Generalitat Valenciana (GV-1170793); CICYT (PB95-0695)

我們研究了極端嗜熱古菌Pyrococcus horikoshii 的嗜熱性酰基磷酸酶acylphosphatase (PhAcP) ，以及與它同源的人類嗜溫性酶(HuCTAcP) 的熱穩定性。我們發現PhAcP的熱穩定性之所以比HuCTAcP高出很多，是由於熔融溫度的焓變值的增加以及變性熱容量的減少。研究蛋白質熱穩定性的其中一個推動力，是運用我們的知識去製造耐高溫的酶，這對工業和生物技術非常重要。通過交換 PhAcP的嗜熱核和 HuCTAcP的嗜溫核以及研究變種的熱穩定性，我們認為蛋白表面是改善熱穩定性工程的首選地區。嗜熱和嗜溫蛋白質之間的主要區別，在於嗜熱蛋白質有更多的表面鹽橋。為了探討表面鹽橋對蛋白熱穩定性的貢獻，我們採用雙突變循環，量化嗜熱蛋白T.celer L30e一表面鹽橋的相互作用能。我們的結果顯示，表面鹽橋對蛋白質穩定性的貢獻是獨立於溫度變化的。此外，表面鹽橋對蛋白質變性熱容量的減少起一定作用。 / We characterized the thermodynamic properties of thermophilic acylphosphatase from Pyrococcus horikoshii (PhAcP) and its mesophilic homologue from human (HuCTAcP) and found that the much higher thermostability of PhAcP was the result of increased enthalpy change at melting temperature and decreased heat capacity change of unfolding. One incentive to study protein thermostability is to apply our knowledge to engineer thermostable enzyme which is of great industrial and biotechnological importance. Through swapping the core of thermophilic PhAcP and mesophilic HuCTAcP and characterizing the thermostability of the resulting variants, we concluded that surface is a preferred region for thermostability engineering. The key difference between thermophilic and mesophilic proteins lies in the surface on which thermophilic proteins have more salt-bridges. To investigate the contribution of surface salt-bridge to protein thermostability, we employed double-mutant cycle to quantify the pair-wise interaction energy of a surface salt-bridge in thermophilic T.celer L30e. Our results showed that surface salt-bridge had a temperature independent contribution to the protein stability and plays a role in the reduction of the heat capacity change of unfolding. / Detailed summary in vernacular field only. / Yu, Tsz Ha. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 89-93). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Content --- p.iv / List of Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.ix / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- Definition of protein stability --- p.1 / Chapter 1.2 --- Contribution to thermostability from the protein core --- p.2 / Chapter 1.2.1 --- Definition of hydrophobic effect --- p.2 / Chapter 1.2.2 --- Why the hydrophobic effect has been recognized as the major driving force for protein folding? --- p.2 / Chapter 1.3 --- Contribution to thermostability from the protein surface --- p.6 / Chapter 1.3.1 --- Electrostatic interactions --- p.7 / Chapter 1.3.2 --- Exposed hydrogen bonds and helix propensity --- p.10 / Chapter 1.3.3 --- Surface loop --- p.11 / Chapter 1.4 --- Protein stability curve --- p.13 / Chapter 1.5 --- The incentive to study protein thermostability --- p.17 / Chapter Chapter 2: --- Materials and Methods --- p.18 / Chapter 2.1 --- Generation of DNA clones --- p.18 / Chapter 2.2 --- Plasmid transformation to competent E. coli strain --- p.18 / Chapter 2.3 --- Expression of recombinant proteins --- p.19 / Chapter 2.3.1 --- T. celer L30e --- p.19 / Chapter 2.3.2 --- Acylphosphatase --- p.20 / Chapter 2.4 --- Protein extraction from E. coli by sonication --- p.20 / Chapter 2.5 --- Protein purification --- p.20 / Chapter 2.5.1 --- T. celer L30e --- p.20 / Chapter 2.5.2 --- Acylphosphatase --- p.22 / Chapter 2.6 --- Circular dichroism experiment --- p.22 / Chapter 2.6.1 --- Thermal denaturation --- p.22 / Chapter 2.6.2 --- Denaturant-induced denaturation --- p.23 / Chapter 2.7 --- Differential scanning calorimetry --- p.24 / Chapter 2.8 --- Enzymatic assay of AcPs using benzoyl phosphate as substrate --- p.25 / Chapter 2.9 --- Crystallization and crystal structure refinement --- p.26 / Chapter Chapter 3: --- Thermodynamic characterization of thermophilic acylphosphatase from Pyrococcus horikoshii and its mesophilic homologue from human --- p.27 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.2 --- Result --- p.31 / Chapter 3.2.1 --- PhAcP has a higher thermostability than HuCTAcP --- p.31 / Chapter 3.2.2 --- PhAcP has an upshifted and broadened PSC compared with the PSC of HuCTAcP --- p.33 / Chapter 3.2.3 --- PhAcP has a highly enhanced ΔH[subscript m] and slightly reduced ΔC[subscript p]. --- p.37 / Chapter 3.3 --- Discussion --- p.41 / Chapter 3.3.1 --- Thermophilic AcPs harness enhanced ΔH[subscript m] and reduced ΔC[subscript p] to attain a higher thermostability. --- p.41 / Chapter 3.3.2 --- Possible structural differences between PhAcP and HuCTAcP that lead to the higher thermostability of PhAcP. --- p.42 / Chapter Chapter 4: --- Protein surface is a preferred region for thermostability engineering --- p.47 / Chapter 4.1 --- Introduction --- p.47 / Chapter 4.2 --- Results --- p.51 / Chapter 4.2.1 --- Construction of the chimera with Thermophilic Surface and Mesophilic Core (T[subscript surf]M[subscript core]), and the chimera with Mesophilic Surface and Thermophilic Core (M[subscript surf]T[subscript core]). --- p.51 / Chapter 4.2.2 --- The crystal structures of the chimera T[subscript surf]M[subscript core] and M[subscriptsurf]T[subscript core] reveal that anticipated interactions are engineered. --- p.54 / Chapter 4.2.3 --- Characterization of the thermodynamic stabilities of the chimeras at different temperatures --- p.56 / Chapter 4.3 --- Discussion --- p.59 / Chapter 4.3.1 --- Engineering a thermophilic surface onto a mesophilic protein enhances thermostability --- p.59 / Chapter 4.3.2 --- Concluding remarks --- p.64 / Chapter 4.4 --- Supplementary Tables --- p.64 / Chapter Chapter 5: --- Stabilizing surface salt-bridge enhances protein thermostability by upshifting the protein stability curve --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.2 --- Results --- p.70 / Chapter 5.2.1 --- Design of variants --- p.70 / Chapter 5.2.2 --- Determination of the pair-wise interaction energy of K46 and E62 by double-mutant cycles --- p.72 / Chapter 5.2.3 --- Surface salt-bridge K46/E62 is stabilizing and its interaction energy is insensitive to temperature changes --- p.75 / Chapter 5.2.4 --- Stabilizing salt-bridge K46/E62 reduces ΔC[subscript p] and upshifts protein stability curve --- p.77 / Chapter 5.3 --- Discussion --- p.80 / Chapter 5.3.1 --- Stabilization effect brought by surface salt-bridge is insensitive to temperature change --- p.80 / Chapter 5.3.2 --- The pair-wise interaction energy of K46-E62 determined by DMC reflects their electrostatic interaction --- p.80 / Chapter 5.3.3 --- Surface salt-bridge contributes to the reduction of ΔC[subscript p] in thermophilic proteins --- p.81 / Chapter 5.3.4 --- Reduced ΔC[subscript p] upshifts and broadens the PSC resulting a higher T[subscript m] --- p.83 / Chapter 5.4 --- Supplementary Figures and Tables --- p.85 / Chapter Appendix --- List of Publications --- p.88 / References --- p.89

