Browsing by Subject "Archaea"
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Item Archaea at the El Tatio Geyser Field : community composition, diversity, and distribution across hydrothermal features and geochemical gradients(2012-05) Franks, Megan A.; Bennett, Philip C. (Philip Charles), 1959-; Bell, Christopher J.; Cardenas, Meinhard B.; Engel, Annette S.; Hawkes, Christine V.Methanogenesis, a metabolic pathway unique to Archaea, is severely inhibited by the reduced form of arsenic (As). Despite this inhibition, methanogenic Archaea are present in some hydrothermal features at the El Tatio Geyser Field (ETGF), a high-arsenic site with 100+ hydrothermal features, including boiling pools, geyers, fumaroles, and springs. The ability of methanogenic Archaea and other microorganisms to withstand elevated arsenic concentrations, and a variety of other extreme environmental conditions at ETGF, may be due to unique adaptations or syntrophic relationships with other microorganisms. ETGF is situated in the Andes Mountains at an altitude of ~4300 meters. UV radiation is elevated in this region and air temperatures fluctuate widely. Most hydrothermal waters discharge at ~85˚C, the local boiling point, and rapidly evaporate due to the arid climate. This concentrates hydrothermal salts and metals, including arsenic (As) and antimony (Sb). Additionally, dissolved inorganic carbon (DIC) concentrations are extremely low in most features and may limit life. Water chemistry analyses done for this study show variability in dissolved constituents between features that are consistent over time. Variations may be due to the source or residence time of waters, and differences in chemistry could be responsible for the presence or absence of methanogenic Archaea at hydrothermal sites. The overlying control on microbial diversity and community composition may be water geochemistry, and potentially specific constituents. The goals of this study were to detect novel microbial taxa at ETGF, including novel methanogens, as well as to document microbial community composition at select hydrothermal features. The distribution and diversity of microorganisms at each feature was analyzed phylogenetically and within an ecological context in order to determine physicochemical and biological controls on community composition. Additionally, a model methanogen was used in laboratory analyses to determine how concentrations and oxidation states affected growth and methane production. This methanogen, Methanothermobacter thermautotrophicus, is found at ETGF, Yellowstone, and other hydrothermal fields, and thrives in high-temperature environments. MPN (most probable number) analyses show that culturable biomass from multiple sites contain metabolically active methanogens. These results support the biogenicity of dissolved methane detected in the field. 16S rRNA surveys of Archaea at four sites show that Archaea are diverse, and archaeal community composition varies across features. Phylogenetic tree construction indicates that Archaea from ETGF group together, suggesting that the isolation and broad environmental constrains on ETGF have some control on phylogenetic diversity. Laboratory analyses of As and Sb concentrations on M. thermautotrophicus suggest that Sb may decrease the inhibition of methanogenesis by As by preventing the formation of As(III) from As(V). Statistical analyses correlating microbial community composition and structure to physicochemical parameters show that archaeal and bacterial communities relate to different variables; with Bacteria correlating to water temperature, and Archaea correlating to dissolved constituents such as hydrogen gas and sulfate.Item Coenzyme B, amino acid, and iron-sulfur cluster biosynthesis in methanogenic archaea(2009-08) Drevland, Randy Michael; Graham, David E.Methane is a greenhouse gas and a major contributor to climate change. Methanogenic Archaea produce more than 1 billion tons of this gas each year through methanogenesis, the anaerobic reduction of CO₂ to methane. Coenzyme B (CoB) is one of eight coenzymes required for methanogenesis and it is unique to methanogens. Therefore, this coenzyme is a potential target for inhibiting methanogenesis. To further elucidate the CoB biosynthetic pathway, genes from Methanocaldococcus jannaschii were cloned and expressed in an effort to identify the CoB homoaconitase. From this study, the MJ0499-MJ1277 pair of proteins was identified as the methanogen isopropylmalate isomerase involved in leucine and isoleucine biosynthesis. The MJ1003-MJ1271 pair of proteins was characterized as the homoaconitase required for CoB biosynthesis. This enzyme exhibited broad substrate specificity, catalyzing the isomerization of cis-unsaturated tri-carboxylates with [gamma]-chains of 1-5 methylenes in length. Previously characterized homoaconitases only catalyzed half of the predicted reactions in the isomerization of homocitrate. The MJ1003-MJ1271 proteins function as the first homoaconitase described to catalyze the full isomerization of homocitrate to homoisocitrate. Also, the CoB homoaconitase was identified as specific for (R)-homocitrate and cis-unsaturated intermediates, contrary to a previous study that suggested the substrate specificity of this enzyme included (S)-homocitrate and trans-homoaconitate. The M. jannaschii isopropylmalate isomerase and homoaconitase share more than 50% sequence identity and catalyze analogous reactions. Site directed mutagenesis of the MJ1271 protein was used to identify residues involved in substrate specificity. Arg26 of MJ1271 was critical for the specificity of the CoB homoaconitase. Mutation of this residue to the analogous residue in the M. jannaschii isopropylmalate isomerase, Val28, altered the substrate specificity of the homoaconitase to include the substrates of isopropylmalate isomerase. These homologs of aconitase require a [4Fe-4S] cluster for coordinating their respective substrates at the enzyme active site. However, methanogens lack most of the proteins required for iron-sulfur cluster assembly. Therefore, genes homologous to the Salmonella enterica ApbC iron-sulfur scaffold protein were characterized from methanogens. The MMP0704, MJ0283, and SSO0460 proteins from Methanococcus maripaludis, M. jannaschii, and Solfolobus solfataricus, respectively, were identified as scaffold proteins involved in methanogen iron-sulfur cluster biosynthesis.