Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 67-74). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 6 uppsatser.

The deep-sea hypersaline lakes in the Red Sea are among the most challenging, extreme, and unusual environments on the planet Earth. Despite their harshness to life, they are inhabited by diverse and novel members of prokaryotes. Methanogenesis was proposed as one of the main metabolic pathways that drive microbial colonization in similar habitats. However, not much is known about the identities of the methane-producing microbes in the Red Sea, let alone the way in which they could adapt to such poly extreme environments. Combining a range of microbial community assessment, cultivation and omics (genomics, transcriptomics, and single amplified genomics) approaches, this dissertation seeks to fill these gaps in our knowledge by studying archaeal composition, particularly methanogens, their genomic capacities and transcriptomic characteristics in order to elucidate their diversity, function, and adaptation to the deep-sea brines of the Red Sea. Although typical methanogens are not abundant in the samples collected from brine pool habitats of the Red Sea, the pilot cultivation experiment has revealed novel halophilic methanogenic species of the domain Archaea. Their physiological traits as well as their genomic and transcriptomic features unveil an interesting genetic and functional adaptive capacity that allows them to thrive in the unique deep-sea hypersaline environments in the Red Sea.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13792号 / 工博第2896号 / 新制||工||1427(附属図書館) / 26008 / UT51-2008-C708 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格 / 学位規則第4条第1項該当

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13852号 / 工博第2956号 / 新制||工||1436(附属図書館) / 26068 / UT51-2008-C768 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生 / 学位規則第4条第1項該当

Ribosomal RNAs (rRNA) undergo several post-transcriptional modifications inside the cell. These modifications can be (1) RNA- independent (enzyme only) and (2) guide RNA-mediated. In the latter mechanism, a group of small, metabolically stable, non-coding RNAs, present as ribonucleoprotein (RNP) particles, modify ribosomal RNAs inside the cell. One of the highly abundant rRNA modifications is pseudouridine (Y) formation. In Archaea and Eukarya, pseudouridine synthases, with the help of small RNAs, form pseudouridines at functionally important regions in rRNA. Cbf5, the pseudouridine synthase, three other core proteins, and a box H/ACA RNA form the ribonucleoprotein complex in sRNP-mediated rRNA pseudouridylation. Certain Ys in rRNAs are evolutionarily conserved from Bacteria to human. Among those, two Ys are present in helix 69 of rRNA and one in helix 90. We successfully deleted Cbf5 in Haloferax volcanii, a haloarchaeon, and showed that the deleted strain was viable. It was the first report where Cbf5 deletion was achieved, because deletion or mutation of cbf5 or of its homologs is lethal in eukaryotes. We also found that the cbf5 deleted strain was unable to produce the three highly conserved Ys in rRNA of H. volcanii (position 1940, 1942 in helix 69, and 2605 in helix 90), whereas the tRNA Ys were intact. To identify the specific structural features of Cbf5 involved in rRNA Ψ formation, we used a cbf5 deleted strain which was complemented with a plasmid borne copy of the gene. Using the crystal structure of Pyrococcous furiosus Cbf5 as template, we created a homology model of H. volcanii Cbf5 (HvCbf5) and identified several residues and motifs/domains of HvCbf5 that might be important to the protein's enzymatic activity. By using an in vivo mutational approach, we confirmed some previously predicted and certain unidentified residues/motifs/domains that serve as positive determinants of rRNA Ys1940, 1942, and 2605 formation inside the cell. A box H/ACA RNA, sR-h45, was bioinformatically predicted before. We confirmed its presence as a double hairpin RNA inside the cell whose level goes down in the absence of Cbf5. We identified that sR-h45 is the guide RNA for sRNP-mediated Ys at the three above mentioned rRNA positions in H. volcanii. Each hairpin of this RNA can independently modify the substrate, both in vivo and in vitro. To characterize the structure of sR-h45, we have used a sR-h45 deleted strain where the function of sR-h45 was complemented with a plasmid-borne copy of the gene. By a combination of in vivo and in vitro mutagenic approaches, we determined specific nucleotides/structures of this RNA, involved in binding to the core proteins and also to the substrate RNA. We also identified that one hairpin of sR-h45 can modify two successive positions (1940 and 1942) in rRNA.