L'explosion des données de séquençage a permis de résoudre la plupart des relations phylogénétiques chez les archées. Néanmoins, de nombreuses questions restent à résoudre à l'échelle du domaine des archées et à l'échelle des trois domaines du vivant. Parmi elles, les relations phylogénétiques au sein du cluster II, notamment la position des archées halophiles extrêmes qui ont été placées à différentes positions dans l'arbre en fonction des marqueurs et des modèles de reconstruction utilisés, ainsi que la position de la racine des archées et la position des eucaryotes à la lumière des lignées d'archées nouvellement séquencées. Au cours de ma thèse, j'ai contribué à (i) affiner la phylogénie du domaine des Archaea en se concentrant sur les relations phylogénétiques au sein du cluster II, en particulier les positions des lignées halophiles extrêmes par rapport aux méthanogènes à travers des analyses dédiées à cette partie spécifique de l'arbre, et (ii) établir une phylogénie globale des archées afin de comprendre leur histoire évolutive ancienne et leur lien avec les eucaryotes à travers une analyse phylogénomique en deux étapes à l'échelle des trois domaines du vivant. D'abord, en utilisant des approches de génomique comparée sur 155 génomes complets appartenant aux Halobacteria, Nanohaloarchaea, méthanogènes de classe II, Archaeoglobales et Diaforarchaea, j'ai identifié 258 protéines portant un signal phylogénétique fiable pour étudier les relations de parente au sein du cluster II. En combinant différentes approches limitant l'impact du signal non phylogénétique sur l'inférence phylogénétique (comme la méthode Slow-Fast et le recodage des acides aminés), j'ai montré que les Nanohaloarchaea branchent avec les Methanocellales et les Halobacteria branchent avec les Methanomicrobiales. Ce jeu de données a ensuite été utilisé pour étudier la position d'une troisième lignée halophile extrême, les Methanonatronarchaeia, qui se positionnent entre les Archaeoglobales et les Diaforarchaea. Ces résultats suggèrent que l'adaptation à la salinité extrême serait apparue au moins trois fois de manière indépendante chez les archées et que les similitudes phénotypiques observées chez les Nanohaloarchaea, Halobacteria et Methanonatronarchaeia résulteraient d'une convergence évolutive, éventuellement accompagnée de transferts de gènes horizontaux. Enfin, ces résultats suggèrent que le groupement basal des Nanohaloarchaea avec d'autres lignées des DPANN serait la conséquence d'un artefact de reconstruction. Pour la deuxième partie de ma thèse, j'ai appliqué une stratégie consistant à analyser séparément les trois domaines du vivant considérés deux à deux, en mettant à jour 72 familles protéiques précédemment identifiées par Raymann et ses collègues (2015) pour inclure toutes les nouvelles lignées d'archées séquencées depuis la publication de cette étude comme les Asgard, les DPANN, les Stygia, les Acherontia, etc. Au total, mon échantillonnage taxonomique comprend 435 archées, 18 eucaryotes et 67 bactéries. Les résultats des analyses par la méthode Slow-Fast soutiennent une racine des Archaea située entre le superphylum basal des DPANN et le reste des archées séparées en deux groupes monophylétiques : les cluster I et cluster II, comme décrits par Raymann et ses collègues (2015), et montrent que la monophylie des Euryarchaeota est liée aux positions évoluant vite. Mes résultats placent les eucaryotes en tant que groupe frère du superphylum des TACK et montrent que leur regroupement avec les Asgard est lié aux positions évoluant vite. Ces résultats ont des implications majeures sur les inférences de la nature du dernier ancêtre commun des archées et sur l'histoire évolutive de ce domaine qui a conduit à l'apparition de la première cellule eucaryote / The burst of sequencing data has helped disentangling most of the phylogenetic relationships in Archaea. Nevertheless, many questions remain to be addressed both at the level of the archaeal domain and at the level of the three domains of life. Among them, the phylogenetic relationships inside the cluster II, in particular the position of extreme halophilic archaeal lineages relatively to the methanogens which have been placed at different positions in the tree based on the different markers and reconstruction models used, as well as the position of the root of the Archaea and the position of the eukaryotes in the light of the newly sequenced archaeal lineages. During my thesis, I have contributed to (i) refine the phylogeny of the archaeal domain by focusing on the phylogenetic relationships among the cluster II Archaea, in particular the positions of the extreme halophilic lineages through dedicated analyses focusing on this specific part of the archaeal tree, and (ii) establish a global phylogeny of the Archaea to understand their early evolutionary history and their link with the eukaryotes through a large-scale two-step phylogenomic analysis at the level of the three domains of life. First, using comparative genomics approaches on 155 complete genomes belonging to the Halobacteria, Nanohaloarchaea, methanogens class II, Archaeoglobales, and Diaforarchaea, I have identified 258 proteins carrying a reliable phylogenetic signal to investigate the position of the extreme halophilic lineages in Archaea. By combining different approaches limiting the impact of non-phylogenetic signal on phylogenetic inference (like the Slow Fast method and the recoding of amino acids), I showed that the Nanohaloarchaea branch with Methanocellales, and Halobacteria branch with Methanomicrobiales. This dataset has been subsequently used to investigate the position of a third extreme halophilic lineage, the Methanonatronarchaeia, which I showed to branch in between the Archaeoglobales and Diaforarchaea. These results suggest that adaption to high salinity emerged at least three times independently in Archaea, and that the phenotypic similarities observed in Nanohaloarchaea, Halobacteria, and Methanonatronarchaeia likely result from convergent evolution, possibly accompanied by horizontal gene transfers. Finally, these results suggest that the basal grouping of Nanohaloarchaea with other DPANN lineages is likely the consequence of a tree reconstruction artefact. For the second part of my thesis, I have applied a strategy consisting in separately analyzing the three domains of life two by two, by updating 72 protein families previously identified by Raymann and colleagues (2015) to include all novel archaeal lineages that were sequenced since the publication of this study like the Asgard, the DPANN, the Stygia, the Acherontia, etc. In total, my taxonomic sampling includes 435 archaea, 18 eukaryotes, and 67 bacteria. The results of the Slow-Fast method supported a root of the Archaea lying between a basal DPANN superphylum and the rest of the Archaea separated into two monophyletic groups: the cluster I and cluster II as described by Raymann and colleagues (2015), and showed that the monophyly of the Euryarchaeota is supported only by the fast-evolving sites. My results also placed the eukaryotes as the sister group to the TACK superphylum and showed that their sister grouping with the Asgard is linked to the fast-evolving sites. These results have major implications on the inferences of the nature of the last common archaeal ancestor and the subsequent evolutionary history of this domain that led to the rise of the first eukaryotic cell

