Les nouvelles technologies de séquençage des génomes ont permis de révéler l’extraordinaire diversité des séquences virales dans des groupes d’hôtes jusque-là largement inexplorés. Ainsi, notre connaissance des virus d’arthropodes, infectant les animaux les plus diversifiés et abondants sur Terre, était jusque-là essentiellement réduite à des espèces d’intérêt économique et médical. Les nouvelles données de diversité virale chez les arthropodes illustrent le besoin d’étendre l’inventaire viral à l’échelle de l’écosystème et d’inclure les virus comme une composante essentielle de leur fonctionnement et de leur évolution.Dans ces travaux de thèse, j’ai développé et appliqué deux approches d’étude de la diversité virale chez des arthropodes, ainsi que de la circulation des virus dans des écosystèmes, en me focalisant sur des espèces d’intérêt agronomique : i) une approche virus-centrée par fouille de bases de données nucléotidiques, en recherchant la présence d’un groupe de petits virus à ADN inféodés aux arthropodes, les densovirus ii) une approche arthropode-centrée, utilisant une méthode séquençage haut débit de génomes viraux (métagénomique virale) pour analyser des communautés virales associées à des arthropodes de différents niveaux trophiques échantillonnés dans des agroécosystèmes.Mes résultats ont permis de :(i) Mettre en évidence que les densovirus sont largement présents dans l’ensemble du règne animal - notamment chez une grande diversité d’arthropodes - et qu’ils sont très diversifiés génétiquement, ce qui a permis de mieux appréhender histoire évolutive de ce groupe de virus ;(ii) Découvrir de nouveaux virus chez certains ravageurs de cultures : le tétranyque tisserand (Tetranychus urticae, Acarien) provenant de populations de laboratoires, ainsi que le puceron vert du pois (Acyrthosiphon pisum, Hémiptère), le phytonome de la luzerne (Hypera postica, Coléoptère) et l’armigère de la tomate (Helicoverpa armigera, Lépidoptère) provenant de populations naturelles échantillonnées dans des cultures de luzerne et des prairies. Ces études ont permis de mettre en évidence la présence de viromes spécifiques de chaque espèce d’arthropode et de caractériser la distribution de certains virus dans des communautés d’arthropodes d’un même écosystème. Plus de 60 nouvelles espèces de virus d’arthropodes et de plantes ont été découvertes. Leurs liens évolutifs avec des espèces de virus connues ont été caractérisés par des analyses phylogénétiques.(iii) Enfin, les travaux menés en (ii) ont également permis d’optimiser la méthodologie permettant d’obtenir et d’analyser des viromes obtenus à partir d’échantillons multiplexés, optimisant notamment l’étape d’attribution taxonomique des séquences obtenues par séquençage à haut débit, réduisant ainsi leur proportion en « matière noire » inhérente aux analyses des viromes. / High throughput sequencing technologies have revealed the extraordinary diversity of viral sequences in hitherto largely unexplored host groups. Thus, our knowledge about arthropod viruses, infecting the most diverse and abundant animals on Earth, was hitherto essentially reduced to species of economical and medical interest. New data on viral diversity in arthropods illustrate the need to expand viral inventory at the scale of the ecosystem and to include viruses as an essential component of their functioning and their evolution.In my thesis, I developed and applied two approaches to study the diversity of viruses in arthropods and how virus circulate in ecosystems, focusing on species of agronomic interest: (i) a virus-centered approach by exploring nucleotidic sequence databases, searching for the presence of a group of small DNA viruses infecting arthropods, the densoviruses (ii) an arthropod-centered approach at the scale of the ecosystem, using a viral metagenomic method to analyze viral communities associated with arthropods from different trophic levels from the same agroecosystems.My results showed that:(i) Densoviruses are spread throughout the animal kingdom - particularly in a wide diversity of arthropods - and are highly diverse genetically, which led to a better understanding of the evolutionary history of this group of viruses;(ii) A number of new viruses can be described in pests: the spider mite (Tetranychus urticae, Acari) from laboratory populations, as well as the green pea aphid (Acyrthosiphon pisum, Hemiptera), the alfalfa weevil (Hypera postica, Coleoptera) and the cotton bollworm (Helicoverpa armigera, Lepidoptera) from natural populations sampled from alfalfa crops and grasslands. These studies also highlighted that specific viromes are associated with each pest species, and I characterized the distribution of some of these viruses in arthropod communities. In total, more than 60 new species of arthropod and plant viruses were discovered. Their evolutionary links with known virus species was characterized by phylogenetic analyzes.(iii) The work realized in (ii) also contributed to optimize a methodology to prepare and analyze viromes from multiplexed samples, that is particularly suitable to optimize the taxonomic allocation of sequences and thus reduce the "dark matter" that is inherent to viral metagenomics analyses.

