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 Characterization of osteoarthritis metabolism: a mass spectrometry based-approach(Montana State University - Bozeman, College of Letters & Science, 2024) Welhaven, Hope Diane Aloha; Co-chairs, Graduate Committee: Brian Bothner and Ronald K. June II; This is a manuscript style paper that includes co-authored chapters.Osteoarthritis (OA) effects 7% of the global population, equating to more than 500 million people worldwide, and is the leading cause of disability. Its multifaceted etiology includes risk factors ranging from genetics, to aging, obesity, sex, race, and joint injury. OA manifests differently across the patient population where symptom severity, rate of progression, response to treatment, pain perception, as well as others vary person to person posing significant challenges for effective management and prevention. At the cellular level, imbalanced matrix catabolism and anabolism contribute to the breakdown of cartilage, underlying bone, and other tissues affected by OA. Leveraging mass spectrometry-based techniques, particularly metabolomics, offers a promising avenue to dissect OA metabolism across musculoskeletal tissues, while considering individual patient-specific risk factors. Therefore, the goals of this research were to: (1) comprehensively characterize OA phenotypes and endotypes and (2) explore OA pathogenesis within the context of disease-associated risk factors. The first area of research focuses on profiling OA phenotypes and endotypes across disease development. These results provide clear evidence of OA-induced metabolic perturbations in OA cartilage and bone and elucidate mechanisms that shift as disease progresses. Several metabolites and pathways associated with lipid, amino acid, matrix, and vitamin metabolism were differentially regulated between healthy and OA tissues and within OA endotypes. The second area of research focuses on the impact of OA risk factors -- sex, injury, obesity, loading -- on the metabolism of circulatory fluids (i.e., serum, synovial fluid) and chondrocytes. Identification of metabolic indicators of disease, such as cervonyl carnitine, and metabolic pathways associated with these risk factors holds potential for improving screening, monitoring disease progression, and guiding preventative strategies. Overall, this work contributes to our current understanding of OA, its diverse metabolic landscape, risk factors and their interactions. Moreover, it lays the groundwork for personalized medicine by providing detailed insights into individualized phenotypic profiles, thereby advancing the prospect of tailored treatment strategies for OA individuals.Item Development and analysis of lipidomics procedures for the causal investigation of Alzheimer's disease(Montana State University - Bozeman, College of Letters & Science, 2022) Koch, Max Richard; Chairperson, Graduate Committee: Edward Dratz; This is a manuscript style paper that includes co-authored chapters.Uncovering sets of molecular features which cause a healthy metabolic state to transition to one of disease, requires extensive experimentation and often presents a difficult analysis. In the case of neurodegenerative diseases, such as Alzheimer's Disease, simply obtaining suitable samples can be a challenging endeavor. Many current 'Omics' techniques excel at profiling a vast array of molecules, such as water-soluble metabolites, lipids, and proteins, in order to compare groups of samples from healthy and diseased organisms. Such approaches primarily use various associations between molecules and disease to identify biomarkers. However, these 'omics' experiments frequently result in intriguing biological hypotheses, but to date have rarely provided mechanistic explanations. How then, can mechanistic explanations be recovered from metabolite or lipid profile data? In our work, we applied these methods to 6 Alzheimer's diseased brain samples and 6 age matched controls. When analyzed via mass spectrometry, lipids which differed significantly between control and disease were identified, but this information was not able to'provide mechanistic insight. The beginning of any 'omics' based experiment starts with the extraction of the desired molecules. In order to assess the efficiency of three different lipid extraction methods, a lipid standard was extracted from a matrix composed of rat liver tissue and analyzed by mass spectrometry. The classic Folch extraction was found to be best at reproducibly extracting a wide range of lipids. Several of the lipids identified from human brains showed oxidative damage. Lastly, 5 statistical measures of dependence and 3 network algorithms were investigated for their ability to reconstruct mechanistic relationships in a dynamic model of arachidonic acid metabolism. Many of the metabolites of arachidonic acid are oxidation products. Under conditions of high noise and relatively few samples, standard measures of correlation, such as Pearson's correlation, Spearman's correlation and Kendall's Tau were found to perform the best. Metrics which incorporate nonlinear metabolic relations and network algorithms were found to be applicable, when sample size is large and the signal to noise ratio is close to l.Item Investigating the role of allostery through changes in protein stability and dynamics(Montana State University - Bozeman, College of Letters & Science, 