Only a few studies have looked at microbial biogeography in soils and whether microorganisms are endemic to an area is still debatable. Tramway Ridge, a geothermal area on Mount Erebus, Antarctica, provides a unique opportunity due to its isolation and extreme conditions to explore the possibilities of microbial endemism and to identify novel Bacteria and Archaea. This site was chosen for a culture-independent study with a preliminary culturing survey for bacterial communities along three temperature gradients (65 C - 2.5'C). In addition, a physico-chemical analysis was undertaken to identify which environmental factors were driving the different diversity along the transects. An automated rRNA intergenic spacer analysis (ARISA) was used to assess the diversity across the transects using Bacteria and Cyanobacteria-specific primers and results showed that temperature and pH were the main drivers for these communities. Due to its unique physico-chemical and ARISA profile, a hot temperature site (T-3A, 65'C) was chosen for further investigation by bacterial and archaeal 16S rDNA clone libraries. Unique rDNA types among the 78 bacterial and 83 archaeal clones were identified by restriction fragment length polymophisms and 18 bacterial and 5 archaeal operational taxonomic units (gt97% identity) were observed. All of the bacterial sequences were deeply branching and loosely affiliated with other recognised bacterial divisions, with 40% of the sequences not affiliated to any genus. The archaeal clones were found to be deep-branching and sequences clustered together within the Crenarcaheota. In addition, two strains of Bacilli were isolated. The novel microorganisms show that the Tramway Ridge communities are unique from organisms found in other environments and show that quotEverything is (not) everywherequot.

