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 The relationship between physiological stress response and variation in omics data(Montana State University - Bozeman, College of Letters & Science, 2021) Steward, Katherine Fay; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Omics analysis is the cornerstone of systems biology. It offers comprehensive assessments of stress, interaction networks and connections to phenotype. Defining a stressed phenotype can be challenging, however, as stress response mechanisms can arise from a range of environmental conditions and experimental perturbations. Previous work from our lab noted the possibility of a relationship between stress in omics data and the variation of that data. This connection has yet to be clearly defined, and the cellular mechanisms responsible for the canalization of omics data remain a mystery. In this work I have taken advantage of the sensitivity of metabolomics and proteomics to detect cellular stress and characterize its relationship to variation. By utilizing coefficient of variation (CV) as a statistic of merit, the depth of the relationship between stress and variation can be uncovered. Once the model was clearly defined, a proteomics dataset with a large proportion of protein coverage was utilized to investigate what pathways might be responsible for the metabolite and protein canalization.Item Proteomics analysis of the metabolic transition between aerobic and anaerobic conditions in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) Refai, Mohammed Yahya; Chairperson, Graduate Committee: Brian Bothner; Nina Paris, Hunter Fausset, Monika Tokmina Lukaszewska were co-authors of the article, 'Proteomics analysis of the transition between aerobic and anaerobic growth conditions in Escherichia coli' submitted to the journal 'Biochimica et biophysica acta' which is contained within this dissertation.As a facultative anaerobe, Escherichia coli has the ability to grow in anaerobic and aerobic environments. Despite detailed characterizations of this model organism in the presence and absence of oxygen, an in-depth understanding of changes to the proteome during transitions from aerobic to anaerobic growth is lacking. This thesis work focuses on elucidating how protein thiol oxidation and reduction change during a facultative anaerobe's transition from aerobic to anaerobic growth conditions, and pathways of thiol-mediated cell signaling. Redox driven changes in cysteine oxidation involved in signaling are referred to as 'thiol switches'. These modulate diverse biological activities ranging from gene expression and protein synthesis to environmental stress response. Surprisingly, little is known about the role of thiol switches during microbial transitions from aerobic and anaerobic growth conditions. To explore this uncharted territory, a mass-spectrometry (MS)-based proteomics workflow was developed and refined. Following extensive protocol optimization for high-throughput MS data processing, normalization, and pattern matching, the analytical pipeline was fine-tuned for the specific proteome-wide analysis of cysteine chemical modifications in E. coli. The approach was based on open-source software and publicly accessible databases, creating a transparent, reproducible, and easily sharable proteomics approach. Herein, the redox state and chemical forms of protein-based thiol switches in E. coli were characterized over time as the bacterium reversibly transitioned between aerobic and anaerobic growth conditions. Unexpectedly, differential alkylation analysis of cysteine-containing E. coli proteins revealed a higher degree of protein thiol oxidation under anaerobic growth conditions, a result not reported for E. coli or any other facultative anaerobe. Our proteome-wide analysis also revealed that cysteine redox potentials vary widely, and several specific E. coli proteins contain highly reactive thiols. These findings provide strong evidence for thiol-based signaling in E. coli in response to environmental changes such as aerobic to anaerobic growth transitions. Characterization of specific redox switches underlying metabolic changes associated with oxygen availability has uncovered a previously unknown E. coli cell signaling mechanism. Since transitioning between aerobic and anaerobic environments is associated with bacterial virulence, this work opens new avenues to target pathogenic facultative anaerobes and to develop novel thiol-based antibacterial therapies.Item Metabolic interactions and activity partitioning in a methanogenic, interdomain biofilm(Montana State University - Bozeman, College of Letters & Science, 2019) Camilleri, Laura Beth; Chairperson, Graduate Committee: Matthew Fields; Kristopher A. Hunt, Aurelien Mazurie, Jennifer Kuehl, Alex Michaud, James Connolly, Egan Lohman, Zack Miller, Adam M. Deutschbauer and Matthew W. Fields were co-authors of the article, 'Differential gene expression of a bacterial-archaeal interdomain biofilm producing methane' submitted to the journal 'Biofilms' which is contained within this dissertation.; B.P. Bowen, C.J. Petzold, T.R. Northen and M.W. Fields were co-authors of the article, 'Activity partitioning in an archaeal-bacterial