by King Chuen Chow. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1982 / Bibliography: leaves 145-160

La digestion anaérobie est un processus naturel de dégradation de la matière organique réalisé par l'action concertée de plusieurs populations microbiennes. Grâce au développement des techniques moléculaires, la connaissance de la diversité de ce monde microbien longtemps ignoré a connue une avancée significative ces dernières années. Dans cette étude, nous avons procédé à une étude moléculaire de la diversité procaryote à 30 autres digesteurs ayant des caractéristiques de fonctionnement variées. Cette première phase nous a permis d'identifier les phyla les plus couramment rencontrés. Nous avons pu mesurer l'étendue de la diversité de cet écosystème complexe et distinguer les phyla les plus diversifiés. Différents types de structures ont été observés : certains phyla sont constitués de phylotypes très fréquemment rencontrés alors que d'autres sont constitués essentiellement de phylotypes endémiques. Suite à cette première phase, nous avons sélectionné 7 digesteurs pour procéder à une exploration plus poussée de la diversité en séquençant un nombre important de clones pour les domaines Archaea et Bacteria. Nous avons ensuite utilisé une palette d'outils statistiques afin de comparer les populations rencontrées et de déterminer quel était leur degré de ressemblance/différence. Pour le domaine Archaea, 7 OTUs majeures ont été identifiées et sont en équilibre. Selon la disponibilité des substrats, certaines peuvent prendre le dessus. Les OTUs dominantes sont affiliées aux Methanosarcinales, Arc I et Methanomicrobiales. La lignée Arc I est particulièrement abondante dans certains digesteurs et est probablement en compétition avec le genre Methanosaeta. Cela implique que Arc I pourrait être un consommateur d'acétate et un acteur majeur de la méthanogenèse acétoclaste. Des expériences de mises en culture permettront d'approfondir nos connaissances sur le métabolisme de cette lignée qui sont limitées à l'heure actuelle. Par ailleurs, l'analyse statistique a révélé que les communautés bactériennes peuvent être décrites comme des modèles à 3 composantes : un tiers de phylotypes appartenant à un groupe noyau, un tiers de phylotypes partagés entre quelques digesteurs et un tiers de phylotypes endémiques. Le groupe noyau est composé de 6 OTUs affiliées aux Chloroflexi, Betaproteobacteria, Bacteroidetes et Synergistes. Parmi ces OTUs, 2 sont couramment retrouvées dans d'autres environnements alors que les 4 autres semblent être typiques d'environnements anaérobies. Ces populations couramment rencontrées dans les digesteurs anaérobies pourraient constitués des acteurs majeurs du procédé de dégradation de la matière organique. Grâce à la large base de données de séquences du gène de l'ARNr 16S, des sondes spécifiques ont été mises au point afin de cibler les groupes majoritaires et de quantifier leur activité au sein des réacteurs. Cette analyse quantitative a mis en évidence les groupes les plus actifs et notamment l'importance de la division candidate WWE1. Ces expériences d'hybridation ont également permis de mettre en évidence une diversité encore non ciblée par les différentes sondes existantes ainsi que les limites de l'utilisation des amorces 0008F et 1390R pour la réalisation des banques de clones. La comparaison des approches qualitative et quantitative a montré que certains groupes semblent être sur-représentés dans les banques de clones par rapport à leur réelle activité. Nous avons donc suivi une approche complète basée sur l'étude de l'ARNr 16S en partant d'un inventaire moléculaire jusqu'à la quantification à l'aide de sondes spécifiques. Cette comparaison des populations rencontrées dans les digesteurs anaérobies est un premier pas vers une meilleure compréhension du procédé. A terme, la clarification des relations existant entre biodiversité, conditions d'exploitation et performances devrait permettre d'atteindre une meilleure maîtrise du procédé de digestion anaérobie. / Anaerobic digestion is a natural process of degradation of organic matter realised by a microbial consortium. With the development of molecular techniques, our knowledge of the diversity of this microbial ecosystem has been extended in the last years. In this study, the molecular study of prokaryotic diversity was performed on 30 digesters with various operating conditions. In this first phase, the most common phyla were identified. We were able to measure the