College of Agriculture
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As the foundation of the land grant mission at Montana State University, the College of Agriculture and the Montana Agricultural Experiment Station provide instruction in traditional and innovative degree programs and conduct research on old and new challenges for Montana’s agricultural community. This integration creates opportunities for students and faculty to excel through hands-on learning, to serve through campus and community engagement, to explore unique solutions to distinct and interesting questions and to connect Montanans with the global community through research discoveries and outreach.
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Item Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04(Elsevier BV, 2023-01) Goemann, Calvin L.C.; Wilkinson, Royce; Henriques, William; Bui, Huyen; Goemann, Hannah M.; Carlson, Ross P.; Viamajala, Sridhar; Gerlach, Robin; Wiedenheft, BlakeAlgae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments – high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Collectively, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequence-based methods.Item Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress(American Society for Microbiology, 2021-02) Zhuang, Weiping; Balasubramanian, Narayanaganesh; Wang, Lu; Wang, Qian; McDermott, Timothy R.; Copie, Valerie; Wang, Gejiao; Bothner, BrianArsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited.Item Metabolic Responses to Arsenite Exposure Regulated through Histidine Kinases PhoR and AioS in Agrobacterium tumefaciens 5A(MDPI, 2020-09) Rawle, Rachel A.; Tokmina-Lukaszewska, Monika; Shi, Zunji; Kang, Yoon-Suk; Tripet, Brian P.; Dang, Fang; Wang, Gejiao; McDermott, Timothy R.; Copie, Valerie; Bothner, BrianArsenite (AsIII) oxidation is a microbially-catalyzed transformation that directly impacts arsenic toxicity, bioaccumulation, and bioavailability in environmental systems. The genes for AsIII oxidation (aio) encode a periplasmic AsIII sensor AioX, transmembrane histidine kinase AioS, and cognate regulatory partner AioR, which control expression of the AsIII oxidase AioBA. The aio genes are under ultimate control of the phosphate stress response via histidine kinase PhoR. To better understand the cell-wide impacts exerted by these key histidine kinases, we employed 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-coupled mass spectrometry (LC-MS) metabolomics to characterize the metabolic profiles of ΔphoR and ΔaioS mutants of Agrobacterium tumefaciens 5A during AsIII oxidation. The data reveals a smaller group of metabolites impacted by the ΔaioS mutation, including hypoxanthine and various maltose derivatives, while a larger impact is observed for the ΔphoR mutation, influencing betaine, glutamate, and different sugars. The metabolomics data were integrated with previously published transcriptomics analyses to detail pathways perturbed during AsIII oxidation and those modulated by PhoR and/or AioS. The results highlight considerable disruptions in central carbon metabolism in the ΔphoR mutant. These data provide a detailed map of the metabolic impacts of AsIII, PhoR, and/or AioS, and inform current paradigms concerning arsenic–microbe interactions and nutrient cycling in contaminated environments.Item Dynamic gut microbiome changes to low-iron challenge(American Society for Microbiology, 2020-11) Coe, Genevieve L.; Pinkham, Nicholas V.; Celis, Arianna I.; Johnson, Christina; DuBois, Jennifer L.; Walk, Seth T.Iron is an essential micronutrient for life. In mammals, dietary iron is primarily absorbed in the small intestine. Currently, the impacts of dietary iron on the taxonomic structure and function of the gut microbiome and reciprocal effects on the animal host are not well understood. Here, we establish a mouse model of low-iron challenge in which intestinal biomarkers and reduced fecal iron reveal iron stress while serum iron and mouse behavioral markers indicate maintenance of iron homeostasis. We show that the diversity of the gut microbiome in conventional C57BL/6 mice changes dramatically during two-weeks on a low-iron diet. We also show the effects of a low-iron diet on microbiome diversity are long-lasting and not easily recovered when iron is returned to the diet. Finally, after optimizing taxon association methods, we show that some bacteria are unable to fully recover after the low-iron challenge and appear to be extirpated from the gut entirely. In particular, OTUs from the Prevotellaceae and Porphyromonadaceae families and Bacteroidales order are highly sensitive to low-iron conditions, while other seemingly insensitive OTUs recover. These results provide new insights into the iron requirements of gut microbiome members and add to the growing understanding of mammalian iron cycling. IMPORTANCE All cells need iron. Both too much iron and too little lead to diseases and unwanted outcomes. Although the impact of dietary iron on human cells and tissues has been well studied, there is currently a lack of understanding about how different levels of iron influence the abundant and diverse members of the human microbiome. This study develops a well-characterized mouse model for studying low-iron levels and identifies key groups of bacteria that are most affected. We found that the microbiome undergoes large changes when iron is removed from the diet but that many individual bacteria are able to rebound when iron levels are changed by to normal. That