Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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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 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 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.