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 Linking geochemistry with microbial community structure and function in sulfidic geothermal systems of Yellowstone National Park(Montana State University - Bozeman, College of Agriculture, 2015) Jay, Zackary James; Chairperson, Graduate Committee: William P. Inskeep; Doug B. Rusch, Susannah G. Tringe, Connor Bailey, Ryan M. Jennings and William P. Inskeep were co-authors of the article, 'Predominant acidilobus-like populations from geothermal environments in Yellowstone National Park exhibit similar metabolic potential in different hypoxic microbial communities' in the journal 'Applied and environmental microbiology' which is contained within this thesis.; Jacob P. Beam, Alice Dohnalkova, Regina Lohmayer, Brynna Bodle, Brita Planer-Friedrich, Margaret Romine and William P. Inskeep were co-authors of the article, 'Pyrobaculum yellowstonensis strain WP30 respires on elemental sulfur and/or arsenate in circumneutral sulfidic geothermal sediments of Yellowstone National Park' submitted to the journal 'Applied and environmental microbiology' which is contained within this thesis.; Doug B. Rusch, Jacob P. Beam, Mark A. Kozubal, Ryan M. Jennings and William P. Inskeep were co-authors of the article, 'The distribution, diversity and function of predominant Thermoproteales phylotypes in Yellowstone National Park' submitted to the journal 'ISME J' which is contained within this thesis.Members of the archaeal phylum Crenarchaeota are often associated with microbial communities in high-temperature (> 70 °C) geothermal springs. Environmental genome sequencing (metagenomics) has revealed that populations of Sulfolobales, Desulfurococcales, and Thermoproteales are abundant in hypoxic elemental sulfur sediments of Yellowstone National Park (YNP) and possess enzyme complexes that are implicated in the cycling of carbon, sulfur, and arsenic. Therefore, the primary objectives of this work were to (i) identify the abundant Desulfurococcales and Thermoproteales sequences in these habitats, (ii) characterize the growth and curate the genome of the first Thermoproteales representative isolated from YNP (Pyrobaculum yellowstonensis strain WP30), and (iii) establish a linkage between geochemistry and microbial community structure and function by identifying key proteins that are important to these populations in situ. The primary Desulfurococcales populations were related to Acidilobus spp. and exhibited similar metabolic potential in near-neutral (pH 4 - 6) hypoxic elemental sulfur sediments and acidic (pH ~3) iron oxide mats. These populations are primarily anaerobic heterotrophs that ferment complex organic carbon and are auxotrophic with regards to numerous vitamins and cofactors. These organisms are often found together with members of the Thermoproteales, which are widely distributed in elemental sulfur sediments, acidic iron oxide mats, and streamer communities. P. yellowstonensis strain WP30 was obtained from a hypoxic elemental sulfur sediment habitat with high concentrations of arsenic. This organism was shown to reduce elemental sulfur and/or arsenate in the presence of yeast extract. The complete genome of str. WP30 contained numerous dimethylsulfoxide molybopterin (DMSO-MPT) proteins, which are inovolved in redox reactions of inorganic constituents (i.e. sulfur and arsenic), and genomic comparisons revealed that this organism is closely related to native Pyrobaculum populations. The distribution of Thermoproteales populations was correlated with pH, while the presence of respiratory complexes (terminal oxidases, DMSO-MPT, and dissimilatory sulfate reductases) was correlated with the presence of key electron donors and acceptors. Intron sequences identified in Thermoproteales 16S rRNA genes and were shown in silico to prevent the binding of 'universal' primers that are often used in environmental surveys. These metagenomic, microbiological, and geochemical studies have advanced the understanding of Crenarchaeota diversity and function in YNP.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 Geobiological interactions of archaeal populations in acidic and alkaline geothermal springs of Yellowstone National Park, WY, USA(Montana State University - Bozeman, College of Agriculture, 2015) Beam, Jacob Preston; Chairperson, Graduate Committee: William P. Inskeep; Zackary J. Jay, Mark A. Kozubal and William P. Inskeep were co-authors of the article, 'Niche specialization of novel thaumarchaeota to oxic and hypoxic acidic geothermal springs in Yellowstone National Park' in the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.; Hans C. Bernstein, Zackary J. Jay, Mark A. Kozubal, Ryan deM. Jennings, Susannah G. Tringe and William P. Inskeep were co-authors of the article, 'Assembly and succession of iron oxide microbial mat communities in acidic geothermal springs' submitted to the journal 'Geobiology' which is contained within this thesis.; Zackary J. Jay, Markus C. Schmid, Margaret F. Romine, Douglas B. Rusch, Ryan deM. Jennings, Mark A. Kozubal, Susannah G. Tringe, Michael Wagner and William P. Inskeep were co-authors of the article, 'In situ ecophysiology of an uncultured lineage of aigarchaeota from an oxic hot spring filamentous 'streamer' community' in the journal 'International Society for Microbial Ecology journal' which is contained within this thesis.Microbial communities in high-temperature acidic and alkaline geothermal springs contain abundant, novel Archaea whose role in biogeochemical cycling and community function in microbial mats is not described. This thesis utilized a complementary suite of analyses that included aqueous and solid phase geochemistry, community genomics, phylogenomics, targeted 16S rRNA gene sequencing, community transcriptomics, and microscopy to elucidate the role of novel archaeal populations in acidic sulfur and iron rich hot springs in Norris Geyser Basin, Yellowstone National Park (YNP), and alkaline microbial 'streamer' communities in Lower Geyser Basin, YNP. Novel members of the archaeal phylum, Thaumarchaeota were