Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
Browse
31 results
Search Results
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 NMR hydrophilic metabolomic analysis of bacterial resistance pathways using multivalent quaternary ammonium antimicrobials in Escherichia coli and Bacillus cereus exposed to DABCO and mannose functionalized dendrimers(Montana State University - Bozeman, College of Letters & Science, 2021) Aries, Michelle Lynne; Chairperson, Graduate Committee: Mary J. Cloninger; This is a manuscript style paper that includes co-authored chapters.Novel antibiotics developed using a new scaffold are needed to combat the rising tide of antibiotic resistant bacteria. Multivalent antibiotics are a relatively new approach that have the potential to greatly increase the efficacy of antibiotics while making it difficult for bacteria to develop resistance. Dendrimers are an attractive framework for the multivalent presentation of antibacterial moieties. Quaternary ammonium compounds (QAC) are a positively charged class of membrane disruptors that are attracted to the large negative charge on phospholipid membranes. Nuclear magnetic resonance (NMR) metabolomics is a quantitative method used for comparison of metabolic profiles of wild type and mutated bacterial samples, enabling the study of bacterial response to antimicrobials. Proton (1 H) NMR hydrophilic metabolomics was used to study gram-negative and gram-positive bacteria upon exposure to 1,4-diazabicyclo-2,2,2-octane (DABCO) with a 16-carbon chain tethered onto a mannose functionalized poly(amidoamine) (PAMAM) dendrimer (denoted as DABCOMD), a membrane disrupting multivalent QAC. Stock Escherichia coli (E. coli) (denoted as wild type) and DABCOMD mutated E. coli (denoted as mutants) were collected in the mid log and stationary phases. The same procedures were used for Bacillus cereus (B. cereus) as for E. coli samples (denoted as unchallenged), except that a DABCOMD challenged sample set was added (denoted as challenged). The challenged sample set procedures were identical to the unchallenged, except DABCOMD was included at 33 % of the MIC value in the growth media for growth curve acquisition and sample collection. The greatest differences observed between the metabolic profiles of the wild type and mutated E. coli samples and between the challenged and unchallenged B. cereus samples were in energy-associated metabolites and membrane-related pathways. The mutants in all sample types were associated with higher levels of spent energy molecules (including AMP and NAD+) and peptidoglycan related compounds (including N-acetylglucosamine). Overall, more changes were observed for B. cereus (gram-positive), especially in challenged mutant B. cereus samples, than for E. coli (gram-negative) samples. Since DABCOMD is a positively charged multivalent membrane disruptor, both B. cereus and E. coli mutated to garner protection by altering their peptidoglycan layer composition, which is energetically costly.Item Response of soil bacterial communities to cropping systems, temporal changes, and environmental conditions in the northern Great Plains(Montana State University - Bozeman, College of Agriculture, 2021) Ouverson, Laura Tindall; Chairperson, Graduate Committee: Fabian D. MenalledSoil bacterial communities are essential components of the soil ecosystem that support crop production. However, agriculture in semiarid drylands and their associated soil bacterial communities face increasingly warmer and drier conditions due to climate change. Two complementary studies were conducted to assess the response of soil bacterial communities to cropping systems, temporal changes, and soil temperature and moisture conditions in semiarid, dryland agricultural systems of the Northern Great Plains. The first study focused on soil bacterial community response to crop phase in contrasting cropping systems (chemical inputs and no-till, USDA-certified organic tilled, and USDA-certified organic sheep grazed) over a growing season. Organic grazed management supported more diverse bacterial communities than chemical no-till, though diversity in all systems decreased over the growing season. Organic grazed bacterial communities were distinct from those in the organic tilled and chemical no-till systems. An interaction between cropping system and crop phase affected community dissimilarity, indicating that overarching management systems and environmental conditions are influential on soil bacterial communities. The second study evaluated soil bacterial communities in a winter wheat - cover crop or fallow rotation. Observations were conducted in the summer fallow and two cover crop mixtures differing by species composition and phenologies, terminated by three different methods (chemical, grazing, or haying), and subjected to either induced warmer/drier or ambient soil conditions. Only the presence and composition of cover crops