Les virus infectant les archées présentent des morphotypes inhabituels et des génomes extrêmement divers. Leur isolation a permis de développer nos connaissances sur la diversité de la virosphère et demeure une piste de recherche primordiale. Durant ma thèse, j’ai isolé et caractérisé de nouveaux virus d'archées et étudié les interactions virus-hôte dans un système modèle bien établi. Des cultures d'enrichissement et des analyses bioinformatiques nous ont permis de décrire de nouveaux virus de crénarchées hyperthermophiles infectant les espèces du genre Pyrobaculum et ainsi de mieux comprendre la diversité architecturale des virus filamenteux. De plus, un provirus a été identifié chez P. oguniense et l’étude de sa réplication a révélé par microscopie électronique des nanostructures pyramidales à la surface des cellules ressemblant à des structures connues de sortie des virions chez des virus de crénarchées. Deux études de protéomique nous ont fourni un aperçu de la dynamique du protéome de Sulfolobus islandicus : l’analyse du protéome de cellules de S. islandicus non infectées a révélé de nombreuses modifications post-traductionnelles, et l’analyse de la régulation des protéines dans des cellules de S. islandicus infectées par le virus SIRV2 a révélé 136 protéines de l'hôte présentant une régulation temporelle significative. L’analyse structurale de SIRV2 a permis d'étudier la résistance des virus crénarchées à des conditions extrêmes et a révélé pour la première fois que la forme A de l'ADN est biologiquement pertinente. Ces résultats ont contribué au développement des connaissances sur la diversité de la virosphère et sur l'évolution des virus d'archées. / Viruses infecting Archaea display unusual morphotypes and highly diverse genomes. Several virus-host systems have emerged enabling the detailed characterization of virus-host interplay in archaea. However, isolation of new archaeal viruses proved to be instrumental for expanding the knowledge on the diversity of the Earth’s virosphere. Therefore, I have focused on two major lines of research: isolation of new archaeal viruses and characterization of the virus-host interactions in a well-established model system. A new Pyrobaculum virus with a unique architecture among DNA viruses was described, expanding our knowledge on the diversity of architectural solutions explored by filamentous viruses. Furthermore, attempts to trigger the replication of a provirus in P. oguninese led to the development of six-fold pyramidal nanostructures on the cell surface, resembling known virion egress structures of archeal viruses. Finally, I focused on the interplay between Sulfolobus islandicus and the rod-shaped virus SIRV2. Two proteomic studies yielded insights into the dynamics and posttranslational modifications (PTMs) of the Sulfolobus proteome. Sulfolobus proteins were found to carry a high degree of PTMs on functionally diverse proteins. The global analysis of the regulation of viral and host proteins in SIRV2-infected S. islandicus cells yielded insights the temporal regulation of host and virus proteins. Structural studies on SIRV2 virion have resulted in the first ever description of A-form DNA being a biologically relevant form of DNA. Together, these results contribute to the knowledge on the diversity of the extant virosphere, and the biology and evolution of archaeal viruses.