2021) Patterson, Angela Jean; Chairperson, Graduate Committee: Brian Bothner; Faiz Ahmad and Jaigeeth Deveryshetty were authors and Jenna R. Mattice, Nilisha Pokhrel, Brian Bothner and Edwin Antony were co-authors of the article, 'Hydrogen-deuterium exchange reveals a dynamic DNA-binding map of replication protein A' in the journal 'Nucleic Acids Research' which is contained within this dissertation.; Zhongchao Zhao, Elizabeth Waymire, Adam Zlotnick, and Brian Bothner were co-authors of the article, 'Dynamics of hepatitis B virus capsid protein dimer regulates assembly through an allosteric network' in the journal 'ACS chemical biology' which is contained within this dissertation.; Paul B.G. van Erp was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and Cas 3 recruitment by the CRISPR RNA-guide cascade complex' in the journal 'ACS chemical biology' which is contained within this dissertation.; Aidan White, Elizabeth Waymire, Sophie Fleck, Sarah Golden, Royce Wilkinson, Blake Wiedenheft and Brian Bothner were co-authors of the article, 'Thermodynamics of CRISPR-anti-CRISPR interactions provides mechanistic insight into inhibition' which is contained within this dissertation.Allostery is the presence of a communication network that links functional sites of a protein that are distal from one another. The existence of an allosteric network can be observed through conformational change or a change in protein dynamics. These networks can be used to provide insight into the mechanistic function of proteins or protein complexes. In this thesis, four protein complexes were studied (RPA, HBV, Cascade, and Csy) and allosteric networks within the complexes were observed by monitoring the changes in protein dynamics upon an energy perturbation. To measure the changes in protein dynamics, hydrogen deuterium exchange mass spectrometry was used. This technique allows for the determination of how often the hydrogen bonding within a protein structure is broken. By tracking the longevity of the hydrogen bonding network that comprises the studied protein's structure, the dynamics of the protein can be studied. In this work, each of the proteins had changes in protein dynamics that were distal from the site of the energy perturbation that had functional impacts on each of the protein complexes. The combined presence of the distal changes in dynamics with an effect on protein function fits the definition of allostery. If allostery is present in these four diverse systems, is it possible that allostery is present in all proteins?Item Analysis of complex samples by mass spectrometry leads to insights into system dynamics(Montana State University - Bozeman, College of Letters & Science, 2021) Peach, Jesse Thomas; Chairperson, Graduate Committee: Brian Bothner; James Larson, Sutton Kanta, Eric Boltinghouse, Rebecca Mueller, Ganesh Balasubramanian, Mohammed Refai, Brent Peyton and Brian Bothner were co-authors of the article, 'Optimization of thermal small molecule and protein mass spectrometry analysis' submitted to the journal 'Analytical biochemistry' which is contained within this dissertation.; Rebecca Mueller, Dana Skorupa, Margaux Mesle, Sutton Kanta, Eric Boltinghouse, Bailey Sharon, Valerie Copie, Brian Bothner and Brent Peyton were co-authors of the article, 'Longitudinal meta-analysis of the Five Sisters Hot Springs in Yellowstone National Park reveals a dynamic thermoalkaline environment' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.; Stephanie M. Wilson, Logan D. Gunderson, Lizzi Frothingham, Tan Tran, Seth T. Walk, Carl J. Yeoman, Brian Bothner and Mary P. Miles were co-authors of the article, 'Temporal metabolic response yields a dynamic biosignature of inflammation' submitted to the journal 'iScience' which is contained within this dissertation.Systems biology offers a holistic approach to biological science. In its most complete form, systems biology requires comprehensive data encompassing all of the parts or molecules across a set of hierarchical networks. To obtain and analyze the comprehensive and large datasets required for systems biology analysis, biologists have taken advantage of new technology and computational tools. Over the last few decades, advances in computational modeling and analysis technology has dramatically increased the efficacy of systems biology and the understanding of the natural world. However, systems biology is still an emerging discipline. The overwhelming scale of potential biological data that has yet to be described, coupled with interpretation and application obstacles, leaves much work to be accomplished. One aspect of systems biology that needs development is the interpretation and analysis of temporal biological data. Temporal data reveals more about biological phenomena than static data as biology is inherently dynamic. This dissertation explores the benefits of temporal profiling of complex samples to make time-resolved conclusions about complicated biological questions. Three research projects are the backbone of this document, with a chapter being devoted to each. Chapter 2 describes the development of a comprehensive method for extraction and mass spectrometry analysis of several different fractions from hot spring sediment. Chapter 3 delves into a multi-omics analysis tracking changes over the course of three years in a thermoalkaline spring system in Yellowstone National Park. It defines how specific extracellular