggered independently of ArsR and increases expression of molybdenum cofactor and ISC [Fe-S] cluster biosynthetic enzymes. This work provides new insights into the metalloprotein response to arsenic and the regulatory role of ArsR and challenges the current understanding of [Fe-S] cluster biosynthesis during stress. Iron is an essential and plentiful metal, yet the most abundant iron mineral on Earth, pyrite (FeS2), was thought to be unavailable to anaerobic microorganisms. It has recently been shown that methanogenic archaea can meet their iron (and sulfur) demands solely from FeS2. This dissertation shows that Methanosarcina barkeri employs different metabolic strategies when grown under FeS2 or Fe(II) and HS- as the sole source of iron and sulfur which changes the native metalloproteome, metalloprotein complex stoichiometry, and [Fe-S] cluster and cysteine biosynthesis strategies. This work advances our understanding of primordial biology and the different mechanisms of iron and sulfur acquisition dictated by environmental sources of iron and sulfur.Item Allosteric activation of the CRISPR-associated transcription factor Csa 3 by cyclic tetra-adenylate (cA 4)(Montana State University - Bozeman, College of Letters & Science, 2020) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin LawrenceThe CRISPR-Cas immune system provides adaptive and heritable immunity to archaea and bacteria to combat viral infection, and is a source of biochemical tools to researchers. This work combines structural biology and biochemical approaches to provide insight into mechanisms prokaryotes use to control the CRISPR-Cas immune system, linking subsystems into a coordinated response. The first structure of S. solfataricus Csa3 determined by Lintner et al. revealed a dimer with a C-terminal wHTH DNA-binding domain and an N-terminal CARF domain with a putative ligand binding site predicted to bind a two-fold symmetric molecule with both negatively charged and hydrophobic/aromatic moieties, such as dinucleoside polyphosphates or nucleic acid molecules. 1 Later, analysis by Topuzlu et al. of the A. fulgidis Csx3 structure containing a 4 base RNA molecule in a binding pocket revealed similarities between Csx3 and the Csa3 CARF domain, and suggested CARF proteins could bind cyclic or pseudosymmetric linear RNA tetranucleotides represented by the ring-shaped RNA density in the Csx3 binding pocket. 2 Functional studies with S. islandicus Csa3 identified that SiCsa3 regulates transcription of acquisition genes (cas1, cas2, and cas4) and several CRISPR loci. 3,4 Additionally, two groups simultaneously showed that the Type III surveillance complexes produce cyclic oligoadenylate messengers, including cyclic tetra-adenylate (cA 4), which allosterically regulate the RNase activity of Csm6 and Csx1, other CARF proteins. 5,6 These advances support the original predictions by Lintner et al. and suggest that Csa3 binds a cyclic RNA as proposed by Topuzlu et al. Binding of cA 4 likely allosterically causes conformation changes in the wHTH, and regulates the protein's transcriptional regulation. We present the crystal structure of S. solfataricus Csa3 complexed with cyclic tetraadenylate (cA 4). cA 4, as predicted, 1 is bound in the CARF domain 2-fold symmetric pocket, which stimulates conformational changes in the C-terminal domain. Additionally, we identify the presence of a palindromic predicted binding motif upstream of the Type I-A(2) acquisition cassette and CRISPR loci C and D, and reveal through EMSA analysis that Csa3 binds dsDNA nonspecifically with high affinity. Finally, ring nuclease activity is not detected in Csa3, suggesting longer term potentiation of the cA 4 in Csa3 than observed for Csx1/Csm6. 5,6Item Morphological adaptations facilitating attachment for archaeal viruses(Montana State University - Bozeman, College of Letters & Science, 2019) Hartman, Ross Alan; Chairperson, Graduate Committee: Mark J. YoungLittle is known regarding the attachment and entry process for any archaeal virus. The virus capsid serves multiple biological functions including: to protect the viral genome during transit between host cells, and to facilitate attachment and entry of the viral genome to a new host cell. Virus attachment is conducted without expenditure of stored chemical energy i.e. ATP hydrolysis. Instead, virus particles depend on diffusion for transportation and attachment from one host cell to another. This thesis examines the attachment process for two archaeal viruses. Sulfolobus turreted icosahedral virus (STIV) is well characterized for an archaeal virus. Still, no information is available concerning STIV attachment or entry. The research presented here shows that STIV attaches to a host cell pilus. Furthermore, combining the previously determined atomic model for the virus, with cryo-electron tomography, a pseudo-atomic model of the interaction between the host pilus and virus was determined. Based on this data, a model is proposed for the maturation of the virus capsid from a noninfectious to an infectious form, by dissociation of accessory proteins. Finally, a locus of genes is identified in the host cell, encoding proteins essential for viral infection, that are likely components of the pili structure recognized by STIV. The isolation of a new archaeal virus, Thermoproteus Piliferous Virus 1 (TSPV1), is also presented here. The TSPV1 virion has numerous fibrous extensions from the capsid, of varying length, that are the first observed for any virus. The capsid 2-3nm fibers likely serve to extend the effective surface area of the virus, facilitating attachment to host cells. Characterization of this new virus was conducted, including genome sequencing and determination of the