<p>Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation.</p><p>For this thesis, a comprehensive study of the cell cycle in the model archaeon <i>Sulfolobus acidocaldarius</i> was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries.</p><p><i>Sulfolobus</i> species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes.</p><p>A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon <i>Methanothermobacter thermautotrophicus</i> was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.</p>

Archaea is the third domain of life, discovered only thirty years ago. In a microscope archaea appear indistinguishable from bacteria, but they have been shown to be more closely related to eukaryotes than to bacteria. Especially central information processing is homologous to that of eukaryotes. The archaea, previously thought to be limited to extreme environments, constitute a large part of life on Earth to an extent that has only begun to be understood. Despite their abundance little is known about several central cell-cycle features, such as cell division and genome segregation. For this thesis, a comprehensive study of the cell cycle in the model archaeon Sulfolobus acidocaldarius was performed, describing the majority of its cell-cycle regulated genes. Several known DNA replication genes, as well as genes previously not known to have a role in the cell cycle, displayed cyclic transcription. Several transcription factors, kinases and DNA sequence elements were identified as cell-cycle regulatory elements. Among the most important findings were putative cell division and genome segregation machineries. Sulfolobus species were discovered to have three origins of replication, constituting the first known prokaryotes with multiple origins. All origins initiate replication in a synchronous manner. Cdc6 proteins were shown to bind to origin recognition boxes conserved across the Archaea domain. Two Cdc6 proteins function as replication initiators, while a third paralog is implicated as a negative factor. Replication was shown to proceed at a rate similar to that of eukaryotes. A particular type of cell cycle organization was found to be unusually conserved in the Crenachaeota phylum. All the studied species displayed a short prereplicative phase and a long postreplicative phase, and cycle between one and two genome copies. Genome sizes were determined for several species. The euryarchaeon Methanothermobacter thermautotrophicus was also studied, and it was shown to initiate genome segregation during, or just after, replication. In contrast to the crenarchaea it never displayed a single genome copy per cell.

