Theses and Dissertations at Montana State University (MSU)
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Item Theory-based demarcation of hot spring microbial mat species from large DNA sequence datasets(Montana State University - Bozeman, College of Agriculture, 2018) Wood, Jason Michael; Chairperson, Graduate Committee: David M. Ward; Eric D. Becraft, Danny Krizanc, Frederick M. Cohan, and David M. Ward were co-authors of the article, 'Ecotype simulation 2: an improved algorithm for efficiently demarcating microbial species from large sequence datasets' submitted to the journal 'BMC bioinformatics' which is contained within this thesis.; Jason M. Wood, Frederick M. Cohan and David M. Ward were co-authors of the article, 'Biogeography of American Northwest Hot Spring A/B'-lineage Synechococcus populations' submitted to the journal 'Frontiers in microbiology' which is contained within this thesis.The identification of closely related, ecologically distinct populations within microbial communities is paramount to understanding the structure and function of these communities. Microbial systematists have long used differences in DNA sequence relatedness to categorize the observed diversity in a community of microbes without including ecological theory to identify whether or not the identified groups are ecologically distinct. Ecotype Simulation, an evolutionary simulation algorithm based on the Stable Ecotype Model of microbial species and speciation, has been used successfully to study the diversification of thermophilic A/B'-lineage Synechococcus living in the effluent channels of alkaline-siliceous hot springs in Yellowstone National Park. However, Ecotype Simulation is an extremely slow program that is unable to handle the quantity of data produced by modern DNA sequencing technologies. I introduce a new version of this algorithm, called Ecotype Simulation 2, that permits the rapid analyses of microbial diversity from very large DNA sequence datasets. Results from this new version of the Ecotype Simulation algorithm compare favorably with results from the old version, but with analyses performed much more quickly on a much greater quantity of sequences sampled. The new algorithm was used to analyze three datasets. First, the biogeography of thermophilic A/B0-lineage Synechococcus living in hot springs of the American Northwest was analyzed. Results suggested a surprising amount of endemism among springs sampled, as well as implications for adaptations to physical and chemical environmental features not seen before. Second, Ecotype Simulation 2 was used to study the history of change in Synechococcus populations, seasonally (winter to summer) and over a twenty-five year period. Results suggested changes in population abundances and distribution seasonally, but stability in population genetic structure over many years. Finally, Ecotype Simulation 2 was used to study the populations of other predominant phototrophic microbes living along temperature and depth gradients in the same microbial mat community. Results suggested that the algorithm and the Stable Ecotype Model can successfully predict ecological diversity within all predominant mat taxa. Ecotype Simulation 2 provides the means for other microbiologists to base their understanding of the communities they study on evolutionary and ecological principals.Item Comparative genomic analyses of Yellowstone hot spring microbial mat Synechococcus spp.(Montana State University - Bozeman, College of Agriculture, 2015) Olsen, Millie Helen Thornton; Chairperson, Graduate Committee: David M. Ward; Shane Nowack, Jason M. Wood, Eric D. Becraft, Kurt LaButti, Anna Lipzen, Joel Martin, Wendy S. Schackwitz, Douglas B. Rusch, Frederick M. Cohan, Donald A. Bryant and David M. Ward were co-authors of the article, 'Comparative genomics of Synechococcus isolates with different light responses and in situ diel transcription patterns of associated putative ecotypes in the Mushroom Spring microbial mat' submitted to the journal 'Frontiers in microbiology' which is contained within this thesis.The question of "What is a microbial species?" has been a highly debated issue in the field of microbiology. Many have accepted a molecular species demarcation approach, that any two organisms with a high enough 16S rRNA sequence similarity are members of the same species. However, the Ward lab has shown that there are many ecologically distinct Synechococcus spp. inhabiting hot springs of the Lower Geyser Basin in Yellowstone National Park, WY, that would be defined as members of the same species using the molecular demarcation approach. Using a theory-based species demarcation approach with a conserved photosystem gene (psaA), evidence of the existence of putative ecotypes, or