affected bacterial community dissimilarity, where mid-season soil bacterial communities were distinct from early season and fallow communities. Bacterial communities responded to an interaction between the presence and composition of cover crops and environmental conditions, but not termination. No treatment effects were observed in bacterial communities in 2019, which could be attributed to above average rainfall. The results of these studies suggest cover crop mixtures including species tolerant to warmer and drier conditions can foster diverse soil bacterial communities compared to fallow soils. Overall, these studies contribute to a better understanding of how soil bacterial communities respond to soil health building practices in the Northern Great Plains. Cropping systems can foster unique soil bacterial communities, but these effects may be moderated by environmental and temporal conditions.Item Biochemical and biophysical characterization of plastic degrading aromatic polyesterases(Montana State University - Bozeman, College of Letters & Science, 2019) Topuzlu, Ece; Chairperson, Graduate Committee: Valerie Copie; Brandon C. Knott and Mark D. Allen were authors and Japheth Gado, Harry P. Austin, Erika Erickson, Bryon S. Donohoe, Nicholas A. Rorrer, Fiona L. Kearns, Graham Dominick, Christopher W. Johnson, Valerie Copie, Christina M. Payne, H. Lee Woodcock, Gregg T. Beckham and John E. McGeehan were co-authors of the article, 'Structural and biochemical characterization of MHETASE' submitted to the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.As the world is producing more plastics than it can recycle, accumulation of manmade polymers in the environment is becoming one of the greatest global threats humanity is facing today. One of the major contributors to the plastics pollution problem is polyethylene terephthalate (PET), an aromatic polyester widely used in the packaging, beverage, garment and carpeting industries. As a response to the onslaught of plastics in the environment, fungi and bacteria are evolving metabolic pathways to convert plastics into useable energy sources. One of these organisms, a bacterium, Ideonella sakaiensis 201-F6, has recently been identified to convert PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG), and to use these compounds for energy and growth. I. sakaiensis' ability to convert PET is made possible by two enzymes, named PETase and MHETase. As a first step, PETase breaks down the insoluble substrate PET into a soluble major hydrolysis product - mono-(2- hydroxyethyl) terephthalate (MHET), which is then further hydrolyzed by MHETase into TPA and EG. Crystal structure of PETase, as well as some of its biochemical features, have been reported several times to date, but MHETase has remained largely uncharacterized. This work focuses on further discovery-driven biophysical and biochemical characterization of PETase, visualization of PETase activity on various polyester surfaces, as well as the structural and biochemical characterizations of the MHETase enzyme. We have found that several aspects of PETase-mediated substrate surface modification hydrolysis mechanisms differ depending on the specific mechanical and material characteristics of the substrate. We have also found that PETase is inhibited by BHET. Additionally, we have solved the crystal structure of MHETase. MHETase consists of an alpha/beta hydrolase domain, and a 'lid' domain, commonly seen in lipases. Molecular dynamics simulations revealed the mechanism of MHETase action. Through bioinformatics approaches, we have also identified mutants of interest for improved MHETase activity. Coincubation of MHETase with PETase affects PET turnover in a synergistic fashion. Taken together, this work provides additional insights into the mechanisms of action of the PETase and MHETase enzymes, which may open new avenue for bioremediation and removing plastics from the environment in a sustainable manner.Item Design, synthesis, and evaluation of novel antimicrobials for the eradication of biofilms(Montana State University - Bozeman, College of Letters & Science, 2020) Walsh, Danica Jade; Chairperson, Graduate Committee: Thomas S. Livinghouse; Thomas Livinghouse was a co-author and corresponding author and Darla M. Goeres, Madelyn Mettler, and Philip S. Stewart were co-authors of the article, 'Antimicrobial activity of naturally occurring phenols and derivatives against biofilm and planktonic bacteria' in the journal 'Frontiers in chemistry' which is contained within this dissertation.; Thomas Livinghouse was a co-author and corresponding author and Greg M. Durling, Yenny Chase-Bayless, Adrienne D. Arnold and Philip S. Stewart were co-authors of the article, 'Sulfenate esters of simple phenols exhibit enhanced activity against biofilms' submitted to the journal 'ACS Omega' which is contained within this dissertation.; Thomas Livinghouse was a co-author and corresponding