biofilm' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methanococcus maripaludis factor causes slowed growth in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Growth effects of sulfopyruvate and sulfoacetate on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon Methanococcus maripaludis S2' submitted to the journal 'Scientific reports' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methane production in Pelosinus fermentans JBW45' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.Biofilms are an ancient survival strategy in which communities of organisms can grow as a cohesive unit, generally attached to a surface and/or at interfaces. Despite the paradigm that 99% of microorganisms grow as a biofilm in the environment, current research methods are largely limited to monoculture planktonic studies. Although more investigations are trying to improve culture complexity by evaluating interactions between two or more populations, experiments are still more readily performed with microorganisms in the planktonic growth mode. The research presented here aims to elucidate the complexity of interactions between two microorganisms from different domains of life that results in enhanced metabolism due to localization of cells in close proximity within an anaerobic biofilm. Desulfovibrio vulgaris Hildenborough (DvH) and Methanococcus maripaludis S2 (Mmp) form a syntrophic mutualism when grown in sulfate-limited media that requires electron flux from DvH to Mmp through what is commonly assumed to be interspecies hydrogen transfer, thereby establishing cross-feeding. The biofilm has been shown to promote a stable and more even carrying capacity for both populations that is likely linked to improved hydrogen transfer (and/or other potential carbon and electron co-metabolites) as compared to planktonic populations. Transcriptomic and proteomic analyses, utilizing RNA-seq and deuterated water respectively, were used to elucidate genes and proteins that contribute to the biofilm growth mode that results in a more efficient metabolism for the syntrophic co-culture (defined by biomass per substrate flux). The results demonstrate the expression of many genes with unknown functions, and others that contribute to cell-cell interactions as well as active proteins in electron processing (e.g., lactate oxidation) in DvH and CO2 reduction (e.g., methanogenesis) in Mmp. A metabolic model of the coculture provided reinforcement for transcriptomic assumptions and aided in the identification of a sulfonate and other amino acids as important syntrophic metabolites. Assessment of biofilm co-culture activity utilizing a new method, Biorthogonal Noncanonical Amino Acid Tagging (BONCAT), showed Mmp was less active in the uptake of a methionine analog as compared to DvH. Alternate assessments confirmed that Mmp was in fact active (based upon methane generation) although translational activity was below the detection limit. Further investigation of the system under sulfate stress showed that the metabolic pairing is more stable than previously thought and could indicate survival strategies that drive the seemingly 'mutualistic' relationship as a forced cooperation. The sulfate stress response coincided with observed lags in DvH growth when grown in Mmp spent medium that was associated with a decoupling of lactate-oxidation and sulfate-reduction. Together the results demonstrate metabolic interactions and activity partitioning within a methanogenic archaeal-bacterial biofilm. The dogma of mutualism being synonymous with equal reciprocity is challenged as it pertains to this model biofilm system. Moreover, this unique bacterial-archaeal biofilm represents interdomain interactions that could represent systems that contributed shared metabolic processes that lead to the development of eukaryotic life.Item Statistical methodology for biological signals in the presence of measurement uncertainty(Montana State University - Bozeman, College of Letters & Science, 2018) Barbour, Christopher Robert; Chairperson, Graduate Committee: Mark Greenwood; Bibiana Bielekova (co-chair); Mark Greenwood, Dominique Zosso and Bibiana Bielekova were co-authors of the article, 'Constructed composite response: a framework for constructing targeted latent variables' submitted to the journal 'Biometrika' which is contained within this dissertation.; Christopher Barbour, Mark Greenwood, Dominique Zosso and Bibiana Bielekova were co-authors of the article, 'Extending CCR methodology to high-dimensional data to develop a sensitive clinical endpoint for multiple sclerosis' submitted to the journal 'Computational and graphical statistics' which is contained within this dissertation.; Peter Kosa, Mika Komori, Makoto Tanigaw, Ruturaj Masvekar, Tianxia Wu, Kory Johnson, Panagiotis Douvaras, Valentina Fossati, Ronald Herbst, Yue Wang, Keith Tan, Mark Greenwood and Bibiana Bielekova were co-authors of the article, 'Molecular-based diagnosis of multiple sclerosis and its progressive stage' in the journal 'Annals of neurology' which is contained within this dissertation.In recent years, increasing amounts of complex biological data are being collected on patients in many branches of medical research. Many of these signals are being collected with