Motile microorganisms navigate through their environment using special molecular machinery in order to sense gradients of various signals: chemotaxis (reactions to chemical compounds) and phototaxis (to light) sensory cascades. Transmembrane receptors play a central role in these cascades as they receive input signals and transmit them inside the cell, where they modulate activity of the kinases CheA, which are tightly bound to their cytoplasmic domains. CheA further phosphorylates the response regulator protein CheY, which regulates the flagella. At the same time, CheA phosphorylates and, by means of this, activates another response regulator, CheB, which, along with the constantly active CheR protein, catalyzes two opposite reactions: methylation and demethylation of the specific glutamic acid residues located at the cytoplasmic domain of the receptors. The latter reactions establish the adaptation mechanism, which allows microbes to sense in a very broad range of the input signal intensities. Many functional, structural and dynamical aspects of the signal propagation through the prokaryotic receptors as well as a mechanism of the signal amplification remain still unclear. In the present thesis we have used various techniques of computational biophysics, chiefly molecular dynamics (MD) simulations, in order to approach these problems. In Chapter 3, we have carried out MD simulations of the isolated linker domain (HAMP) from the E. coli Tsr chemoreceptor. The MD simulations revealed highly dynamical nature of this domain, which allows for interconversion between several metastable states. These metastable states feature a number of structural and dynamical properties, which were previously reported for HAMP domains of various receptors obtained from different organisms. It allowed us to reconcile numerous experimental data and to hypothesize that different HAMP domains share similar mechanism of their action. In Chapter 4, we have performed MD simulations of the whole cytoplasmic domain of the Tsr chemoreceptor. The simulations revealed a mechanism for the inter-domain coupling between the HAMP domain and a part of the cytoplasmic domain adjacent to the HAMP, the adaptation subdomain, by means of the regulated unfolding of a short linker region termed the stutter. Also, we have found that the reversible methylation/demethylation of the cytoplasmic domain affects its flexibility and symmetry. Altogether, these findings suggest a mechanism of signal propagation at the level of an individual chemoreceptor dimer. In Chapter 5, we have built a model of the trimer-of-dimers of the archaeal phototaxis receptor complex (NpSRII:NpHtrII). Subsequent MD simulations revealed an important role of dynamics in signal transduction and, potentially, in the kinase activation. In Chapter 6, we have reconstructed a whole transmembrane lattice formed by the NpSRII:NpHtrII complexes. The concave shape of the obtained lattice naturally explains polar localization of the receptor arrays in prokaryotic cells. At the same time, additional MD simulations of an individual unit of this lattice (a dimer of the photosensor) revealed global motional modes in its transmembrane region, which presumably co-occur with its activation and can spread across the tightly packed transmembrane arrays allowing for the signal amplification.

O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo. / The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.

O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo. / The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.

No description available.

Les virus sont les entités biologiques les plus abondantes dans les océans (∼1031 particules). Ils colonisent tous les écosystèmes de la planète y compris les environnements extrêmement acides, chauds et salins, environnements où les archées sont les organismes dominants. Les virus infectant les Crenarchées hyperthermophiles présentent des morphologies exceptionnelles et aussi une très faible proportion de gènes possédant des homologues avec de fonction connue. Parmi ces virus, le virus ATV (Acidianus two-Tailed virus), infecte les archées hyperthermophiles du genre Acidianus. ATV a la propriété unique de présenter un important développement structural complètement indépendante de son hôte, à l'extérieur de celui-Ci. Les virions d'ATV développent de longues queues à chaque extrêmité de sa capside, mais seulement à des températures proches de celles de l'habitat de son hôte, 85°C. Le sujet de ma thèse a porté sur l'étude structurale de protéines du virion d'ATV. J'ai résolu la structure cristalline de la protéine ATV-273, qui possède un nouveau fold α/β. J'ai aussi déterminé la forme de l'enveloppe de cette protéine par SAXS. J'ai montré qu'il est possible de placer deux dimères d'ATV-273, observés dans la structure cristalline, dans cette enveloppe. Ce résultat est aussi en accord avec l'état d'oligomérisation en solution déterminé par chromatographie d'exclusion stérique couplée à la diffusion de la lumière. La fonction de cette protéine reste cependant inconnue. / Viruses are the most abundant biological entity in the oceans (∼1031 particles) and remarkably, viruses populate every ecosystem on the planet including the extreme acidic, thermal, and saline environments where archaeal organisms dominate. The viruses infecting hyperthermophilic Crenarchaea revealed exceptional morphologies and also a very low proportion of genes with recognizable functions and homologues. Among these viruses we find ATV (Acidianus two-Tailed virus). ATV is a virus infecting hyperthermophilic archaea of the genus Acidianus, which has the unique property of undergoing a major morphological development outside and independently of the host cell. Virions develop long tails at each pointed end of the initial lemon-Shaped particle, at temperatures close to those of the host natural habitat, 85 °C. The subject of my thesis has focused on the virion proteins of ATV. I have solved the crystal structure of ATV-273 that revealed a new α/β fold. I have also obtained a SAXS envelope where it is possible to fit two crystal dimers, in agreement with the oligomerization state in solution as determined by size-Exclusion chromatography coupled to multi angle light scattering. The function of this protein, however, could be not determined. Moreover, a negative staining electron microscopy model was obtained for the AAA+ ATPase ATV-618, which belongs to the MoxR familiy and presents sequence high similarity with the AAA-ATase RavA from Escherichia coli K12. I have shown that this thermostable AAA-ATPase enzyme assumes a hexameric ring organisation in the presence of ATP.

Thesis (Ph.D. in Biophysics & Genetics, Program in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 90-98). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;