small molecules correlate with microbial fitness. Specifically, how unique nitrogen and sulfur containing molecules in the sediment drive archaeal abundance and diversity. The final chapter introduces the concept of a 'dynamic biosignature', a set of metabolites that have similar responses to known biomarkers, in this case pro-inflammatory cytokines. A cohort of metabolites was identified that provided mechanistic insight into the inflammatory response. Overall, this dissertation provides examples of systems biology analysis and provides evidence that static, single time-point datasets fail to capture that which is the essence of biology - change.Item Relating protein structure to function: how protein dynamics maximizes energy gained by electron transfer in an anaerobic energy conservation mechanism(Montana State University - Bozeman, College of Letters & Science, 2019) Berry, Luke Montgomery; Chairperson, Graduate Committee: Brian Bothner; Angela Patterson, Natasha Pence, John Peters and Brian Bothner were co-authors of the article, 'Hydrogen deuterium exchange mass spectrometry of oxygen sensitive proteins' in the journal 'Bio-protocols' which is contained within this dissertation.; Saroj Poudel, Monika Tokmina-Lukaszewska, Daniel R. Colman, Diep M.N. Nguyen, Gerrit J. Schut, Micheal W.W. Adams, John W. Peters, Eric S. Boyd and Brian Bothner were co-authors of the article, 'H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle' in the journal 'Biochimica et biophysica acta (BBA) - general subjects' which is contained within this dissertation.; Monika Tokmina-Lukaszewska, Derek F. Harris, Oleg A. Zadvornyy, Simone Raugei, John W. Peters, Lance C. Seefeldt and Brian Bothner were co-authors of the article, 'Combining in-solution and computational methods to characterize the structure-function relationship of the nitrogengase systems' which is contained within this dissertation.; Hayden Kallas, Derek F. Harris, Monika Tokmina-Lukaszewska, Simone Raugei, Lance C. Seefeldt and Brian Bothner were co-authors of the article, 'Iron protein docking effects on MOFE protein dynamics: function of negative cooperativity and the regulation of electron trasfer' which is contained within this dissertation.Reduced ferredoxin (Fd) plays a critical role in anaerobic metabolism by acting as an alternative source of energy to adenosine triphosphate (ATP). The reduction potential of Fd is low (-450 mV) making it difficult to reduce individually. However, it has recently been discovered that a unique mechanism known as electron bifurcation allows anaerobic organisms to reduce Fd without suffering a loss of energy. Electron bifurcation was originally discovered in complex III of the electron transport chain, and increased the efficiency of the proton motive force without an overall change in the electron flow, minimizing energy loss. EB accomplishes this is by coupling a favorable (exergonic) and unfavorable (endergonic) reduction reaction. The exergonic reaction produces a singly reduced cofactor with a sufficiently negative reduction potential to allow the endergonic process to proceed. This allows anaerobic organisms to couple the formation of NADH, with the reduction of Fd. A detail of interest in the bifurcating mechanism is how these enzymes regulate the flow of electrons down the exergonic and endergonic branches to prevent multiple electrons from traveling down the exergonic branch. It is hypothesized that changes in the protein conformation alter the distance between cofactors altering the rate of electron transfer. To fully understand how changes in a protein's conformation regulates electron transfer in electron bifurcation we used a suite of in-solution techniques, such as H/D exchange and chemical cross-linking coupled to mass spectrometry to characterize the structure and dynamics of the model bifurcating enzyme, NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn), during the different steps of electron bifurcation. Additionally we also set out to use these techniques to characterize the structure and dynamics of the nitrogenase systems in order to obtain biophysical evidence of negative cooperativity in the various nitrogenase systems.Item Pyrolysis of thermal protection system materials: molar yields of volatile products derived from in situ mass spectrometric measurements(Montana State University - Bozeman, College of Letters & Science, 2018) Bessire, Brody Kelly; Chairperson, Graduate Committee: Timothy Minton; Sridhar A. Lahankar and Timothy K. Minton were co-authors of the article, 'Pyrolysis of phenolic impregnated carbon ablartor (PICA)' in the journal 'ACS applied materials and interfaces' which is contained within this thesis.; Timothy K. Minton was a co-author of the article, 'Decomposition of phenolic impregnated carbon ablator (PICA) as a function of temperature and heating rate' in the journal 'ACS applied materials and interfaces' which is contained within this thesis.; Timothy K. Minton was a co-author of the article, 'Pyrolysis of epoxy-novolac materials as a function of time and temperature' submitted to the journal 'The journal of analytical and applied pyrolysis' which is contained within this thesis.Mass spectrometric techniques have been developed to measure the molar yields of pyrolysis products from ablative resins and composite materials at heating rates that are relevant to flight conditions. Thermal decomposition mechanisms