extend of the diversity of this complex ecosystem and distinguish the most diversified phyla. Different types of structures were observed: some phyla are constituted of frequently observed phylotypes while others are mainly composed of endemic phylotypes. In a second phase, 7 digesters were selected to pursue the exploration of the diversity at a deeper level with a large number of clones sequenced for Archaea and Bacteria domain. A selection of statistic tools was used to compare the populations encountered and determine their level of similarity/difference. For Archaea domain, seven OTUs were found to be dominant and are in equilibrium. Depending on the availability and the concentration of the substrates, some OTUs of the seven may become more abundant. These dominant OTUs are affiliated with Methanosarcinales, Methanomicrobiales and Arc I phylogenetic groups. We highlighted the abundance of the lineage Arc I which is probably in competition with Methanosaeta species. This implies that Arc I could be an acetate consumer and therefore a major actor in acetoclastic methanogenesis. New cultivation experiments are being conducted to explore the metabolic ability of Arc I that are still unclear. Moreover, statistical analysis revealed that the Bacteria community can be described as a three component model: one third making up a core group of phylotypes defined as OTUs commonly found in most of the digesters, one third are phylotypes shared between a few digesters, and another third are endemic phylotypes. The core group is composed of 6 OTUs affiliated with the less diversified phyla: Chloroflexi, Betaproteobacteria, Bacteroidetes and Synergistetes. Other phyla such as Firmicutes, Alpha and Deltaproteobacteria are composed of a majority of endemic sequences. Among these OTUs 2 are commonly found in other environments while the other four seem to be specific of anaerobic environments. These populations commonly found in anaerobic digesters could be major actors of the degradation of the organic matter. Based on the large database of 16S rDNA sequences specific probes were designed to target the dominant groups and quantify their activity inside the reactors. This quantitative analysis underlined the most active groups and especially the importance of the candidate division WWE1. These hybridization experiments also revealed a diversity that is not yet targeted by the existent probes and the limits in the use of primers 0008F and 1390R for the clones libraries construction. The comparison of qualitative and quantitative approach showed that some groups seem to be over-represented in clones libraries compared to their real activity. We followed a full-cycle RNA approach from a molecular inventory to quantification with specific probes. This comparison of anaerobic digester populations is a first step toward a future understanding on microbial resource management in order to manage complex microbial system. Ultimately, the relationship between biodiversity, operating conditions and digester efficiency could be established for a better process engineering in anaerobic digestion.

No description available.

N-linked glycosylation is a posttranslational modification involving the attachment of a carbohydrate to an asparagine (Asn) residue located within an Asn-X-Ser/Thr consensus sequence. This process is well understood in Eucarya and, more recently, in Bacteria. However, information about the equivalent process in Archaea is limited. This lack of knowledge includes data on glycan structures, assembly, and attachment. Assembly is believed to involve the sequential addition of monosaccharides to a membrane-embedded lipid carrier using a series of glycosyltransferases. Once the glycan is completed, it is transported across the membrane via a flippase and transferred to the protein using an oligosaccharyltransferase (Stt3p homolog). Working with the archaeon Methanococcus maripaludis, a novel glycan N-linked to the flagellin proteins of this organism was identified. Mass spectrometry has identified the glycan to have a mass of 1036.4 Da and secondary fragmentation pattern analysis of the intact glycan indicates that in addition to an N-acetylglucosamine residue (203 Da) and a di-N-acetylhexosamine (258 Da), there is an additional terminal mass of 575 Da suggesting that the glycan may be a tetrasaccharide. Genes suspected to be involved in flagellin glycosylation were targeted for in-frame deletion. The deletion of a number of these genes resulted