protein identity for the capsid fibers. The research presented here provides a substantial advancement in our knowledge of the attachment process for archaeal viruses. Attachment to host pili is now emerging as a common theme for archaeal viruses. Furthermore, the isolation of the new archaeal virus TSPV1 demonstrates a novel strategy to increase the probability of interaction between a virus and host cell.Item Analysis of archaeal viruses and characterization of their communities in Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2014) Bolduc, Benjamin Ian; Chairperson, Graduate Committee: Mark J. Young; Daniel P. Shaughnessy, Yuri I. Wolf, Eugene Koonin, Francisco F. Roberto, and Mark J. Young were co-authors of the article, 'Identification of novel positive-strand RNA viruses by metagenome analysis of archaea-dominated Yellowstone hot springs' in the journal 'Journal of virology' which is contained within this thesis.; Jennifer Wirth, Aurélien Mazurie, and Mark J. Young were co-authors of the article, 'Viral community composition in Yellowstone acidic hot springs assessed by network analysis' submitted to the journal 'ISME Journal' which is contained within this thesis.; Mark J. Young was a co-author of the article, 'Characterization of viral communities in Yellowstone hot springs by deep-sequencing' which is contained within this thesis.Viruses infecting the Archaea - the third domain of life - are the least understood of all viruses. Despite only 100 archaeal viruses being described, work on these viruses revealed a remarkable level of morphological and genetic diversity unmatched by their bacterial and eukaryotic counterparts, whose numbers range over 6000. Study of these archaeal viruses could gain insight into fundamental aspects of biology and reveal underlying evolutionary connections spanning the three domains of life, including the origin of life. In addition, we understand very little about their community structures in natural environments. To address these daunting tasks, a viral metagenomics approach was undertaken using next generation sequencing technologies. Despite this, only a fragmented view of the viral communities is possible in natural ecosystems. Therefore, this dissertation sought to apply a network-based approach in combination with viral metagenomics to not only describe natural viral communities, but to find and characterize the first RNA viruses out of acidic, high-temperature hot springs in Yellowstone National Park, USA. These hot springs harbor low complexity cellular communities dominated by several species of hyperthermophilic Archaea. The results of this dissertation show that this approach can identify distinct viral populations and provide insights into the viral community. Furthermore, the viral communities of these hot springs are relatively stable over the course of the sampling time period. In addition, a number of viral clusters - each representing a viral family at the taxonomic level - are likely previously uncharacterized DNA viruses infecting archaeal hosts. This approach demonstrates the utility of combining viral community sequencing with a network analysis to understand viral community structures in natural ecosystems. Additional analysis of these viral metagenomes led to the identification of novel RNA viral genome segments. Since no RNA virus infecting Archaea is known to exist, this dissertation also sought to more fully characterize these sequences. Genes for RNA-dependent RNA polymerases, a hallmark of positive-strand RNA viruses were identified, suggesting the existence of novel positive-strand RNA viruses likely replicating in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses infecting eukaryotes and are even more distant from known bacterial RNA viruses.Item Proteomic analyses of Sulfolobus solfataricus : an extremophilic archaeon(Montana State University - Bozeman, College of Letters & Science, 2003) Barry, Richard CorneliusItem X-ray crystallographic studies of sulfolobus turetted icosahedral virus (STIV) : a hyperthermophilic virus from Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2006) Larson, Eric Thomas; Chairperson, Graduate Committee: C. Martin LawrenceSulfolobus turreted icosahedral virus (STIV) was isolated from acidic hot springs of Yellowstone National Park and was the first hyperthermophilic virus described with icosahedral capsid architecture. Structural analysis of the STIV particle and its major capsid protein suggests that it belongs to a lineage of viruses that predates the division of the three domains of life. Functional predictions of the viral proteins are hindered because they lack similarity to sequences of known function. Protein structure, however, may suggest functional relationships that are not apparent from the sequence. Thus, we have initiated crystallographic studies of STIV and expect to gain functional insight into its proteins while illuminating the viral life cycle. These studies may also provide genetic, biochemical, and evolutionary insight into its thermoacidophilic host and the requirements for life in these harsh environments. The first three proteins studied in structural detail are A197, B116, and F93. As anticipated, these structures suggest possible functions. The structure of A197 reveals a glycosyltransferase GT-A fold.Item Chemical approaches to probe environmental stress in Archaea(Montana State University - Bozeman, College of Letters & Science, 2009) Tarlykov, Pavel Victorovich; Chairperson, Graduate Committee: Brian BothnerLittle is known about strategies and mechanisms employed by thermophilic organisms to adapt to environmental stress. Sulfolobus solfataricus is a thermophile that belongs to Archaea, the third domain of life, and