author and Greg Durling, Adrienne Arnold, Whitney Braiser, Luke Berry, Darla M. Goeres and Philip S. Stewart were co-authors of the article, 'Enhanced antimicrobial activity of prodrug phenols against biofilms and planktonic bacteria' which is contained within this dissertation.The majority of microorganisms live in association with surfaces as biofilms. Biofilm communities are encased in a robust, extracellular matrix that reduces their susceptibility to antimicrobial agents. This poses a health concern due to the potential for pathogenic bacteria to cause serious infections. For example, hospital-acquired infections are among the top ten leading causes of death in the U.S. and are responsible for nearly 23,000 deaths per year. The goal of my research is to develop efficient antimicrobial agents capable of eradicating biofilms. In this project, I have focused on three different derivatizations of small, phenolic compounds in effort to increase efficacy towards biofilms. An initial study compared the potency of small, naturally occurring phenols and their corresponding allyl, propyl, and methallyl derivatives against bacteria. This study showed that in parent and derivative pairs potency increased towards free floating cells but decreased towards biofilms. This illustrated the importance of evaluating antimicrobial efficacy toward biofilms when the bacteria they intend to treat has the propensity to form biofilms. This was in contrast to a second studyishowing that trichloromethylsulfenate ester derivatives generally increased potency towards both biofilms and planktonic cells. In a third study, we found that iminodiacetoxy-methylester (AM) appendages increase potency towards planktonic cells and biofilms. AM appendages are ester groups that are employed as part of a prodrug design. Prodrugs are biologically inactive compounds until metabolized. Ester groups are commonly used in prodrug intracellular dyes, where, once inside the cell, ester groups are cleaved enzymatically, resulting in a negatively charged dye that is retained in the cell. Similarly, after the cleavage event, the AM antimicrobial compound will concentrate within the cell. This design serves two functions to increase potency: increasing permeability towards the biofilm matrix and achieving cellular retention. We have shown that the efficacy of antimicrobial agents towards biofilms can be increased through this strategic design. This class of prodrugs presents a wide array of potential applications, from controlling hospital-acquired infections to incorporation into household cleaning products and addresses the need for novel treatments of pathogenic bacteria.Item Insect microbe interactions: honey bee antiviral defense mechanisms and characterization of Spiroplasma colonizing wheat stem sawfly(Montana State University - Bozeman, College of Agriculture, 2017) Brutscher, Laura Marie; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Virus and DSRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense' in the journal 'Scientific reports' which is contained within this thesis.; Curtis Fowler, David K. Weaver and Carl J. Yeoman were co-authors of the article, 'Identification and characterization of a Spiroplasma sp. (Ixodetis clade) associated with the wheat stem sawfly (Cephus cinctus)' submitted to the journal 'Microbial ecology' which is contained within this thesis.Insects play important roles in ecosystems throughout the world. There are many beneficial insects, including those that pollinate plants in diverse landscapes, while other insects are considered agricultural pests. Regardless of ecological role, insects are hosts for microbial symbionts and pathogens. Some microorganisms (e.g., viruses) are harmful to insect health, but many microbial symbionts aid in host biological processes. The projects herein describe the interplay between insects and microbes; specifically (1) honey bee host - virus interactions and (2) identification and characterization of a wheat stem sawfly-associated Spiroplasma. Honey bees (Apis mellifera) are pollinators of numerous agricultural crops and other plant species. Since 2006, there have been high annual losses of honey bee colonies in the U.S. (~33%) and throughout the world. Colony deaths are influenced by multiple factors including RNA virus infections. Honey bee antiviral defense involves several immune pathways, including dsRNA mediated responses, (i.e., RNA interference and non-sequence-specific dsRNA-triggered responses), but their relative importance in antiviral defense is not well understood. To investigate honey bee antiviral defense, bees were infected with model virus in the absence or presence of dsRNA, which reduced virus abundance. Transcriptome-level analysis determined hundreds of genes were differentially expressed in response to co-treatment of dsRNA and virus, including immune-related genes. RNAi-mediated gene knockdown of two putative antiviral