The membrane-spanning phospholipids of the domain Archaea are postulated to provide membrane stability; this thesis reports the design and synthesis of a synthetic membrane-spanning macrocyclic lipid to test this hypothesis. Protected glutaric anhydride reacted with 10-undecyn-1-ol to produce a glutarate monoester. Copper-mediated azide-alkyne coupling (CuAAC) using 1,5-diazido-3-oxapentane(bis-azide) afforded a dicarboxylicacid with a hydrophobic chain of sufficient length to span a 35 Å bilayer membrane. The carboxylic acids were each esterified with an equivalent of 10-undecyn-1-ol. After optimization an 87% yield was obtained in the closure of the 72-membered macrocycle with the bis-azide using CuAAC. Deprotection and coupling with p-nitrophenyl phosphorodichloridate completed the synthesis. Two other phospholipids, a linear bolaamphiphile derived from the precursor to the second click reaction, and a linear amphiphile created from a glutarate bis-dodecyl ester, were also synthesized to provide controls for probing the orientation of the macrocyclic phospholipid in the bilayer membrane of vesicles. The amphiphile, linear bolaamphiphile, and macrocyclic bolaamphiphile were synthesized in 4, 5, and 6 steps with yields of 19, 4, and 5% respectively. The hydrophilic head group of the macrocyclic phospholipid was designed to release p-nitrophenolate in basic conditions from the p-nitrophenylphosphate head group to produce an absorbance at 400 nm. This assay was expected to elucidate the membrane-spanning orientation of the phospholipid in the bilayer membrane of vesicles. The final target compound failed to release p-nitrophenolate under basic conditions and underwent phosphate elimination to produce an α, β-unsaturated ester instead. Although the macrocyclic lipid produced associates with membranes and may be membrane-spanning, this lipid design was unable to reveal its membrane orientation. / Graduate

Il est largement accepté qu'il existe une relation évolutive entre Archées et Eucaryotes, mais la nature exacte de cette relation reste vivement débattue. Au cours de cette thèse, je me suis servie de la grande quantité de données génomiques disponibles pour étudier ce problème à travers deux approches phylogénomiques complémentaires : (i) l'analyse d'un système cellulaire archéen particulier possédant une relation évolutive avec les eucaryotes, et (ii) une analyse phylogénomique à large échelle étendue aux trois domaines du vivant. Dans la première étude, j'ai conduit une analyse détaillée d'un système cellulaire possédant un lien entre Archées et Eucaryotes, la réplication de l'ADN. J'ai réalisé une analyse phylogénomique exhaustive des composants de la réplication de l'ADN chez tous les génomes complets d'archées. Cela m'a permis de les classer précisément en terme d'orthologie, de paralogie, de transferts horizontaux de gènes, et de copies issues d'éléments mobiles. Mes résultats fournissent un panorama complet de la diversité de la réplication de l'ADN parmi les différentes lignées, and m'ont permis d'inférer l'existence d'une machinerie de réplication de l'ADN de type moderne chez le dernier ancêtre commun archéen. J'ai ainsi été capable de clarifier l'histoire évolutive qui a forgée cette machinerie cellulaire clef au cours de la diversification des archées. Mon étude m'a permis de mettre en avant un nouveau jeu de marqueurs porteurs d'information sur les relations évolutives, non encore résolues, des différentes archées. De plus, j'ai analysé, pour la première fois, le signal phylogénétique porté pour les composants de la réplication de l'ADN. Ce signal est fortement en accord avec celui porté par deux autres machineries informationnelles clefs, la traduction et la transcription, renforçant ainsi l'existence d'un arbre archéen robuste. Enfin, la plupart des composants inférés comme étant présents chez l'ancêtre archéen sont partagés avec les eucaryotes, permettant de discuter des relations évolutives entre Archées et Eucaryotes... / It is widely accepted that there exist an evolutionary relationship between Archaea and Eukaryotes, but the exact nature of this relationship is hotly debated. In this thesis I have taken advantage of the large available genomic data to investigate the issue through two complementary phylogenomic approaches: (i) the analysis of a specific archaeal cellular system with an evolutionary link to eukaryotes, and (ii) a large-scale phylogenomic analysis at the level of the three domains of life. In the first study, I carried out a detailed analysis of a cellular system with an evolutionary link between Archaea and Eukaryotes, DNA replication. I performed an exhaustive phylogenomic analysis of the components of DNA replication in all complete archaeal genomes. This allowed me to accurately assign them in terms of orthology, paralogy, horizontal gene transfers, and copies originating from mobile elements. My results provide a full picture of the diversity of DNA replication among different lineages, and allowed me to infer the presence of a modern-type DNA replication machinery in the last archaeal common ancestor. I was able to clarify the evolutionary history that shaped this key cellular machinery during archaeal diversification. My study allowed me to highlight a new set of markers that provide information on yet unclear evolutionary relationships within archaea. In addition, I analyzed, for the first time, the phylogenetic signal carried by DNA replication components. This is highly consistent with that harbored by two other key informational machineries, translation and transcription, strengthening the existence of a robust organismal tree for the Archaea. Finally, most of the components inferred to have been present in the archaeal ancestor are shared with eukaryotes, allowing discussion on the evolutionary relationships between Archaea and Eukaryotes...