predicted ecologically distinct species, has been found in the microbial mat, distributed along both temperature and light gradients. Isolates representative of these ecologically distinct populations have also been shown to have distinct temperature adaptations and light adaptations. I obtained the genomes of these isolates, which include representatives of populations with different temperature distributions and different vertical distributions, and replicate isolates within individual putative ecotypes. Using these genome sequences, I compared the psaA gene phylogeny and multi-locus sequence phylogenies with a phylogeny created using genes shared among the genomes to explore the effects of recombination on phylogenies of closely-related organisms. I then explored the underlying genetic mechanisms of the niche adaptations of these ecotypes by (i) comparing the isolate gene content, diel transcription patterns, and positive selection evidence of putative ecotypes with different vertical distributions in the mat and different light adaptations, (ii) comparing the gene content and evidence of positive selection among isolates representative of populations with different temperature distributions, and (iii) comparing the gene content and evidence of positive selection among replicate isolates within individual putative ecotypes. I found that, while recombination may have caused the inaccurate demarcation of genetically distinct isolates into a single PE, there is genomic evidence that species of Synechococcus that are ecologically distinct from one another exist, along both temperature and vertical gradients. Members of a species are ecologically homogenous, though there is evidence of some genetic heterogeneity within a species.Item Geobiological interactions of archaeal populations in acidic and alkaline geothermal springs of Yellowstone National Park, WY, USA(Montana State University - Bozeman, College of Agriculture, 2015) Beam, Jacob Preston; Chairperson, Graduate Committee: William P. Inskeep; Zackary J. Jay, Mark A. Kozubal and William P. Inskeep were co-authors of the article, 'Niche specialization of novel thaumarchaeota to oxic and hypoxic acidic geothermal springs in Yellowstone National Park' in the journal 'The International Society for Microbial Ecology journal' which is contained within this thesis.; Hans C. Bernstein, Zackary J. Jay, Mark A. Kozubal, Ryan deM. Jennings, Susannah G. Tringe and William P. Inskeep were co-authors of the article, 'Assembly and succession of iron oxide microbial mat communities in acidic geothermal springs' submitted to the journal 'Geobiology' which is contained within this thesis.; Zackary J. Jay, Markus C. Schmid, Margaret F. Romine, Douglas B. Rusch, Ryan deM. Jennings, Mark A. Kozubal, Susannah G. Tringe, Michael Wagner and William P. Inskeep were co-authors of the article, 'In situ ecophysiology of an uncultured lineage of aigarchaeota from an oxic hot spring filamentous 'streamer' community' in the journal 'International Society for Microbial Ecology journal' which is contained within this thesis.Microbial communities in high-temperature acidic and alkaline geothermal springs contain abundant, novel Archaea whose role in biogeochemical cycling and community function in microbial mats is not described. This thesis utilized a complementary suite of analyses that included aqueous and solid phase geochemistry, community genomics, phylogenomics, targeted 16S rRNA gene sequencing, community transcriptomics, and microscopy to elucidate the role of novel archaeal populations in acidic sulfur and iron rich hot springs in Norris Geyser Basin, Yellowstone National Park (YNP), and alkaline microbial 'streamer' communities in Lower Geyser Basin, YNP. Novel members of the archaeal phylum, Thaumarchaeota were identified in oxic iron oxide mats and hypoxic elemental sulfur sediments in acidic geothermal springs. These two different groups of Thaumarchaeota likely utilize organic carbon as electron donors and exhibited metabolic capacities based on the presence and absence of oxygen (e.g., heme copper oxidases). The assembly and succession of iron oxide mats in acidic geothermal springs showed later colonization (> 40 d) of Thaumarchaeota. Early colonizers (< 7 d) of Fe(III)-oxide mats include Hydrogenobaculum spp. (Aquificales) and the iron-oxidizing Metallosphaera yellowstonensis (7 - 14 d), which accrete copious amounts of Fe(III)-oxides. Interaction of Hydrogenobaculum and M. yellowstonensis is important to mat formation and subsequent later colonization of heterotrophic archaea (> 40 d). The succession of these communities follows a repeatable pattern, which exhibits interplay among oxygen flux, hydrodynamics, and microbial growth. The biogeochemical and