Scholarly Work - Chemistry & Biochemistry
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/8714
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Item Longitudinal analysis of the Five Sisters hot springs in Yellowstone National Park reveals a dynamic thermoalkaline environment(Springer Science and Business Media LLC, 2022-11) Peach, Jesse T.; Mueller, Rebecca C.; Skorupa, Dana J.; Mesle, Margaux M.; Kanta, Sutton; Boltinghouse, Eric; Sharon, Bailey; Copie, Valerie; Bothner, Brian; Peyton, Brent M.Research focused on microbial populations of thermoalkaline springs has been driven in a large part by the lure of discovering functional enzymes with industrial applications in high-pH and high temperature environments. While several studies have focused on understanding the fundamental ecology of these springs, the small molecule profiles of thermoalkaline springs have largely been overlooked. To better understand how geochemistry, small molecule composition, and microbial communities are connected, we conducted a three-year study of the Five Sisters (FS) springs that included high-resolution geochemical measurements, 16S rRNA sequencing of the bacterial and archaeal community, and mass spectrometry-based metabolite and extracellular small molecule characterization. Integration of the four datasets facilitated a comprehensive analysis of the interwoven thermoalkaline spring system. Over the course of the study, the microbial population responded to changing environmental conditions, with archaeal populations decreasing in both relative abundance and diversity compared to bacterial populations. Decreases in the relative abundance of Archaea were associated with environmental changes that included decreased availability of specific nitrogen- and sulfur-containing extracellular small molecules and fluctuations in metabolic pathways associated with nitrogen cycling. This multi-factorial analysis demonstrates that the microbial community composition is more closely correlated with pools of extracellular small molecules than with the geochemistry of the thermal springs. This is a novel finding and suggests that a previously overlooked component of thermal springs may have a significant impact on microbial community composition.Item Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis(2020-02) Steward, Katherine F.; Eilers, Brian J.; Tripet, Brian P.; Fuchs, Amanda; Dorle, Michael; Rawle, Rachel A.; Soriano, Berliza; Balasubramanian, Narayanaganesh; Copie, Valerie; Bothner, Brian; Hatzenpichler, RolandBioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.Item Copper modulates sex-specific fructose hepatoxicity in nonalcoholic fatty liver disease (NALFD) Wistar rat models(2020-04) Morrell, Austin; Tripet, Brian P.; Eilers, Brian J.; Tegman, Megan; Thompson, Damon; Copie, Valerie; Burkhead, Jason L.This study aimed to characterize the impact of dietary copper on the biochemical and hepatic metabolite changes associated with fructose toxicity in a Wistar rat model of fructose-induced liver disease. Twenty-four male and 24 female, 6-week-old, Wister rats were separated into four experimental dietary treatment groups (6 males and 6 females per group), as follows: (1) a control diet: containing no fructose with adequate copper (i.e., CuA/0% Fruct); (2) a diet regimen identical to the control and supplemented with 30% w/v fructose in the animals' drinking water (CuA/30% Fruct); (3) a diet identical to the control diet but deficient in copper content (CuD/0% Fruct) and (4) a diet identical to the control diet but deficient in copper content and supplemented with 30% w/v fructose in the drinking water (CuD/30% Fruct). The animals were fed the four diet regimens for 5 weeks, followed by euthanization and assessment of histology, elemental profiles and identification and quantitation of liver metabolites. Results from 1H nuclear magnetic resonance metabolomics revealed mechanistic insights into copper modulation of fructose hepatotoxicity through identification of distinct metabolic phenotypes that were highly correlated with diet and sex. This study also identified previously unknown sex-specific responses to both fructose supplementation and restricted copper intake, while the presence of adequate dietary copper promoted most pronounced fructose-induced metabolite changes.Item Exposure of Methicillin-Resistant Staphylococcus aureus to Low Levels of the Antibacterial THAM-3ΦG Generates a Small Colony Drug-Resistant Phenotype(2018-06) Weaver, Alan J.; Peters, Tami R.; Tripet, Brian P.; Van Vuren, Abigail; Rakesh; Lee, Richard E.; Copie, Valerie; Teintze, MartinThis study investigated resistance against trishexylaminomelamine trisphenylguanide (THAM-3ΦG), a novel antibacterial compound with selective microbicidal activity against Staphylococcus aureus. Resistance development was examined by culturing methicillin resistant S. aureus (MRSA) with sub-lethal doses of THAM-3ΦG. This quickly resulted in the formation of normal (WT) and small colonies (SC) of S. aureus exhibiting minimal inhibitory concentrations (MICs) 2× and 4× greater than the original MIC. Continuous cell passaging with increasing concentrations of THAM-3ΦG resulted in an exclusively SC phenotype with MIC >64 mg/L. Nuclear magnetic resonance (NMR)-based metabolomics and multivariate statistical analysis revealed three distinct metabolic profiles for THAM-3ΦG treated WT, untreated WT, and SC (both treated and untreated). The metabolome patterns of the SC sample groups match those reported for other small colony