Im Rahmen des BioPara Netzwerkes: „Gesamterfassung von biochemischen und metagenomischen Parametern in Biogasanlagen und deren Korrelation zur Produkteffizienz“ erfolgte die in dieser Arbeit durchgeführte Isolierung und Charakterisierung von prozessrelevanten methanogenen Archaeen aus frischen Schlammproben einer kommerziellen Biogasanlage. Insgesamt wurden sechs verschiedene Arten der Gattungen Methanobacterium, Methanoculleus sowie Methanosarcina isoliert und als Reinkultur kultiviert. Darunter wurden mit Methanobacterium aggregans und Methanosarcina flavescens bis dahin unbekannte Spezies identifiziert und umfassend charakterisiert. Außerdem erfolgte mit Methanoculleus bourgensis und M. flavescens die Anreicherung der beiden abundantesten methanoarchaealen Mikroorganismen des beprobten Biogasreaktors. Weiterführend wurde ein anaerobes Testsystem etabliert, um die Methanproduktivität der analysierten Biogasanlagen im Labormaßstab abzubilden. Mit diesem Testsystem wurden die hydrogenotrophen und methylotrophen Wege der Methanogenese als potentiell raten-limitierende Schritte des Biogasprozesses in NawaRo-Anlagen analysiert. Die hydrogenotrophe Methanogenese verlief in allen beprobten Biogasreaktoren nicht an der maximalen Kapazitätsgrenze und stellte daher keinen raten-limitierenden Schritt dar. Weiterführend wurde die methylotrophe Methanogenese bei hohen Methanosarcina-Abundanzen als nicht-ratenlimitierender Prozess identifiziert und durch die Zugabe von Isolat E03.2 zu inaktivierten Schlammproben der untersuchten Biogasanlagen, ergaben sich Hinweise auf die Anwesenheit von Methoxygruppen. Aufgrund der fundamentalen Bedeutung des Wasserstoff-Stoffwechsels in NawaRo-Biogasanlagen, wurden u.a. Wasserstoff-abhängige Gesamt- sowie methanoarchaeale Enzymreaktionen aus frischen Schlammproben quantifiziert und als mögliche Parameter zur Prozessüberwachung analysiert. Dabei konnte eine positive Korrelation der Gesamt-Hydrogenaseaktivität mit der Methanproduktivität in den untersuchten Biogasanlagen gezeigt werden. Die Gesamt-Hydrogenaseaktivität ist somit einen aussagekräftiger Parameter zur Prozesskontrolle.

Is the nucleolus a sine qua non condition of eukaryotes? The present project starts from this central question to contribute to our knowledge about the origin and the evolution of the cells. The nucleolus is a cryptic organelle that plays a central role in cell function. It is responsible for the orchestration of ribosomal RNA expression, maturation and modification in the regulatory context of cellular homeostasis. Ribosomal expression is undoubtedly the greatest transcriptional and regulatory activity of any cell. The nucleolus is not just a conventional organelle –membrane-limited-, but a magnificent transcriptional puff: a dichotomy between structure and process, form and function. What is the minimum nucleolus? Evolution should bring some light into these questions. Evolutionary cell biology (ECB) has raised increasing attention in the last decades. Is this a new discipline and an historical opportunity to combine functional and evolutionary biology towards the insight that cell evolution underlies organismic complexity? In the post-genomic era, we have developed the potential of combining high throughput acquisition of data with functional in situ and in sillico approaches: integration understood as omics approaches. Can this provide a real consilience between evolutionary and functional approaches? The reduced number of model organisms and cultivation techniques still excludes the majority of the extant diversity of cells from the scope of experimental inquiry. Furthermore, at the conceptual level, the simplification of evolutionary processes in biosciences still limits the conformation of a successful disciplinary link between functional and evolutionary biology. This limits the formulation of questions and experiments that properly address the mechanistic nature of cellular events that underlie microbial and organismic diversity and evolution. Here we provide a critical and comparative review to the historical background of ECB. This project takes the lessons learned from ECB and attempts to find a homologue structure of the eukaryotic nucleolus within the Archaea. We found nanometric structures in S. solfactarius that either are positive to specific nucleolar techniques such as Nucleolar organizer regions NOR silver staining. These is structures are novel and its significance should be revised on the evolutionary cell biology perspective.