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. / text

The past few decades have witnessed an array of advances in DNA science including the introduction of genomics and bioinformatics. The quest for complete genome sequences has driven the development of microarray and massively parallel sequencing technologies at a rapid pace, yielding numerous scientific discoveries. My thesis applies several of these genome-scale technologies to understand genomic response to perturbation as well as chromatin structure, and it is divided into three major studies. The first study describes a method I developed to identify drug targets by overexpressing human genes in yeast. This chemical genomic assay makes use of the human ORFeome collection and oligonucleotide microarrays to identify potential novel human drug targets. My second study applies genome resequencing of yeast that have evolved resistance to antifungal drug combinations. Using massively parallel genomic sequencing, I identified novel genomic variations that were responsible for this resistance and it was confirmed in vivo. Lastly, I report the characterization of chromatin structure in a non-eukaryotic species, an archaeon. The conservation of the nucleosomal landscape in archaea suggests that chromatin is not solely a hallmark of eukaryotes, and that its role in transcriptional regulation is ancient. Together, these 3 studies illustrate how maturation of genomic technology for research applications has great utility for the identification of potential human and antifungal drug targets and offers an encompassing glance at the structure of genomes.

Aquarium biofilters are designed to promote the growth and activity of nitrifying microorganisms, which are primarily responsible for the removal of toxic nitrogen-cycle intermediates. Ammonia is a natural waste product excreted by fish that is lethal to aquatic life at relatively low concentrations. Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in biofilters of mature freshwater aquaria with low-ammonia conditions. However, no study has investigated the early establishment of AOA and AOB within biofilter communities, especially when aquarium ammonia concentrations are elevated. My thesis research investigated the relative abundance of AOA and AOB in freshwater aquarium biofilters through early aquarium establishment. AOA and AOB genes were detected in DNA extracts from the biofilters of 14 start-up freshwater aquaria with increasing fish biomass loads (Experiment 1), as well as from 12 biofilters of start-up aquaria treated with AOA and AOB supplements (Experiment 2). In start-up aquaria, early ammonia concentrations increased with fish biomass, and AOB amoA genes were strongly detected over AOA marker genes in all filters without initial AOA inoculation. Inoculation of AOA-dominated supplements into newly established biofilters improved early ammonia oxidation rates in comparison to filters supplemented with AOB or those lacking supplements. Inoculated AOA thrived in filter biofilm during and beyond stabilization of low-ammonia conditions in aquaria. Microbial activity experiments demonstrated that AOA were present and active in the biofilters eight months after inoculation, when aquaria were fully established. In addition, AOB and AOA populations were monitored in new aquaria in three unregulated home environments. Thaumarchaeal 16S rRNA genes were detected in all aquarium filters within one month of aquarium development. In one filter, AOA were the only ammonia-oxidizers detected in the biofilm during aquarium development, suggesting that AOA were the sole contributors to nitrification in this aquarium. The results from these experiments suggest that AOA may be key players in early aquarium nitrification once introduced into the aquarium environment. Further, this research provides insight into the ecology of AOB and AOA in engineered freshwater environments

The past few decades have witnessed an array of advances in DNA science including the introduction of genomics and bioinformatics. The quest for complete genome sequences has driven the development of microarray and massively parallel sequencing technologies at a rapid pace, yielding numerous scientific discoveries. My thesis applies several of these genome-scale technologies to understand genomic response to perturbation as well as chromatin structure, and it is divided into three major studies. The first study describes a method I developed to identify drug targets by overexpressing human genes in yeast. This chemical genomic assay makes use of the human ORFeome collection and oligonucleotide microarrays to identify potential novel human drug targets. My second study applies genome resequencing of yeast that have evolved resistance to antifungal drug combinations. Using massively parallel genomic sequencing, I identified novel genomic variations that were responsible for this resistance and it was confirmed in vivo. Lastly, I report the characterization of chromatin structure in a non-eukaryotic species, an archaeon. The conservation of the nucleosomal landscape in archaea suggests that chromatin is not solely a hallmark of eukaryotes, and that its role in transcriptional regulation is ancient. Together, these 3 studies illustrate how maturation of genomic technology for research applications has great utility for the identification of potential human and antifungal drug targets and offers an encompassing glance at the structure of genomes.