Diseases in marine animals are emerging at an increasing rate. Disease forecasting enabled by virus surveillance presents a proactive solution for managing emerging diseases. Broad viral surveys aid in disease forecasting by providing baseline data on viral diversity associated with various hosts, including many that are not associated with disease. However, these viruses can become pathogens due to expansion in host or geographic range, as well as when changing conditions shift the balance between commensal viruses and the host immune system. Therefore, it is extremely valuable to identify and characterize viruses present in many different hosts in a variety of environments, regardless of whether the hosts are symptomatic or not. The lack of a universal gene shared by all viruses makes virus surveillance difficult, because no single assay exists that can detect the enormous diversity of viruses. Viral metagenomics circumvents this issue by purifying viral particles directly from host tissues and sequencing the nucleic acids, allowing for virus identification. However, virus identification is only the first step, which should ideally be followed by complete sequencing of the viral genome to identify genes of interest and develop assays to reveal viral prevalence, tropism, ecology, and pathogenicity. This dissertation focuses on the discovery of novel viruses in marine animals, characterization of complete viral genomes, and the development of subsequent diagnostic assays for further analysis of virus ecology. First, viral metagenomics was used to explore the viruses present in the healthy Weddell seal (Leptonychotes weddellii) population in Antarctica, which led to the discovery of highly prevalent small, circular single-stranded DNA (ssDNA) viruses. The lack of knowledge regarding the viruses of Antarctic wildlife warrants this study to determine baseline viral communities in healthy animals that can be used to survey changes over time. From the healthy Weddell seals, viral metagenomics led to the discovery of 152 novel anellovirus genomes, encompassing two anellovirus species. Characterizing these viruses is important for understanding the prevalence and diversity of ssDNA viruses, which have only recently been described in marine animals. Furthermore, since emerging diseases can be caused by changing conditions affecting host susceptibility to a virus that was previously not related to disease (opportunistic pathogen), having baseline data allows for quick identification of the pathogen. In addition to determining baseline data, viral metagenomics can explore the role of viruses in disease. A novel virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in the Atlantic sea star Asterias forbesi displaying symptoms of sea star wasting disease (SSWD). AfaCV was the first circular replicase-encoding ssDNA (CRESS-DNA) virus discovered in echinoderms, but it was only present in 10% of SSWD sea stars indicating it is not involved in the development of the disease. This dissertation also focuses on elucidating the role of two previously characterized viruses, chelonid fibropapillomatosis-associated herpesvirus (CHHV5; Chelonid herpesvirus 5, ChHV5) and Zalophus californianus anellovirus (ZcAV), in animal health. PCR amplicon sequencing was used to obtain large portions of the 132 kb genome of ChHV5, the putative etiological agent of the neoplastic sea turtle disease, fibropapillomatosis. Obtaining the genome of ChHV5 from Florida green, Kemp’s ridley, and loggerhead sea turtles provides data for phylogenetic analysis across geographic locations and sea turtle species, as well as a reference for designing downstream molecular assays to examine viral latency. ZcAV was first described from the lungs of captive sea lions involved in a mortality event. PCR could not detect ZcAV in the blood of infected animals, and since sea lions are a protected species, it is not possible to obtain lung biopsies from live sea lions to determine ZcAV prevalence or its role in sea lion health. To answer these important questions, an enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to ZcAV in serum from wild sea lion populations. This newly developed ELISA showed that sea lions mount an immune response to ZcAV, and was used to determine the prevalence of ZcAV among wild sea lion populations. This dissertation makes an important contribution to marine science through discovery and characterization of viruses present in healthy and diseased marine animals. Several different methods were used for virus whole-genome sequencing including viral metagenomics, PCR amplicon sequencing, and target enrichment. These findings were expanded upon by developing and using PCR assays and a serological assay to screen for virus prevalence. These methods have implications for viral surveillance and understanding the role of novel viruses in animal health.

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.

