Chairperson, Graduate Committee: Williams P. Inskeep.Kozubal, Mark Andrew2013-06-252013-06-252010https://scholarworks.montana.edu/handle/1/1668The 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.enOxidationMicrobial matsGeneticsDissertationCopyright 2010 by Mark Andrew Kozubal