genes increased virus abundance and supported their antiviral role. Additional investigation of these and other genes will improve our understanding of dsRNA-mediated antiviral defense in honey bees. In contrast, wheat stem sawflies (Cephus cintus) are major wheat pests in the Northwest United States. Strategies that target endosymbionts of sawflies could reduce wheat crop losses. Hereunto, the microbes that colonize wheat stem sawfly have not been explored. Targeted DNA sequencing determined sawflies were colonized by a Spiroplasma species that has greatest 16S rRNA sequence identity with Ixodetis clade species. Metagenomic sequencing identified several Spiroplasma encoded genes involved in metabolism, which may be important to the sawfly host. Further characterization of honey bee-virus interactions and the role of Spiroplasma in sawfly health may contribute to limiting threats to global crop production and will further scientific understanding of non-model insect-microbe interactions.Item A molecular, structural, and cellular multiple-level study aimed at understanding the unique reaction catalyzed by the last enzyme in the heme-biosynthesis pathway of gram-positive bacteria, coproheme decarboxylase (CHDC)(Montana State University - Bozeman, College of Letters & Science, 2018) Celis Luna, Arianna I.; Chairperson, Graduate Committee: Jennifer DuBois; Bennett R. Streit, Garrett C. Moraski, Timothy D. Lash, Gudrun S. Lukat-Rodgers, Kenton R. Rodgers and Jennifer L. DuBois were co-authors of the article, 'Unusual peroxide-dependent, heme-transforming reaction catalyzed by hemQ' in the journal 'Biochemistry' which is contained within this thesis.; George H. Gauss, Bennett R. Streit, Krista Shisler, Garrett C. Moraski, Kenton R. Rodgers, Gudrun S. Lukat-Rodgers, John W. Peters and Jennifer L. DuBois were co-authors of the article, 'A structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (hemQ)' in the journal 'Journal of the American Chemical Society' which is contained within this thesis.; Dissertation contains two articles of which Arianna I. Celis Luna is not the main author.Heme b is one of nature's most ancient and versatile co-factors and is essential for aerobic life. As such, heme b is synthesized by almost every living organism and plays a major role in bacterial virulence. A pathway for heme b biosynthesis, which is unique to some of the most primitive gram-positive bacteria including many important pathogens, was recently discovered. This pathway, now known as the coproprophyrin-dependent (CPD) branch, ends in a step catalyzed by an unusual enzyme known alternately as coproheme decarboxylase (ChdC) or HemQ. This research aimed to understand ChdC function at the molecular, structural, and cellular levels. Using the ChdC enzyme from Staphylococcus aureus (SaChdC) and a variety of biochemical and analytical tools (conventional and stopped-flow UV-Vis spectroscopy, resonance Raman, HPLC, LC-MS, site-directed mutagenesis, EPR, and X-ray crystallography), the work presented here describes how the coproheme substrate is accommodated in the SaChdC active site and poised for reactivity. The cumulative results show that ChdC catalyzes the oxidative decarboxylation of coproheme III to generate heme b in a sequential and clock-wise fashion, generating harderoheme III in the process. This reaction is H 2 O 2-dependent and the mechanism involves the formation of the high-valent Fe(IV) intermediate (Compound I) and a tyrosine radical (Tyr °). The coproheme-bound ChdC structure revealed a helical-loop that is flexible and moves in towards the active site in the presence of substrate. This loop is hypothesized to act as an 'active site gate' which mediates substrate entry and product egress. Due to the cytotoxicity of heme and its porphyrin precursors, we proposed that the metabolite flux in this pathway is controlled by transient protein-protein interactions. Using the UV-Vis characteristics of porphyrins and phenotype characterization of the deltachdC knock-out strain of S. aureus complemented with ChdC point mutants, we present preliminary evidence for an interaction between ChdC the preceding enzyme of the pathway, CpfC. The same approaches also implicated potential interactions between ChdC and an unidentified heme-chaperone, which delivers heme to its final cellular destination. We propose that this chaperone is HemW. Experiments to test this hypothesis are outlined. This work elucidates yet different way that nature has equipped cells to perform radical chemistry in order to accomplish essential molecule transformations, such as that of decarboxylation and the simultaneous generation of CO 2, and emphasizes the importance of substrate/product post-catalysis cellular trafficking.Item The coenzyme M biosynthetic pathway in proteobacterium Xanthobacter autotrophicus Py2(Montana State University - Bozeman, College of Letters & Science, 2018) Partovi, Sarah