can be found in unusual habitats, such as the hot springs of Yellowstone National Park. This archaeon can tolerate high temperature, extreme acidity and high concentrations of heavy metals and other toxic substances. Thus, S. solfataricus has been chosen by many researchers as a model system for biochemical, structural, and genetic studies. In this work S. solfataricus has been exposed to hydrogen peroxide as a natural mild oxidant and arsenic as a common toxic metalloid. One of the aims was to quantitatively define the regulation of proteins upon treatment with hydrogen peroxide or arsenic species in different time periods and concentrations. In this sense, two-dimensional gel electrophoresis approach in conjunction with novel chemical tagging probes has been applied to detect changes on the level of regulation and chemical modification of individual proteins within the whole proteome in response to the stressors. Proteins expression levels have been monitored, redox-sensitive and phosphoproteomic profiles of the S. solfataricus proteome have been identified. Synthesis of the results has allowed a general scheme for how S. solfataricus fights H₂O₂- and As-induced stress. Lists of mapped proteins have been created and potential biomarkers for oxidative stress have been identified, which can guide further research to better understand mechanisms of proteomic response to the environmental stress in Archaea on the example of thermophilic archaeon S. solfataricus.Item Crenarchaeal virus-host systems : structure-function studies of crenarchaeal viruses and prokaryotic adaptive immunity(Montana State University - Bozeman, College of Letters & Science, 2011) Lintner, Nathanael Greenleaf; Chairperson, Graduate Committee: C. Martin Lawrence; Valerie Copie (co-chair)Virus-host interactions are one of the most important drivers of microbial ecology and evolution. Among the three domains of life, the least is known about viruses infecting members of the domain Archaea. This work combines a traditional hypothesis-driven molecular and biochemical approach with a structural genomics-like approach of pursuing a large number of structural targets to gain a detailed molecular understanding of a model Crenarchaeal virus-host system, Sulfolobus turreted icosahedral virus (STIV) and its Sulfolobus host. This work specifically focuses on the viral protein, A81, a putative transcriptional regulator associated with the prokaryotic adaptive immune system, CRISPR-Cas, and a large protein-RNA complex involved in CRISPR-mediated DNA interference. A81 is a protein-of-unknown-function encoded by the STIV genome. The structure of STIV-A81 reveals a unique ring-shaped octameric assembly. While structural homology-based searches fail to reveal a function for A81, the central pore has a size and charge consistent with a potential interaction with single-stranded nucleic acid. The structure of the CRIPSR-associated protein, Csa3 reveals a putative 2-domain transcription factor. The N-terminal domain is a variation on the di-nucleotide binding-domain that orchestrates dimer formation. There is a conserved 2-fold symmetric pocket on the dimer axis that likely represents a regulatory ligand-binding site implying a small-molecule regulator of CRISPR/Cas. The C-terminal domain is a winged helix-turn-helix common among transcription factors. The domain architecture of Csa3 suggests a small molecule regulator of CRISPR/Cas in the Crenarchaea. The CRISPR-associated complex for antiviral defense (CASCADE) is predicted to be central to CRISPR-mediated DNA-interference in many bacteria and Archaea. We isolated components of an archaeal CASCADE from Sulfolobus solfataricus. Csa2 expressed in S. solfataricus co-purifies with Cas5a, Cas6, Csa5 and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein, forms a stable complex with Cas5a. A recombinant Csa2-Cas5a-complex is sufficient to bind crRNA and complementary ssDNA. Transmission electron microscopy reveals an extended helical complex of variable length, perhaps due to substochiometric amounts of capping factors. Csa2 displays a crescent-shaped fold including a modified RNA-recognition motif (RRM) plus two additional domains present as insertions into the RRM. A preliminary model for this and other CASCADEs is proposed.Item X-ray crystallographic studies of the proteins from sulfolobus spindle-shaped viruses (SSVs)(Montana State University - Bozeman, College of Letters & Science, 2009) Menon, Smita Kesavankutty; Chairperson, Graduate Committee: C. Martin LawrenceViruses populate virtually every ecosystem on the planet. Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world. However, compared to eukaryotic and bacterial viruses, our knowledge of viruses infecting the archaea is limited. Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, structural studies can reveal distant evolutionary relationships and provide functional insights that are not apparent from the primary amino acid sequence alone. Several such structural studies have already contributed to our understanding of the Sulfolobus Spindle-shaped viruses (Fuselloviridae). Here we report the structure of two proteins, SSV1 F112 and SSVRH D212. Biochemical, proteomic and structural studies of F112 reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Continuing these efforts, a second structure was also determined where the overall fold and conservation of active site residues place D212 within the PD-(D/E)XK nuclease superfamily. Notably, the structure of F112 contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses and the evolutionary implications of such distribution are discussed. Here we review and describe our progress towards understanding these viruses at a molecular level.