Publications by Colleges and Departments (MSU - Bozeman)
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Item Characterization of an Endophytic Gloeosporium sp. and Its Novel Bioactivity with “Synergistans”(2014-12) Schaible, George A.; Strobel, Gary A.; Mends, Morgan Tess; Geary, Brad; Sears, JoeGloeosporium sp. (OR-10) was isolated as an endophyte of Tsuga heterophylla (Western hemlock). Both ITS and 18S sequence analyses indicated that the organism best fits either Hypocrea spp. or Trichoderma spp., but neither of these organisms possess conidiophores associated with acervuli, in which case the endophytic isolate OR-10 does. Therefore, the preferred taxonomic assignment was primarily based on the morphological features of the organism as one belonging to the genus Gloeosporium sp. These taxonomic observations clearly point out that limited ITS and 18S sequence information can be misleading when solely used in making taxonomic assignments. The volatile phase of this endophyte was active against a number of plant pathogenic fungi including Phytophthora palmivora, Rhizoctonia solani, Ceratocystis ulmi, Botrytis cinerea, and Verticillium dahliae. Among several terpenes and furans, the most abundantly produced compound in the volatile phase was 6-pentyl-2H-pyran-2-one, a compound possessing antimicrobial activities. When used in conjunction with microliter amounts of any in a series of esters or isobutyric acid, an enhanced inhibitory response occurred with each test fungus that was greater than that exhibited by Gloeosporium sp. or the compounds tested individually. Compounds behaving in this manner are hereby designated “synergistans.” An expression of the “median synergistic effect,” under prescribed conditions, has been termed the mSE50. This value describes the amount of a potential synergistan that is required to yield an additional median 50 % inhibition of a target organism. In this report, the mSE50s are reported for a series of esters and isobutyric acid. The results indicated that isoamyl acetate, allyl acetate, and isobutyric acid generally possessed the lowest mSE50 values. The value and potential importance of these microbial synergistic effects to the microbial environment are also discussed.Item Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing(2014-04) Beliaev, Alexander S.; Romine, Margaret F.; Serres, Margrethe; Bernstein, Hans C.; Linggi, Bryan E.; Markillie, Lye M.; Isern, Nancy G.; Chrisler, William B.; Kucek, Leo A.; Hill, Eric A.; Pinchuk, Grigoriy E.; Bryant, Donald A.; Wiley, H. Steven; Fredrickson, Jim K.; Konopka, AllanWe used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph–heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic–heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.Item IL-1alpha signaling is critical for leukocyte recruitment after pulmonary Aspergillus fumigatus challenge(2015-01) Caffrey, Alayna K.; Lehmann, Margaret M.; Zickovich, Julianne M.; Espinosa, Vanessa; Shepardson, Kelly M.; Watschke, Christopher P.; Hilmer, Kimberly M.; Thammahong, Arsa; Barker, Bridget M.; Rivera, Amariliz; Cramer, Robert A.; Obar, Joshua J.Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine lung.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.Item Polar and alpine microbiology in a changing world(2014-08) Priscu, John C.; Laybourn-Parry, Johanna; Häggblom, MaxHigh altitude and high latitude regions on Earth are experiencing rapid changes in climate. Ecological impacts resulting from these changes are now being observed at all ecosystem levels and larger deviations and more significant impacts are anticipated in the future. Satellite data show dramatic reductions in the extent and thickness of sea ice at both poles, and rising temperatures are causing alpine glaciers worldwide to shrink in area and volume. By virtue of their relatively rapid growth rates and metabolic diversity, we can expect microorganisms to be the first responders to fluctuating climatic conditions. Because microorganisms are keystone players in elemental transformations, variations in their abundance and diversity will initiate a cascade of impacts throughout entire ecosystems. Clearly, knowledge of the distribution, biodiversity and functional roles of microorganisms inhabiting polar and alpine environments is essential to our under-standing of ecosystem processes in a changing climate.Item B cells modulate systemic responses to Pneumocystis lung infection and protect on-demand hematopoiesis via T cell-independent, innate mechanism when type-I-IFN-signaling is absent(2015-02) Hoyt, T. R.; Kochetkova, I.; Dobrinen, E.; Meissner, NicoleHIV infection results in a complex immunodeficiency due to loss of CD4+ T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag−/−) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR−/−) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag−/− mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag−/− mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4+ T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.Item [FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation(2014-11) Peters, John W.; Schut, Gerrit J.; Boyd, Eric S.; Mulder, David W.; Shepard, Eric M.; Broderick, Joan B.; King, Paul W.; Adams, Michael W. W.The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.Item