in mutants that had a downward shift in flagellin molecular mass as evidenced by immunoblotting with anti-flagellin antisera. The annotation of these genes indicates that they are involved in the assembly of the glycan and its subsequent transfer to the target protein. Mutants that carried a deletion of an oligosaccharyltransferase (MMP1424) had flagellins that appeared completely unmodified by immunoblotting. Furthermore, mutants that had deletions in one of two glycosyltransferase genes (MMP1080, MMP1079) produced flagellins of intermediate molecular mass. Subsequent complementation of these glycosyltransferase mutations partially or fully restored flagellins to wild-type mass. Examination by electron microscopy showed that two mutants (ΔMMP1424, ΔMMP1079) were unable to assemble flagellar filaments while one glycosyltransferase mutant (ΔMMP1080) still produced flagellar filaments. Collectively, these data begin to reveal the pathway and requirement of N-linked glycosylation in archaea using M. maripaludis as a model. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-09-27 11:14:57.497

Les ADN topoisomérases sont des enzymes capables de moduler la torsion de la double hélice d’ADN afin de rendre compatible sa topologie avec les différents processus cellulaires impliquant l’ADN. Les hyperthermophiles possèdent au moins une topoisomérase particulière, la reverse gyrase qui est constituée à la fois d’un domaine topoisomérase IA etd’un domaine hélicase de type SF2. Mon sujet de thèse a eu pour objectif de déterminer principalement l’implication des ADN topoisomérases IA dans les différents processus cellulaires de Sulfolobus solfataricus. Ce crenarchaeon hyperthermophile possède, en plus, d’une ADN topoisomérase de type II (Topo VI), trois ADN-topoisomérases IA dont une « classique » (TopA) et deux reverse gyrases (TopR1 et TopR2). Notre approche a permis d’estimer, pour la première fois, le nombre de TopR1 et de TopR2 par cellule en fonction des différentes conditions testées. L’étude des variations quantitatives des ADN topoisomérases a clairement mis en évidence que TopR1 et TopR2 sont régulées différemment ce qui renforce l’hypothèse d’une spécialisation de leurs fonctions. Nous avons ainsi montré que TopR1 est responsable du maintien de l’homéostasie du surenroulement de l’ADN. Si la Topo VI de par son activité antagoniste est impliquée dansce même contrôle homéostatique, elle ne fait pas l’objet d’une régulation quantitative. De plus, nous avons mis en évidence que TopR1 était liée à la vie à haute température. Enfin, nos résultats suggèrent que TopR2 serait pour sa part impliquée dans la stabilité des génomes. L’identification des partenaires protéiques respectifs des quatre ADN topoisomérases de S. solfataricus permettra d’avoir une vision globale des réseaux de régulation permettant derésoudre les différentes des contraintes topologiques générées au cours de la vie de cet hyperthermophile. / DNA topoisomerases act in all DNA metabolism processes to control the DNA topology. Hyperthermophiles possess at least a particular topoisomerase, the reverse gyrase composed of a DNA topoisomerase IA domain and a helicase SF2 domain within the same polypeptide. The general objective of my thesis was to determine the involvement of each DNA topoisomerase in different cellular processes of S. solfataricus. This hyperthermophilic crenarchaeon possesses in addition to a type II DNA topoisomerase (Topo VI), three DNA topoisomerases IA : a classical one (TopA) and two reverse gyrases (TopR1 and TopR2). Our experimental approach allowed to estimate for the first time the number of TopR1 and TopR2 per cell in relation to different conditions. The study of quantitative variations of each DNA topoisomerase clearly showed that TopR1 and TopR2 are differently regulated suggesting that they are involved in distinct cellular processes. Indeed, we showed that TopR1 is the main actor of the homeostatic control of the DNA supercoiling. If the Topo VI with its antogonistic activity is involved in this homeostatic control, there is no regulation at the level of protein quantity. In addition we evidenced that TopR1 is somehow linked to the life at high temperature. Our results suggest that TopR2 is involved in genome stability. The identification of the respective potential partners of the four DNA topoisomerases of S. solfataricus will allow to get a more detailed understanding of the DNA topology regulation during the hyperthermophilic life style.