said, a select few members, referred to as “iron-sensitive” bacteria seem to be lost. This study begins to identify individual members of the mammalian microbiome most affected by changes in dietary iron levels.Item Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis(2019-08) Carlson, Alyssa K.; Rawle, Rachel A.; Wallace, Cameron W.; Brooks, Ellen G.; Adams, Erik; Greenwood, Mark C.; Olmer, Merissa; Lotz, Martin K.; Bothner, Brian; June, Ronald K."Objective Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. Design: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. Results: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. Conclusion: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.Item TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy(2019-06) McLoughlin, Michael R.; Orlicky, David J.; Prigge, Justin R.; Krishna, Pushya; Talago, Emily A.; Cavigli, Ian R.; Eriksson, Sofi; Miller, Colin G.; Kundert, Jean A.; Sayin, Volkan I.; Sabol, Rachel A.; Heinemann, Joshua; Brandenberger, Luke O.; Iverson, Sonya V.; Bothner, Brian; Papagiannakopoulos, Thales; Shearn, Colin T.; Arner, Elias S. J.; Schmidt, Edward E.Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr–double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.Item Virus-like Particle-Induced Protection against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function(2012-07) Rynda-Apple, Agnieszka; Dobrinen, Erin; McAlpine, Mark; Read, Amanda; Harmsen, Ann L.; Richert, Laura E.; Calverley, Matthew; Pallister, Kyler; Voyich, Jovanka M.; Wiley, James A.; Johnson, Ben; Young, Mark J.; Douglas, Trevor; Harmsen, Allen G.The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms.Item Inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses(2009-09) Wiley, James A.; Richert, Laura E.; Swain, Steve D.; Harmsen, Ann L.; Barnard, Dale L.; Randall, Troy D.; Jutila, Mark A.; Douglas, Trevor; Broomell, Chris; Young, Mark J.; Harmsen, Allen G.Background Destruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage. Methodology/Principal Findings Mice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms. Conclusions/Significance The immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.Item Something old, something new, something borrowed; how the thermoacidophilic archaeon Sulfolobus solfataricus responds to oxidative stress(2009-09) Maaty, Walid S.; Wiedenheft, Blake A.; Tarlykov, Pavel V.; Schaff, Nathan; Heinemann, Joshua V.; Robison-Cox, James; Dougherty, Amanda; Blum, Paul; Lawrence, C. Martin; Douglas, Trevor; Young, Mark J.; Bothner, BrianTo avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H2O2) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 µM H2O2. Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had significant changes in abundance. A recently characterized ferritin-like antioxidant protein, DPSL, was the most highly regulated species of mRNA and protein, in addition to being post-translationally modified. As expected, a number of antioxidant related mRNAs and proteins were differentially regulated. Three of these, DPSL, superoxide dismutase, and peroxiredoxin were shown to interact and likely form a novel supramolecular complex for mitigating oxidative damage. A scheme for the ability of this complex to perform multi-step reactions is presented. Despite the central role played by DPSL, cells maintained a lower level of protection after disruption of the dpsl gene, indicating a level of redundancy in the oxidative stress pathways of S. solfataricus. This work provides the first “omics” scale assessment of the oxidative stress response for an archeal organism and together with a network analysis using data from previous studies on bacteria and eukaryotes reveals evolutionarily conserved pathways where complex and overlapping defense mechanisms protect against oxygen toxicity.Item Marsarchaeota are an aerobic archaeal lineage abundant in geothermal iron oxide microbial mats(2018-05) Jay, Zackary J.; Beam, Jacob P.; Dlakic, Mensur; Rusch, Douglas B.; Kozubal, Mark A.; Inskeep, William P.The discovery of archaeal lineages is critical to our understanding of the universal tree of life and evolutionary history of the Earth. Geochemically diverse thermal environments in Yellowstone National Park provide unprecedented opportunities for studying archaea in habitats that may represent analogues of early Earth. Here, we report the discovery and characterization of a phylum-level archaeal lineage proposed and herein referred to as the \'Marsarchaeota\', after the red planet. The Marsarchaeota contains at least two major subgroups prevalent in acidic, microaerobic geothermal Fe(III) oxide microbial mats across a temperature range from similar to 50-80 degrees C. Metagenomics, single-cell sequencing, enrichment culturing and in situ transcriptional analyses reveal their biogeochemical role as facultative aerobic chemoorganotrophs that may also mediate the reduction of Fe(III). Phylogenomic analyses of replicate assemblies corresponding to two groups of Marsarchaeota indicate that they branch between the Crenarchaeota and all other major archaeal lineages. Transcriptomic analyses of several Fe(III) oxide mat communities reveal that these organisms were actively transcribing two different terminal oxidase complexes in situ and genes comprising an F-420-dependent butanal catabolism. The broad distribution of Marsarchaeota in geothermal, microaerobic Fe(III) oxide mats suggests that similar habitat types probably played an important role in the evolution of archaea.