Stable isotopes track biogeochemical processes under seasonal ice cover in a shallow, productive lake(Springer, 2014) Gammons, Christopher H; Henne, William; Poulson, Stephen; Parker, Stephen R.; Johnston, Tyler B.; Dore, John E.; Boyd, Eric S.Biogeochemical dynamics under seasonal ice cover were investigated in the shallow (<10 m) water column of highly productive Georgetown Lake, western Montana, USA. This high altitude (1,800 m) reservoir is well-mixed in summer, but becomes strongly stratified under ice cover (mid-November–mid-May). A rapid drop in dissolved oxygen (DO) concentration and rise in dissolved inorganic carbon (DIC) concentration was observed after the onset of ice, with a corresponding increase in δ18O-DO and decrease in δ13C-DIC, likely caused by respiration (R) of organic carbon. Photosynthesis/respiration ratios (P/R) estimated from simultaneous measurement of DO and δ18O-DO were near unity prior to ice formation but then systematically decreased with time and depth in the lake under ice cover. P/R in the water column was higher at a shallower monitoring site compared to a deeper site near the dam outlet, which may have been important for over-winter survival of salmonids. By March, the bottom 3 m of the water column at both sites was anoxic, with the bottom 1 m being euxinic. Elevated concentrations of dissolved sulfide, ammonium, phosphate, Fe2+, and Mn2+ in deep water suggest coupling of organic carbon degradation with reduction of a number of electron acceptors (e.g., Fe3+,NO3-, SO24-). The concentrations and δ34S values of H2S in the deep water and SO2i in the shallow water were similar, indicating near-complete reduction of sulfate in the euxinic zone. Late in the winter, an influx of isotopically heavy DIC was noted in the deep water coincident with a buildup of dissolved CH4 to concentrations >1 mM. These trends are attributed to acetoclastic methanogenesis in the benthic sediments. This pool of dissolved CH4 was likely released from the lake to the atmosphere during spring ice-off and lake turnover.Item Bile Salts Affect Expression of Escherichia coli O157:H7 Genes for Virulence and IronAcquisition, and Promote Growth under Iron Limiting Conditions(2013-09) Hamner, Steve; McInnerney, Kathleen; Williamson, Kerry S.; Franklin, Michael J.; Ford, Tim E.Bile salts exhibit potent antibacterial properties, acting as detergents to disrupt cell membranes and as DNA-damaging agents. Although bacteria inhabiting the intestinal tract are able to resist bile’s antimicrobial effects, relatively little is known about how bile influences virulence of enteric pathogens. Escherichia coli O157:H7 is an important pathogen of humans, capable of causing severe diarrhea and more serious sequelae. In this study, the transcriptome response of E. coli O157:H7 to bile was determined. Bile exposure induced significant changes in mRNA levels of genes related to virulence potential, including a reduction of mRNA for the 41 genes making up the locus of enterocyte effacement (LEE) pathogenicity island. Bile treatment had an unusual effect on mRNA levels for the entire flagella-chemotaxis regulon, resulting in two- to four-fold increases in mRNA levels for genes associated with the flagella hook-basal body structure, but a two-fold decrease for “late” flagella genes associated with the flagella filament, stator motor, and chemotaxis. Bile salts also caused increased mRNA levels for seventeen genes associated with iron scavenging and metabolism, and counteracted the inhibitory effect of the iron chelating agent 2,2’-dipyridyl on growth of E. coli O157:H7. These findings suggest that E. coli O157:H7 may use bile as an environmental signal to adapt to changing conditions associated with the small intestine, including adaptation to an iron-scarce environment.Item Complete genome of Ignavibacterium album, a metabolically versatile, flagellated, facultative anaerobe from the phylum Chlorobi(2012-05) Liu, Zhenhua; Frigaard, N. U.; Vogl, K.; Iino, T.; Ohkuma, M.; Overmann, J.; Bryant, Donald A.Prior to the recent discovery of Ignavibacterium album (I. album), anaerobic photoautotrophic green sulfur bacteria (GSB) were the only members of the bacterial phylum Chlorobi that had been grown axenically. In contrast to GSB, sequence analysis of the 3.7-Mbp genome of I. album shows that this recently described member of the phylum Chlorobi is a chemoheterotroph with a versatile metabolism. I. album lacks genes for photosynthesis and sulfur oxidation but has a full set of genes for flagella and chemotaxis. The occurrence of genes for multiple electron transfer complexes suggests that I. album is capable of organoheterotrophy under both oxic and anoxic conditions. The occurrence of genes encoding enzymes for CO2 fixation as well as other enzymes of the reductive TCA cycle suggests that mixotrophy may be possible under certain growth conditions. However, known biosynthetic pathways for several amino acids are incomplete; this suggests that I. album is dependent upon on exogenous sources of these metabolites or employs novel biosynthetic pathways. Comparisons of I. album and other members of the phylum Chlorobi suggest that the physiology of the ancestors of this phylum might have been quite different from that of modern GSB.