No description available.

La conversion des résidus U en pseudouridine (?) est la modification la plus fréquente dans les ARN. Cette réaction est catalysée par des ARN:?-synthases qui fonctionnent soit seules, soit au sein de particules snoRNP H/ACA composées de 4 protéines et d’un snoRNA. Nous avons démontré que l’ARN:?-synthase Pus7p de S. cerevisiae est capable d’agir sur les ARNt cytoplasmiques (position 13 dans 10 ARNt et position 35 dans le pré-ARNtTyr contenant un intron) (Behm-Ansmant et al., 2003). En parallèle, j’ai recherché les requis en séquence et en structure pour qu’un ARN soit substrat de Pus7p. J’ai également réalisé une étude préliminaire de l’importance des domaines additionnels de Pus7p par rapport à l’enzyme TruD d’E. coli. Comme les systèmes enzymatiques responsables de la formation de trois résidus ? du snRNA U2 de S. cerevisiae avaient été identifiés, nous avons pu débuter une étude de l’importance fonctionnelle de ces résidus pour la réaction d’épissage en utilisant l’approche globale par puces à ADN que l’équipe de J. Beggs (Edinburgh University) avait mise au point pour S. cerevisiae. Nous avons ainsi observé une baisse de l’efficacité d’épissage de certains ARN pré-messagers lorsque plusieurs gènes codant des U2 snRNA:?-synthases sont inactivés simultanément. L’ARN:?-synthase Pus7p est présente dans les 3 domaines du vivant et en particulier chez les archaea où l’activité de cette enzyme n’avait jamais été étudiée. Nous avons découvert l’existence de 2 systèmes enzymatiques différents pour la modification d’une même position d’un ARNt chez 2 espèces d’archaea thermophiles : une enzyme seule chez Pyrococcus abyssi contre un système de guide sRNP H/ACA chez Sulfolobus solfataricus dans lequel la protéine Pus7 est mutée au niveau d’acides aminés requis pour l’activité. Finalement, nous avons produit chacune de ces enzymes en grandes quantités et nous avons réalisé des tests de cristallisation. Des cristaux de Pus7 de P. abyssi ont été obtenus et une mesure complète de données de diffraction de rayons X a été réalisée au laboratoire à 2,5 Å de résolution. / thesis

The archaeal flagellum is a unique motility structure. Although functionally similar to the bacterial flagellum, the archaeal flagellum shares more similarities to the bacterial type IV pilus. Using the methanogenic archaea Methanococcus voltae and Methanococcus maripaludis as model organisms, the structural and post-translational requirements for flagellation have been investigated. Known to contain glycosylated flagellin proteins, the N-glycosylation pathway was studied in M. voltae. A number of possible glycosylation component genes, including glycosyl transferases, flippases and an oligosaccharyl transferase were inactivated or deleted in M. voltae and their resulting phenotypes were characterized. Four glycosyl transferases were identified as involved in the assembly of the M. voltae glycan structure, with three of these enzymes, AglA, AglC1 and AglC2, experimentally verified. As well, the oligosaccharyl transferase, AglB, has also been experimentally confirmed and was found to be the homolog of the bacterial and eukaryotic equivalents, PglB and Stt3p, respectively. Disruption of glycan synthesis or attachment resulted in very poorly or non-flagellated cells, implicating for the first time that the glycan structure on archaeal flagellins is necessary for proper flagella assembly and/or stability. These findings also represent the first proven N-glycosylation genes within the domain Archaea. New markerless, in-frame deletion methodology has allowed for advanced studies of a demonstrated and putative set of flagella-related genes in M. maripaludis. This collection of 11 co-transcribed genes, consisting of three flagellin genes (flaB1-flaB3), six genes of unknown function (flaC-flaH) and two genes implicated in flagellin subunit export (flaI and flaJ), make up the fla operon in this organism. Each gene from flaB1-flaI was systematically targeted for deletion and complementation to determine its necessity for flagellation. The analysis showed that both major flagellins, FlaB1 and FlaB2 are required for flagellation, while the minor flagellin, FlaB3, was required for the hook-like region of the flagella filament. FlaC, FlaF, FlaG and FlaH were shown for the first time to be essential for flagellation, while a naturally-occurring, truncated version of FlaD was found not to be required. These results continue to develop our understanding of the archaeal flagellum and the components necessary for its assembly and/or structure. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-07-11 16:50:37.119