a certain amount of imprecision in the attained measurements. Two such areas in multiple sclerosis (MS) research are clinical scale development and proteomics analysis. Scales are often constructed from multiple outcome measures to create a combined metric that is a better measure of the true trait of interest than any of the original components. When the interest is in creating a scale that is sensitive to changes over time, developing it using cross-sectional data may not tune the projection to detect changes over time optimally. The proposed methodology, coined the Constructed Composite Response (CCR), was developed to maximize detected longitudinal change. A simulation study, and analysis of a motivating dataset, demonstrated that the CCR methodology performs better at capturing longitudinal change than traditional techniques. Including sparsifying constraints, motivated by penalized regression models, improved the performance of the CCR in high- dimensional data. In proteomics data, undesirable sources of variation are often present. Examples include temporal fluctuation in control samples and technical variability from multiple assay runs. When developing a molecular classifier of MS, a novel variable screening procedure was implemented to eliminate proteins with high levels of these unwanted sources. A simulation study compared this with traditional screening approaches and findings are discussed. Future extensions and directions of research are also discussed.Item Application of the thiosulfonate switch technique and a modified biotin switch technique protocols to detect protein S-nitrosothiols in mouse liver lysates in 1D and 2D gel studies(Montana State University - Bozeman, College of Letters & Science, 2015) Miller, Colin Gregory; Chairperson, Graduate Committee: Paul Grieco; Brian Bothner (co-chair)While the role of nitric oxide (NO) in cell signaling and liver growth has been well documented, the identification of S-nitrosylated proteins, one of the major NO transport mechanisms within the cell, remains a challenge. Classically, the implementation of biotin labeling, known as the biotin switch technique (BST), with streptavidin-agarose bead pulldown and subsequent immunoblotting, has offered the best results for identifying S-nitrosocysteine residues within proteins. However, this technique has come under scrutiny for its use of ascorbate as a reducing agent. Numerous published accounts have shown ascorbate's poor reducing potential especially for S-nitrosoproteins. To this end, the Grieco lab has shown that pure S-nitrosylated proteins can be transformed into S-phenylsulfonylcysteine residues, which can be readily converted into mixed disulfides thus allowing for labeling of pure nitrosylated proteins at pH's as low as pH 4. This protocol is referred to as the thiosulfonate switch technique (TST). The Grieco lab has also modified the biotin switch technique to incorporate electrophilic maleimide and orthopyridyl disulfide (OPSS) dyes for fluorescence labeling of the S-nitroso proteome. To examine the scope and limitations of the TST vis-a-vis cell lysates, the TST protocol and the modified BST protocol, both employing novel Z-CyDyes developed in the Grieco Laboratory, have been used to specifically label S-nitrosylated proteins in complex liver lysates. The successful labeling of mouse liver lysates, employing UV and ascorbate SNO knockout negative controls, is demonstrated in both 1D and 2D gel studies. Also reported herein is the creation of novel second generation maleimide dyes (SO3-Cy-Mal) based on Z-CyDyes.Item Toward resolving the human neocortex epileptic proteome(Montana State University - Bozeman, College of Letters & Science, 2013) Keren-Aviram, Gal; Chairperson, Graduate Committee: Edward DratzEpilepsy is a common and often devastating neurological disorder, which is not well understood at the molecular level. Exactly why some brain regions produce epileptic discharges and others do not is not known. Patients who fail to respond to antiseizure medication can benefit from surgical removal of brain regions that produce epileptic activities. The tissue removed in these surgeries offers an invaluable resource to uncover the molecular and cellular basis of human epilepsy. Here, we report a proteomic study, as part of a Systems Biology of Epilepsy Project, which utilizes in vivo electrophysiologically-characterized human brain samples from the neocortex of 6 patients with refractory epilepsy, to determine whether there are common proteomic patterns in human brain regions that produce epileptic discharges. This study is unique in that comparison of protein expression was made within same patient, between nearby epileptic and non-epileptic (or less epileptic) brain regions, as defined by their interictal (between seizure) spike frequencies. Protein spots were resolved from three subcellular fractions, using two-dimensional differential-in-gel-electrophoresis, revealing 31 spots that changed significantly and were identified by liquid-chromatography tandem mass-spectrometry. Interestingly, glial fibrillary acidic protein was found to be consistently down regulated in high spiking brain tissue and glial fibrillary acidic protein levels showed strong negative