identified in oxic iron oxide mats and hypoxic elemental sulfur sediments in acidic geothermal springs. These two different groups of Thaumarchaeota likely utilize organic carbon as electron donors and exhibited metabolic capacities based on the presence and absence of oxygen (e.g., heme copper oxidases). The assembly and succession of iron oxide mats in acidic geothermal springs showed later colonization (> 40 d) of Thaumarchaeota. Early colonizers (< 7 d) of Fe(III)-oxide mats include Hydrogenobaculum spp. (Aquificales) and the iron-oxidizing Metallosphaera yellowstonensis (7 - 14 d), which accrete copious amounts of Fe(III)-oxides. Interaction of Hydrogenobaculum and M. yellowstonensis is important to mat formation and subsequent later colonization of heterotrophic archaea (> 40 d). The succession of these communities follows a repeatable pattern, which exhibits interplay among oxygen flux, hydrodynamics, and microbial growth. The biogeochemical and micromorphological signatures may be important for the interpretation of ancient Fe(III)-oxide geothermal deposits. Interactions between Archaea and Aquificales are also important in oxic, alkaline 'streamer' communities, which contain a novel Aigarchaeota population and Thermocrinis spp. This Aigarchaeota population (Candidatus "Calditenuis aerorheumensis") exhibits a filamentous morphology and was intricately associated with Thermocrinis spp. C. aerorheumensis is an aerobic chemoorganotroph. Oxygen is the predominant electron acceptor of C. aerorheumensis, and mRNA transcripts were elevated for heme copper oxidase complexes. Organic carbon electron donors may come from bacteria in close proximity and/or dissolved organic carbon. Archaeal interactions with Aquificales contribute to higher-order level properties (e.g., biomineralization, metabolite sharing) that are important in the formation of hot spring microbial mats and streamer communities.Item Proteomic analyses of Sulfolobus solfataricus : an extremophilic archaeon(Montana State University - Bozeman, College of Letters & Science, 2003) Barry, Richard CorneliusItem Proteomics and in vivo labeling of protein thiols in Sulfolobus solfataricus during exposure to antimony(Montana State University - Bozeman, College of Agriculture, 2014) Mathabe, Patricia Mmatshetlha Kgomotso; Chairperson, Graduate Committee: Brian Bothner; Walid S. Maaty, Benjamin D Reeves, Tegan Ake, Mohammed Refai, Timothy R. McDermot, Paul A Grieco, Mark J. Young, and Brian Bothner were co-authors of the article, 'Proteomics and in vivo labeling of proteinthiols in Sulfolobus solfataricus during exposure to antimony' which is contained within this thesis.Antimony (Sb) has a long history in both the chemical and social literature. As a metalloid it is often found in the environment with Arsenic (As). Extended exposure in humans causes heart disease, lung disease, diarrhea, severe vomiting and ulcers. In plants, it inhibits early crop growth. A large body of data on bacterial response and mechanisms for detoxification also exists. In contrast, knowledge about how archaeal species respond to Sb is much less extensive. The model crenarchaeal organism Sulfolobus solfataricus, can survive in environments with high antimony concentrations, and the genetic and biochemical mechanisms responsible for antimony tolerance have yet to be reported. As a first step in bringing to light the biological response of S. solfataricus to antimony, a set of proteomic and chemical tagging experiments were undertaken. Two-dimensional differential gel electrophoresis (2D-DIGE) showed a limited response from intracellular and membrane proteins with respect to their abundance. In contrast, chemical targeting of cysteine residues revealed that extensive oxidation had occurred to both cytosolic and membrane proteins upon exposure to antimony. To remove any possible experimental artifacts that could alter the oxidation state of protein thiols, a method for labeling cytoplasmic proteins in live S. solfataricus cells was developed. This method used the recently described Z-dye probes for quantitative comparisons. Together, our results suggest that Sb response is primarily focused on a general stress factors likely stemming from oxidative damage to proteins. No evidence for a specific transport or bioconversion was present. Cysteine residues in membrane proteins displayed the most significant oxidative changes. The demonstration that chemical biology approaches can be applied to prokaryotic cells, even those growing at extremes of temperature and Ph, should have broad appeal for microbiologists well beyond those investigating archaea.Item Sulfolobus as a model organism for the study of diverse biological interests : forays into thermal virology and oxidative stress(Montana State University - Bozeman, College of Letters & Science, 2006) Wiedenheft, Blake Alan; Chairperson, Graduate Committee: Mark Young; Trevor Douglas (co-chair)My research interests have focused on two distinct aspects of Sulfolobus biology: virology and oxidative stress. My major contribution to the emerging field of thermal virology has been the isolation, characterization and comparative genomic analysis of a spindle-shaped virus partical (SSV RH) infecting the thermoacidophilic archaeal host Sulfolobus solfataricus (18). Insights from this comparative genomic analysis have served as a platform for targeted structural studies, as well as providing molecular tools used to follow the viral life cycle in culture and for assessing the ecological significance of these viruses in the environment (9, 19-24). My research endeavors in oxidative stress arose from an early interest in iron metabolism and protective mechanisms that allow life to cope with the paradoxical role that iron plays in biological systems. Pursuit of this interest has lead to the discovery of a new class of proteins termed, "DPS-Like" (7, 17, 24, 25). These previously unrecognized proteins function as antioxidants and are widely distributed across both prokaryotic domains of life.Item 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.