Archaeal protein trafficking is a poorly understood process that is only beginning to unfold. Integral to this process are the various signal peptidases. Two types of archaeal signal peptidases are identified thus far: signal peptidase I (SPI) and the preflagellin peptidase. SPI is responsible for processing the majority of secreted proteins. Sequence analysis of archaeal SPI enzymes indicates a Sec-11 type enzyme with two conserved Aspartic acid and a Histidine in place of the conserved Lysine in bacteria. Site directed mutagenesis and in vitro assays identified three conserved residues (Ser52, His122 and Asp148) critical for M. voltae SPI activity, distinguishing the archaeal enzyme from its bacterial and eukaryal counterparts. The archaeal preflagellin peptidase is a type IV prepilin peptidase-like enzyme, initially characterized for its essential role in preflagellin processing prior to flagellar filament assembly. Unusual substrates have been proposed for this enzyme, including preflagellins of peculiar signal peptide lengths in certain archaeal species, as well as sugar binding proteins with extremely short signal peptides in S. solfataricus. In this thesis, in vitro comparisons of the signal peptide length requirements for the two different preflagellin peptidases, FlaK in M. voltae and PibD in S. solfataricus, are presented. While a signal peptide length cut-off of 5 amino acids was found for FlaK below which preflagellins remained unprocessed, substrates with shorter (4 and 3 aa) signal peptides were recognized and properly cleaved by PibD, suggesting a diversification of the preflagellin peptidases among the archaeal species. The ability of FlaK and PibD to complement FlaK activity in an M. maripaludis flaK mutant was evaluated. M. maripaludis flaK is a markerless, stable mutant displaying pili as the sole cell surface appendage, providing the unique opportunity to closely study this structure without the interference of flagella. Here, purification and characterization of the archaeal pili is described for the first time in any archaeon. A putative pilus gene cluster with characteristics of type IV pilus genes was identified in M. maripaludis. In-frame deletion of the putative major pilin gene, MMP0237, resulted in a nonpiliated phenotype that could be restored by complementation, providing a direct link of this gene to piliation. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-09-28 12:59:16.801

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós Graduação em Biologia Molecular, 2016. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2017-02-01T16:02:21Z No. of bitstreams: 1 2016_DeborahVasconcellosAmbrósio.pdf: 3329953 bytes, checksum: e66459c673c7c9890002220c0a4f0bf5 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2017-02-01T21:11:26Z (GMT) No. of bitstreams: 1 2016_DeborahVasconcellosAmbrósio.pdf: 3329953 bytes, checksum: e66459c673c7c9890002220c0a4f0bf5 (MD5) / Made available in DSpace on 2017-02-01T21:11:26Z (GMT). No. of bitstreams: 1 2016_DeborahVasconcellosAmbrósio.pdf: 3329953 bytes, checksum: e66459c673c7c9890002220c0a4f0bf5 (MD5) / A classificação dos organismos em três domínios, proposta por Woese e colaboradores em 1990, revelou a importância do domínio Archaea e desde então, vários trabalhos têm sido realizados a fim de melhor entender este grupo que se encontra amplamente distribuído no nosso planeta, sendo importante para vários processos ecológicos incluindo os ciclos do Carbono e Nitrogênio. A maior parte do conhecimento deste domínio, incluindo a sua diversidade no bioma Cerrado, está descrita principalmente por métodos independentes de cultivo devido à grande dificuldade de obtenção de culturas em laboratório. Este trabalho propõe a obtenção de culturas laboratoriais de archaeas mesófilas do Cerrado, a