Eve; Chairperson, Graduate Committee: John W. Peters; Florence Mus, Andrew E. Gutknecht, Hunter A. Martinez, Brian P. Tripet, Bernd Markus Lange, Jennifer L. DuBois and John W. Peters were co-authors of the article, 'Coenzyme M biosynthesis in bacteria involves phosphate elimination by a unique member of the aspartase/fumarase superfamily' submitted to the journal 'Journal of biological chemistry' which is contained within this thesis.The metabolically versatile bacterium Xanthobacter autotrophicus Py2 has been the focus of many studies within the field of bioenergy sciences, as it contains two unique CO 2 fixing enzymes, and can utilize unconventional substrates such as propylene and acetone as the sole supplemented carbon source while fixing CO 2 in the process. Unexpectedly, coenzyme M (CoM) was found to play a crucial role as a C 3 carrier in the pathway for propylene metabolism in the late 1990s. Previously, CoM was thought to be present solely as a C 1 carrier in methanogenic archaea for nearly 30 years. Though CoM biosynthesis has been characterized in methanogenic archaea, bacterial CoM biosynthesis remained uncharacterized. In X. autotrophicus Py2, four putative CoM biosynthetic enzymes encoded by xcbB1, C1, D1, and E1 have been identified through informatics and proteomic approaches. XcbB1 is homologous to the archaeal ComA which catalyzes the addition of sulfite to phosphoenolpyruvate, and forms the initial intermediate, phosphosulfolactate, in one of the methanogen CoM biosynthetic pathways. The remaining genes do not encode homologues of any of the previously characterized enzymes in methanogen CoM biosynthesis, suggesting bacteria have a unique pathway. The production of phosphosulfolactate by ComA homolog XcbB1 was verified, indicating that bacterial CoM biosynthesis is initiated in an analogous fashion to the PEP-dependent methanogenic archaeal CoM biosynthesis pathway. XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS), and XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to D-cysteine desulfhydrases. Direct demonstration of activities for XcbB1 and C1 strengthens their hypothetical assignment to a CoM biosynthetic pathway, and puts firm contraints on our proposed functions for XcbD1 and E1. Known AFS members catalyze beta-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. We demonstrate herein that XcbC1 catalyzes a beta-elimination reaction on the substrate phosphosulfolactate to yield sulfoacrylic acid and inorganic phosphate. To our knowledge, beta-elimination reactions releasing phosphate is unprecedented among the AFS, indicating XcbC1 is a unique phosphatase. This work will serve as the framework for future studies aimed at uncovering the final stages of the biosynthetic pathway. By elucidating the XcbB1 and XcbC1 reactions, we have made significant strides towards understanding bacterial CoM biosynthesis which evaded characterization in previous years.Item Introducing the ArsR regulated arsenic stimulon(Montana State University - Bozeman, College of Agriculture, 2017) Saley, Tara Carolyne; Chairperson, Graduate Committee: Timothy McDermottThe United States EPA ranks arsenic as the number one environmental toxin. Since microorganisms are significant drivers of arsenic toxicity and mobility in nature, it is important to understand how microbes detect and react to arsenic. The microbial arsenic resistance operon (ars) is critical for sensing arsenic in the environment and controlling the cellular response to this toxin. The ars operon is minimally comprised of arsRBC, which codes for an ArsR transcriptional repressor, arsenite effluxer, and an arsenate reductase, respectively, with the operon negatively regulated by the transcriptional repressor, ArsR. Our model organism Agrobacterium tumefaciens 5A carries two ars operons, with each containing two arsR genes. We conducted an RNASeq study to examine the regulatory roles of the encoded four ArsR regulatory proteins as a function of +/- arsenite. We report that the regulatory influence of the ArsR proteins extends well beyond the ars operon, with both activation and repression effects. In addition to the expected arsenic resistance response, many cellular functions were impacted, including: phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, and iron homeostasis. Each of the ArsR proteins uniquely influenced different sets of genes and an arsR regulatory hierarchy was observed, wherein ArsR1 is auto regulatory and negatively regulates arsR4, ArsR4 activates arsR2, and ArsR2 negatively regulates arsR3. ArsR3 is the least active with respect to number of genes regulated. To summarize, this study provides a more complete understanding of how microbial gene expression and biogeochemical cycling may be influenced by arsenic in the environment.Item Effects of substratum topography on bacterial adhesion(Montana State University - Bozeman, College of Engineering, 1996) Scheuerman, Teresa Rush