variants (SCV) of S. aureus. Supplementation of the SCV with menadione resulted in almost complete recovery of growth rate. This auxotrophism was corroborated by NMR analysis revealing the absence of menaquinone production in the SCV. In conclusion, MRSA rapidly acquires resistance to THAM-3ΦG through selection of a slow-growing menaquinone auxotroph. This study highlights the importance of evaluating and monitoring resistance to novel antibacterials during development.Item Multi-level Omics Analysis Provides Insight to the Ignicoccus hospitalis-Nanoarchaeum equitans Association(2017-09) Rawle, Rachel A.; Hamerly, Timothy; Tripet, Brian P.; Giannone, Richard J.; Wurch, Louie; Hettich, Robert L.; Podar, Mircea; Copie, Valerie; Bothner, BrianBACKGROUND Studies of interspecies interactions are inherently difficult due to the complex mechanisms which enable these relationships. A model system for studying interspecies interactions is the marine hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans. Recent independently-conducted 'omics' analyses have generated insights into the molecular factors modulating this association. However, significant questions remain about the nature of the interactions between these archaea. METHODS We jointly analyzed multiple levels of omics datasets obtained from published, independent transcriptomics, proteomics, and metabolomics analyses. DAVID identified functionally-related groups enriched when I. hospitalis is grown alone or in co-culture with N. equitans. Enriched molecular pathways were subsequently visualized using interaction maps generated using STRING. RESULTS Key findings of our multi-level omics analysis indicated that I. hospitalis provides precursors to N. equitans for energy metabolism. Analysis indicated an overall reduction in diversity of metabolic precursors in the I. hospitalis-N. equitans co-culture, which has been connected to the differential use of ribosomal subunits and was previously unnoticed. We also identified differences in precursors linked to amino acid metabolism, NADH metabolism, and carbon fixation, providing new insights into the metabolic adaptions of I. hospitalis enabling the growth of N. equitans. CONCLUSIONS This multi-omics analysis builds upon previously identified cellular patterns while offering new insights into mechanisms that enable the I. hospitalis-N. equitans association. GENERAL SIGNIFICANCE Our study applies statistical and visualization techniques to a mixed-source omics data set to yield a more global insight into a complex system, that was not readily discernable from separate omics studies.Item Biochemical association of metabolic profile and microbiome in chronic pressure ulcer wounds(2015-05) Ammons, Mary Cloud B.; Morrissey, Kathryn; Tripet, Brian P.; Van Leuvan, James T.; Han, Anne; Lazarus, Gerald S.; Zenilman, Jonathan M.; Stewart, Philip S.; James, Garth A.; Copie, ValerieChronic, non-healing wounds contribute significantly to the suffering of patients with co-morbidities in the clinical population with mild to severely compromised immune systems. Normal wound healing proceeds through a well-described process. However, in chronic wounds this process seems to become dysregulated at the transition between resolution of inflammation and re-epithelialization. Bioburden in the form of colonizing bacteria is a major contributor to the delayed headlining in chronic wounds such as pressure ulcers. However how the microbiome influences the wound metabolic landscape is unknown. Here, we have used a Systems Biology approach to determine the biochemical associations between the taxonomic and metabolomic profiles of wounds colonized by bacteria. Pressure ulcer biopsies were harvested from primary chronic wounds and bisected into top and bottom sections prior to analysis of microbiome by pyrosequencing and analysis of metabolome using 1H nuclear magnetic resonance (NMR) spectroscopy. Bacterial taxonomy revealed that wounds were colonized predominantly by three main phyla, but differed significantly at the genus level. While taxonomic profiles demonstrated significant variability between wounds, metabolic profiles shared significant similarity based on the depth of the wound biopsy. Biochemical association between taxonomy and metabolic landscape indicated significant wound-to-wound similarity in metabolite enrichment sets and metabolic pathway impacts, especially with regard to amino acid metabolism. To our knowledge, this is the first demonstration of a statistically robust correlation between bacterial colonization and metabolic landscape within the chronic wound environment.Item Untargeted Metabolomics Studies Employing NMR and LC–MS Reveal Metabolic Coupling Between Nanoarcheum Equitans and Its Archaeal Host Ignicoccus Hospitalis(2014-11) Hamerly, Timothy; Tripet, Brian P.; Tigges, Michelle M.; Giannone, Richard J.; Wurch, Louie; Hettich, Robert L.; Podar, Mircea; Copie, Valerie; Bothner, BrianAbstract Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC–MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with informa-tion about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hosp-italis that are impacted by the presence of N. equitans, including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equi-tans’s consumption of a significant fraction of the metab-olite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans. Combining LC–MS and NMR metabolomics datasets improved cov-erage of the metabolome and enhanced the identification and quantitation of cellular metabolites.