partir de sedimentos de um córrego da reserva ecológica do IBGE, localizada em Brasília-DF. Os micro-organismos foram cultivados em meios sólido e líquido, confeccionados a partir da mistura de água e sedimentos deste córrego. O meio de cultura foi adicionado de cloreto de amônio, para favorecer o crescimento de oxidantes de amônia, bem como diferentes agentes antimicrobianos. As amostras foram incubadas em estufa a 28ºC e analisadas semanalmente quanto ao crescimento, sendo realizados repiques quando necessário. As análises de microscopia revelaram colônias compostas por células diminutas, de diferentes formatos e tamanhos que variam de 0,5 a 3μm. Porém, não foi possível relacionar os aspectos morfológicos às archaeas presentes no cultivo. O DNA das colônias obtidas foi submetido a ensaios de PCR com iniciadores específicos para os genes que codificam o rRNA 16S de Archaea e Bacteria e o gene amoA de Archaea, seguido de análises de bioinformática. O resultado das análises revelou um co-cultivo entre diferentes archaeas dos filos Euryarchaeota, Thaumarchaeota e Bathyarchaeota (Miscelaneous Crenachaeotic Group-MCG) com bactérias de quatro gêneros distintos, pertencentes às famílias Brucellaceae e Burkholderiaceae, sendo a maioria das sequências de Archaea afiliadas ao grupo MCG. As sequências de DNA referentes ao gene amoA, obtidas a partir das culturas laboratoriais, se afiliaram a um grupo relacionado ao grupo I.1b do filo Thaumarchaeota, não se associando a qualquer representante previamente cultivado. O sequenciamento dos fragmentos de DNA relativos aos genes de rRNA 16S e amoA de Archaea obtidos a partir das amostras do sedimento revelaram a presença de organismos dos mesmos filos encontrados no cultivo. As análises de α-diversidade das sequências do gene rRNA 16S indicam que a comunidade do córrego Roncador pode ser considerada rica, com uma cobertura estimada de 58,33% para o nível de espécie. / The classification of living organisms in three domains, proposed by Woese and collaborators in 1990, revealed the importance of Archaea and since then, a number of studies were developed in order to better understand this widely distributed group, with important roles in many ecological processes including the nitrogen and carbon cycles. The knowledge of this domain, including its diversity in the Cerrado biome, is mostly described by culture independent methods due to the difficulty of obtaining cultures in artificial media. This work describes the cultivation and characterization of Cerrado’s mesophilic archaea, from a stream sediment of the IBGE ecological reserve located in Brasília-DF. The microorganisms were cultivated in solid and liquid media prepared with a mixture of stream’s sediment and water. In order to improve the growth of ammonia oxidizers, ammonium chloride was added to the media. Antimicrobial agents were also added to prevent bacterial and fungal growth. The samples were incubated at 28ºC and analyzed weekly for growth. Microscopy analyses showed small cells with different shapes and sizes, from 0,5 to 3μm, which were submitted to DNA extraction procedures and subjected to PCR assays with primers directed to the 16S rRNA gene of Archaea and Bacteria, as well as the archaeal amoA gene. The amplicons were submitted to automatic DNA sequencing procedures, and the results revealed that all colonies consisted in co-cultures of different types of archaea from the Euryarchaeota, Thaumarchaeota and Bathyarchaeota (Miscelaneous Crenachaeotic Group-MCG) phyla and Bacteria from four distinct genres of the Brucellaceae and Burkholderiaceae families. The majority of the archaeal 16S rRNA sequences were affiliated to the MGG group. The sequences of the archaeal amoA gene from the cultures were affiliated to a group associated to the I.1b Thaumarchaeal group, and showed no affiliation to any previously cultured members. Archaeal rRNA 16S and amoA gene sequences from the stream sediment were analyzed and the results showed the presence of organisms from the same phyla found in the cultures. α-diversity analysess of the rRNA 16S sequences showed that the archaeal community of the stream can be considered rich with an estimated coverage of 58,33% for the species level.