La métagénomique vise à étudier le contenu génétique et génomique d'un échantillon provenant d'un environnement naturel. Cette discipline récente s'attache à étudier les génomes de différents organismes provenant d'un même milieu. La métagénomique pose de nouvelles questions, tant d'un point de vue biologique qu'informatique. Les masses de données générées par les études métagénomiques et la complexité des milieux étudiés, nécessitent de développer de nouvelles structures de données et de nouveaux algorithmes dédiés. Parmi les différentes approches existantes en métagénomique, la métagénomique comparative consiste à comparer plusieurs métagénomes afin d'en connaître les divers degrés de similarité. Lorsque cette comparaison se base uniquement sur le contenu brut des échantillons, sans faire appel à des connaissances externes, on parle de métagénomique comparative de novo. L'objectif des travaux que nous proposons est de développer une méthode permettant d'extraire les séquences similaires de deux jeux de données métagénomiques, où chaque jeu peut être composé de centaines de millions de courtes séquences. La comparaison proposée consiste à identifier les séquences d'un premier jeu similaires à au moins une séquence d'un second jeu. Afin d'être rapide et économe en mémoire, l'implémentation de notre méthode a nécessité la conception d'une nouvelle structure d'indexation, basée sur le filtre de bloom. Le logiciel final, nommé Compareads, a une consommation mémoire faible (de l'ordre de quelques go) et peut calculer l'intersection de deux échantillons de 100 millions de séquences chacun en une dizaine d'heures. Notre méthode est une heuristique qui génère un faible taux de faux positifs. Le logiciel Compareads est dédié à l'analyse de grands jeux de données métagénomiques. À l'heure actuelle, il est le seul outil capable de comparer de tels jeux. Compareads a été appliqué sur plusieurs projets métagénomiques. Notre outil produit des résultats robustes, biologiquement exploitables et en accord avec diverses méthodes fondamentalement différentes. Il est actuellement utilisé de manière intensive sur les échantillons provenant de l'expédition tara oceans. Sur ce projet, notre méthode à permis de mettre en évidence que les grands systèmes océaniques influent sur la répartition globale des micro-organismes marins. / Metagenomics studies overall genomic information of multiple organisms coming from the same biotope. The information is generally provided by next generation sequencing technologies (NGS). Typical data are samples of short reads (i.e. reads of few hundred base pairs). To study such metagenomics information, we developed an original method for extracting similarities between two samples of reads. More precisely, this approach locates the set of common reads present in two samples. In order to fit with current memory capacities and to be time efficient, we used a modified Bloom filter data structure. Finding the common reads between multiple samples and crossing this information with the location of samples leads to visualize some biological processes like ubiquitous species or effect of water stream caring some species. Finally, the tool can also be used as a filter on metagenomics datas to remove for example only one specie. Our software, Compareads, is actually used on the Tara Oceans project where it shows that global dynamic of oceans seems to play a part on the dispersion of marine microorganisms.

Philosophiae Doctor - PhD / The estimated 5 × 10³⁰ prokaryotic cells inhabiting our planet sequester some 350–550 Petagrams (1 Pg = 1015 g) of carbon, 85–130 Pg of nitrogen, and 9–14 Pg of phosphorous, making them the largest reservoir of those nutrients on Earth (Whitman et al. 1998). However, reports suggest that only less than 1% of these microscopic organisms are cultivable (Torsvik et al. 1990; Sleator et al. 2008). Until recently with the development of metagenomic techniques, the knowledge of microbial diversity and their metabolic capabilities has been limited to this small fraction of cultivable organisms (Handelsman et al. 1998). While metagenomics has undoubtedly revolutionised the field of microbiology and biotechnology it has been generally acknowledged that the current approaches for metagenomic bio- rospecting / screening have limitations which hinder this approach to fully access the metabolic potentials and genetic variations contained in microbial genomes (Beloqui et al. 2008). In particular, the construction of metagenomic libraries and heterologous expression are amongst the major obstacles. The aim of this study was to develop an ultra-high throughput approach for screening enzyme activities using uncloned metagenomic DNA, thereby eliminating cloning steps, and employing in vitro heterologous expression. To achieve this, three widely used techniques: cell-free transcription-translation, in vitro compartmentalisation (IVC) and Fluorescence Activated Cell Sorting (FACS) were combined to develop this robust technique called metagenomic in vitro compartmentalisation (mIVC-FACS). Moreover, the E. coli commercial cell-free system was used in parallel to a novel, in-house Rhodococcus erythropolis based cell-free system. The versatility of this technique was tested by identifying novel beta-xylosidase encoding genes derived from a thermophilic compost metagenome. In addition, the efficiency of mIVC-FACS was compared to the traditional metagenomic approaches; function-based (clone library screening) and sequence-based (shotgun sequencing and PCR screening). The results obtained here show that the R. erythropolis cell-free system was over thirty-fold more effective than the E. coli based system based on the number of hits obtained per million double emulsions (dE) droplets screened. Six beta-xylosidase encoding genes were isolated and confirmed from twenty-eight