correlation with spiking frequency. We next developed a two-step analysis method to select for frequently changing spots among the patients and identified 397 of those proteins. Spots of interest were clustered by protein expression patterns across all samples. This analysis predicted proteomic changes due to both histological differences and molecular pathways by examination of gene ontology clusters. Our experimental design and proteomic data analysis predicts novel glial and vascular changes and changes in cytoskeleton and neuronal projections that provide new insights into the structural and functional basis of neocortical epilepsy.Item 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 Synthesis of zwitterionic cyanine dyes for use in proteomics(Montana State University - Bozeman, College of Letters & Science, 2012) Epstein, Mark Galen; Chairperson, Graduate Committee: Paul GriecoThe CyDye family of fluorescent dyes are the tools currently in use today for applications in two dimensional difference gel electrophoresis (2D-DIGE) techniques. The lysine labeling CyDyes are limited by problems with over labeling resulting in protein precipitation and isoelectric point (pI) drift at high pH's. These limitations have been addressed by a family of highly water soluble and pI balancing zwitterionic BODIPY dyes, which were previously synthesized in the Grieco group. The absorbance maxima of the BODIPY fluorophores were tuned through extension of the pi system to produce a three color, spectrally resolved dye set. However the fluorescence of the green emitting BOPIDY suffered at pH's less than 3.5 and greater than 11, while the red emitting BODIPY was susceptible to Michael addition changing its emission profile. To address the limitations of the BODIPY family of dyes, a new family of zwitterionic 2DDIGE dyes based on the established CyDye fluorophores have been synthesized. A complete three dye zwitterionic minimal labeling set which features a cysteic acid motif, titratable amine functionality and an NHS activated ester group reactive towards lysine residues has been synthesized: Z-Cy2 (QY= 6.8% ± 0.1, epsilon= 155,000), Z-Cy3 (QY= 11.1% ± 0.4, epsilon= 124,500), Z-Cy5 (QY= 43.3% ± 0.6, epsilon= 217,600). In addition, a complete three dye zwitterionic saturation labeling set which incorporates a cysteic acid motif and maleimide functionality reactive towards cysteine residues has also been synthesized: Z-Cy2-Mal (QY= 6.6 % ± 0.1, epsilon= 104,500), Z-Cy3-Mal (QY= 12.4 % ± 0.5, epsilon= 127,700), Z-Cy5-Mal (QY= 40.2 % ± 0.4, epsilon= 217,400).Item Type-2 diabetes and innate immunity : new connections revealed by multi-dimensional fractionation of blood plasma prior to proteomic analysis(Montana State University - Bozeman, College of Letters & Science, 2010) Laffoon, Scott Bradley; Chairperson, Graduate Committee: Edward DratzWe compared levels of protein isoforms in human blood plasma from patients with newly diagnosed and untreated type-2 diabetes (T2DM) with non-diabetic controls in samples obtained from US NIH. We immunodepleted fourteen of the most abundant proteins from pooled plasma samples and separated the depleted samples into six fractions by reverse-phase liquid chromatography at 80°C. Proteins from these fractions were labeled with new high quantum yield, hydrophilic and spectrally resolved fluorescent detection dyes developed at MSU and resolved on large-format (24cm x 20cm) two-dimensional gels. By fluorescence analysis of 2D gels, using >1.4 fold change and p<0.05 acceptance criteria, we have identified five T2DM associated proteins and isoforms, including: two isoforms of zinc-alpha glycoprotein (ZAG), one isoform of serum amyloid A-1 (SAA-1) preprotein, one isoform of cysteine-rich secreted protein-3 (CRISP-3), one isoform of haptoglobin, and an A1-apolipoprotein fragment. Complement factor H related-5 (CFHR-5) is the likely identification of a sixth protein found significantly down in T2DM. Changes in the plasma levels of CRISP-3 and CFHR-5 strengthen the hypothesis that T2DM is a disease involving innate immunity. Three of these proteins are known to specifically bind to the transport protein, human serum albumin (HSA). Also, CRISP-3 is a specific and high-affinity ligand of alpha 1 beta glycoprotein, which is an HSA binder. To investigate HSA binding properties, we quantitatively measured the binding of a dye probe by HSA at neutral pH. These measurements revealed that HAS binding of the probe correlates with several metabolic parameters of central importance to the diagnosis of T2DM, including fasting plasma glucose (FPG). Therefore, this assay may reveal altered properties of HSA that could be developed for the clinical assessment of individuals' metabolic status. We sought modifications of HSA or altered cargo of HSA that may cause the difference in binding. 1D gels of plasma proteins reacted with maleimide dye showed no changed levels of the oxidation state of HSA's lone thiol, Cys-34. However, 1D blots of plasma proteins reacted with the oxidative carbonyl probe, hydrazide-biotin conjugate, and probed with luminol reactive HRP-neutravidin showed a surprising anti-correlation of HSA oxidation with hemoglobin A1c, an indicator of glycemic control.