micromorphological signatures may be important for the interpretation of ancient Fe(III)-oxide geothermal deposits. Interactions between Archaea and Aquificales are also important in oxic, alkaline 'streamer' communities, which contain a novel Aigarchaeota population and Thermocrinis spp. This Aigarchaeota population (Candidatus "Calditenuis aerorheumensis") exhibits a filamentous morphology and was intricately associated with Thermocrinis spp. C. aerorheumensis is an aerobic chemoorganotroph. Oxygen is the predominant electron acceptor of C. aerorheumensis, and mRNA transcripts were elevated for heme copper oxidase complexes. Organic carbon electron donors may come from bacteria in close proximity and/or dissolved organic carbon. Archaeal interactions with Aquificales contribute to higher-order level properties (e.g., biomineralization, metabolite sharing) that are important in the formation of hot spring microbial mats and streamer communities.Item The distribution of cultivated and uncultivated cyanobacteria and green non-sulfur bacteria in hot spring microbial mats(Montana State University - Bozeman, College of Agriculture, 1993) Ruff-Roberts, Alyson L.Item Molecular analysis of hot spring microbial mats to study bacterial diversity and physiology(Montana State University - Bozeman, College of Agriculture, 1996) Nold, Stephen CharlesItem Fermentation and anaerobic decomposition in a hot spring microbial mat(Montana State University - Bozeman, College of Agriculture, 1984) Anderson, Karen LeighItem Glycolate production and consumption in a hot spring cyanobacterial mat(Montana State University - Bozeman, College of Agriculture, 1985) Bateson, Mary MargaretItem Identification and distributions of dominant bacterial populations in hot spring Synechococcus mats(Montana State University - Bozeman, College of Agriculture, 1997) Ferris, Michael JosephItem The fate of fermentation products and glycollate in hot spring microbial mats with emphasis on the role played by Chloroflexus aurantiacus(Montana State University - Bozeman, College of Agriculture, 1983) Tayne, Timothy AldenItem Geomicrobiology of iron oxyhydroxide mats in acidic geothermal springs of Yellowstone National Park, Wyoming, United States of America(Montana State University - Bozeman, College of Agriculture, 2010) Kozubal, Mark Andrew; Chairperson, Graduate Committee: Williams P. Inskeep.The microbial community structure and function in acidic, high-temperature ironoxidizing geothermal springs of Yellowstone National Park was investigated utilizing a variety of complementary approaches including microbial cultivation and characterization, geochemical analysis of aqueous and solid phases, phylogenetic and functional gene analysis, comparative genomics, and protein sequence modeling. Cultivation efforts resulted in the isolation of an Fe(II)-oxidizing chemolithotroph Metallosphaera yellowstonii MK1 T. The distribution and relative abundance of MK1-like 16S rRNA gene sequences was evaluated in 14 acidic geothermal springs containing Fe(III)-oxide microbial mats. Highly related MK1-like sequences (>99% sequence similarity) were consistently observed in Fe(III)-oxide mats across a temperature range of 55 to 80 °C. Quantitative PCR confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community of selected sites. Four additional isolates were obtained from thermophilic Fe(III) microbial mats including Sulfobacillus sp. MK2, Sulfolobus sp. MK3, Acidicaldus sp. MK5 and Crenarchaeota sp. MK4, which represents a new taxonomical lineage at the class level or higher. A draft genome has been assembled for M. yellowstonii strain MK1 and comparative studies indicate significant similarity to Metallosphaera sedula in regards to putative genes involved in iron and sulfur oxidation, carbon fixation, oxygen reduction and heavy metal resistance. Analysis of gene sequences reveal 7 heme copper oxidases (subunit I) and a variety of genes with possible importance in Fe(II) oxidation including the foxA-J gene cluster, a cbsA cytochrome b 558/566, and a novel sequence coding for a putative blue multi-copper protein (mco). Expression screens and reverse transcriptaseqPCR on samples from three ASC environments and in cultures grown autotrophically show that the fox gene cluster and mco are important when Fe(II) serves as the electron donor. Protein sequence analysis of foxC indicates a novel lysine-lysine or lysine arginine heme b binding domain and is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco indicates a novel multicopper blue protein with two plastocyanin type I copper domains with only three homologous sequences found in Genbank. Both putative proteins likely play an important role in electron transport from Fe(II) to oxygen through Fox Proteins.