of phenolic and an epoxy-novolac resin systems are reviewed. New insights into the thermal decomposition mechanisms of PICA (Phenolic Impregnated Carbon Ablator) and epoxy-novolac D.E.N. 438 (Dow Epoxy-Novolac) are proposed and are based on the measurements of molar yields from these materials. Molar yield data have been provided in the appendices of this thesis for use in material response models. The thermal decomposition of phenolic impregnated carbon ablator (PICA) has been investigated with the objective of measuring molar yields of pyrolysis products at heating rates that are relevant to MSL flight conditions. The relative molar yields of 14 pyrolysis gases were obtained in conjunction with mass loss measurements. These measurements allowed for the calculation of absolute molar yields and mass yields as a function of temperature and heating rate, as well as the simulation of TGA curves. Pyrolysis product yields change as a function of heating rate, and this behavior has been attributed to two mechanisms that compete during the initial stages of thermal decomposition. The results of this study are now available for use in material response models. The thermal decomposition of an epoxy-novolac resin system has also been investigated. Samples of D.E.N. 438 were cured using NMA (methyl-5-norbornene-2,3-dicarboxylic anhydride) as a crosslinking agent and BDMA (N-benzyldimethylamine) as a catalyst. A radiative heating method was developed to minimize experimental uncertainties that may emerge from thermal gradients that are established across the samples as they experience high rates of heating. The molar yields of the six dominant pyrolysis products were measured at a heating rate of 8°C s -1. The molar yields of pyrolysis products provide new insight, and a new thermal decomposition mechanism is proposed.Item Mass spectrometry based lipidomics as a tool in the search for biomarkers and mechanisms of disease(Montana State University - Bozeman, College of Letters & Science, 2016) Willems, Daniel Lee; Chairperson, Graduate Committee: Edward Dratz; Nicholas E. Goocey and Edward A. Dratz were co-authors of the article, 'A highly reproducible and efficient lipid extraction protocol enhanced using 3D printing of centrifuge adapters for optimum glass vials' submitted to the journal 'Lipids' which is contained within this thesis.; Max Koch, Nicholas E. Goocey, Blaine R. Roberts and Edward A. Dratz were co-authors of the article, 'Lipidomic analysis of human brain cortex in alzheimer's disease reveals aberrant levels of acetylcholine precursor speices' submitted to the journal 'American journal of alzheimer's disease and other dementias' which is contained within this thesis.; Max Koch, Nicholas E. Goocey, Blaine R. Roberts and Edward A. Dratz were co-authors of the article, 'Lipidomics reveals aberrent metabolism of lipid molecules in alzheimer's disease cerebral cortex' which is contained within this thesis.Lipidomics studies a highly diverse class of compounds insoluble in water and soluble in organic solvents. Lipids are a major component of cells and tissues, take part in a rich network of metabolic reactions, and are implicated in many disease mechanisms. Lipidomics complements genomics, proteomics and the more common metabolomic analysis of hydrophilic metabolites and can provide new insights into disease mechanisms. The problem approached in this thesis was to compare different methods of sample preparation for lipidomics and apply lipidomics to the study of two major health problems: Nonalcoholic Fatty Liver Disease (NAFLD) and Alzheimer's Disease (AD). Excessive dietary intake of sucrose and fructose, common in the Western Diet, increases deposition of triacylglycerides in the liver and leads to cognitive decline in experimental animals. NAFLD increases the risk of type 2 diabetes, obesity and AD. The high diversity and hydrophobicity of lipids complicates their separation, detection and analysis. However, modern chromatography and mass spectrometry instrumentation and techniques are greatly improving the capability of lipidomic analysis. A lipid extraction protocol was optimized for reproducibility and yield, and was used to extract lipids from rat liver under sucrose stress in a model of human NAFLD and human brain cortex from Alzheimer's Disease (AD) compared to controls. The samples were analyzed using mass spectrometry. The NALFD study did not yield the expected results, instead these data provided a foundation for designing future experiments in progress and to validate the methods used in the AD study. The AD studies showed that several phosphatidylcholine species are down regulated along with acetyl-CoA, which may be the source of low levels of the neurotransmitter acetylcholine in AD. Two different chromatography methods were used to seek a higher coverage of different lipids. Differences in the lipids in AD and controls were evident in the omega-6 and omega-3 fatty acids. The precursors of long omega-3s synthesis were increased while the products EPA and DHA were decreased. In a similar fashion, precursors to long omega-6s were found to be decreased, while the products were increased. This suggests that the omega-6 synthesis pathway may be outcompeting the omega-3 synthesis.Item Development of a protein-based sensor assay for rapid classification of complex biological samples(Montana State University - Bozeman, College of Letters & Science, 2016) Hamerly, Timothy Kyle; Chairperson, Graduate Committee: Brian Bothner; Joshua Heinemann, Monika Tokmina-Lukaszewska, Elizabeth R. Lusczek, Kristine E. Mulier, Greg J. Beilman and Brian Bothner were co-authors of the article, 'Bovine serum albumin as a molecular sensor for the discrimmination of complex metabolite samples' in the journal 'Analytica chemica acta' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Adding metrics to the aging of whiskey using a protein sensor assay' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Analysis of wine using the protein sensor assay' which is contained within this dissertation.; Brian Bothner was a co-author of the article, 'Investigations into the use of a protein sensor assay for metabolite analysis' in the journal 'Applied biochemistry and biotechnology' which is contained within this dissertation.Metabolomics, one of the core 'omics' fields within the umbrella of systems biology, is the study of the small molecules which can be used to characterize the state of an organism. Metabolites are constantly being transformed inside a cell in direct response to stimuli around them. This makes the metabolome the most dynamic of all the omics fields and is considered to be a direct readout of the cells state at any given time. Although highly informative, the metabolome is inherently difficult to study, with thousands of known metabolites, any of which could be important for classifying a cell into a healthy or diseased state. Techniques such as mass spectrometry are well suited to study the metabolome and have been used to successfully classify cells by identify markers for a given disease state. However, current methods require lengthy analysis times due in part to the complexity of the metabolome. The research presented in this dissertation highlights a new and promising methodology which improves classification and speeds marker discovery. Making use of a protein found in animals which has evolved to selectively bind metabolites, an assay was developed which better classified samples compared to current methods used in the field of metabolomics. This improved classification was achieved with an overall decrease in analysis time. The implementation of this method in the study of complex biological systems would have an immediate impact in academic and medical research.Item The application of mass spectrometry-based 'omics technologies to investigate environmental interactions of microbial systems(Montana State University - Bozeman, College of Letters & Science, 2015) Tigges, Michelle Marie; Chairperson, Graduate Committee: Brian Bothner; Michelle Tigges and Heidi Smith were main authors, and Juliana D'Andrill, Al Parker, Brian Bothner and Christine Foreman were co-authors of the article, 'Lability of environmental dissolved organic matter drives dynamic microbial processing' submitted to the journal 'Proceedings of the National Academy of Sciences' which is contained within this thesis.Microorganisms interact with their surroundings and each other. However, these interactions are complex and difficult to understand. This research presents the utilization of simplified environmental microbial systems from the extremes of life to gain insight into the roles of microbes in diverse processes including biogeochemical cycling and viral infection. The complex mixture of organic compounds within aquatic systems, known as dissolved organic matter (DOM), is an integral component of the global carbon cycle. It is a carbon source for microbial activity and impacts biogeochemical and ecological processes. However, little is known about the release and bioconversion of these compounds. This thesis presents a liquid chromatography coupled mass spectrometry (LCMS) based exometabolomics approach to chemically characterize the interaction between DOM and the representative microbial species that transform it. This work illustrates for the first time the ability to measure the relative abundance of molecular constituents of DOM during heterotrophic bacterial processing. Processing was shown to be dynamic over time, even with only single organism interactions. A LCMS based proxy was established to predict the lability of DOM carbon sources, and the labile nature of the source was shown to be a significant factor in DOM processing by single organisms. Further, the temporal interaction of two ecologically relevant beta-Proteobacteria with DOM from the Cotton Glacier, Antarctica highlight the importance of understanding the diversity of single organism DOM interactions to interpret community level bacterial interactions. LCMS-based 'omics techniques can also be utilized to characterize the changes in protein expression associated with viral infection of hyperthermophilic archaea. Viral-host interactions in Sulfolobus archaeal systems are poorly understood, and exhibit a diversity of regulation patterns. LCMS-based shotgun proteomics was utilized to characterize the temporal response of Sulfolobus islandicus to infection by Sulfolobus islandicus rod-shaped virus (SIRV2). The strengths and weaknesses of label-free protein quantitation techniques were assessed, enabling the detection of the regulation of SIRV2 proteins over time and identification of key host responses to infection. Together these studies show the impact of LCMS based 'omics technologies in bringing new insights into environmental microbial interactions.