positive dE droplets. Most of the droplets that were isolated from the same gate encoded the same enzyme, indicating that this technique is highly selective. A comparison of the hit rate of this screening approach with the traditional E. coli based fosmid library method shows that mIVC-FACS is at least 2.5 times more sensitive. Although only a few hits from the mIVC-FACS screening were selected for confirmation of beta-xylosidase activity, the proposed hit rate suggests that a significant number of positive hits are left un-accessed through the traditional clone library screening system. In addition, these results also suggest that E. coli expression system might be intrinsically sub-optimal for screening for hemicellulases from environmental genomes compared to R. erythropolis system. The workflow required for screening one million clones in a fosmid library was estimated to be about 320 hours compared to 144 hours required via the mIVC-FACS screening platform. Some of the gene products obtained in both screening platforms show multiple substrate activities, suggesting that the microbial consortia of composting material consist of microorganisms that produce enzymes with multiple lignocellulytic activities. While this platform still requires optimisation, we have demonstrated that this technique can be used to isolate genes encoding enzymes from mixed microbial genomes. mIVC-FACS is a promising technology with the potential to take metagenomic studies to the second generation of novel natural products bio-prospecting. The astonishing sensitivity and ultra-high throughput capacity of this technology offer numerous advantages in metagenomic bio-prospecting. / National Research Foundation (NRF)

Les zoonoses constituent plus des deux tiers des pathologies virales qui concernent l’homme. Le développement et la démocratisation des outils de métagénomique en font de bons outils d’inventaire et de surveillance de virus potentiellement émergents.Dans un premier temps j’ai développé et validé un protocole expérimental de purification des viromes à ARN qui permettait le maintien de l’infectivité des particules virales. Ce protocole a ensuite été appliqué pour caractériser les communautés virales d’arthropodes hématophages et de prélèvements de faune sauvage. J’ai par la suite réalisé l’inventaire des communautés virales de viande de singe fumée illégalement importée en France et confisquée par les douanes, qui a révélé la présence de nombreux bactériophages, dont certains pourraient infecter des bactéries potentiellement pathogènes pour l’homme.Enfin j’ai caractérisé les communautés virales de culicoïdes collectés au Sénégal, ce qui a permis de mettre en évidence la présence de nombreux virus géants à ADN infectant les amibes. Le séquençage des viromes à ARN a quant à lui révélé la présence d'un certain nombre d'arbovirus qui pourraient constituer un risque d’émergence pour la santé humaine. Du fait de nombreux facteurs intrinsèques et extérieurs à l’agent infectieux, la prédiction des futures émergences de virus zoonotiques est très compliquée voire utopique, mais elle reste un challenge crucial et d’actualité. La stratégie de réalisation d’inventaires des communautés virales présentes dans les différents acteurs des cycles de transmission zoonotique est un premier pas indispensable dans la connaissance des risques potentiels d’émergence en population humaine. / Zoonoses are responsible of more than two thirds of human viral infections. The development of high-throughput sequencing tools and their application in metagenomics allow inventorying the viral communities of various reservoirs in order to detect the emergence of viruses before their infection to humans. In this context, I characterized the viral communities of simian bushmeat illegally imported into France and of Culicoides biting midges, recognized vectors of several viruses of human and veterinary medicine importance. I have first developed a protocol for the purification of RNA viromes which allowed maintaining the infectivity of viral particles. This protocol was subsequently applied to characterize viral communities of bloodsucking arthropods and wildlife samples. In a second part I realized the inventory of viral communities of smoked simian bushmeat illegally imported into France and confiscated by the French customs. This study revealed the presence of a wide diversity of bacteriophages, in which some of them could infect bacteria potentially pathogenic for humans.Finally I characterized the viral communities of Culicoides biting midges collected in Senegal, which revealed the presence of sequences related to several giant DNA viruses infecting amoeba. Sequencing of the RNA virome revealed the presence of several arboviruses that could constitute a risk of emergence of zoonoses for humans.The prediction of future emerging zoonotic viruses is very difficult, if not impossible. However the characterization of viral communities present in the different actors of zoonotic transmission cycle is a first step to evaluate potential risks of transmission to humans.

Les virus sont les entités biologiques les plus abondantes et diversifiées sur Terre et leur diversité est encore très peu connue. Récemment, la métagénomique virale a facilité l'exploration de cette diversité. Néanmoins, la plupart des viromes environnementaux générés à ce jour proviennent de régions tempérées et la plupart des viromes humains proviennent d’échantillons de selles, sang ou de prélèvements oro-naso-pharyngés. L'objectif de mon travail de thèse était d’apporter de nouvelles connaissances sur les communautés virales d’environnements et d’échantillons humains les moins étudiés en utilisant une approche de métagénomique virale.La première partie de cette thèse est une revue des principaux outils d'analyse des métagénomes viraux. La deuxième partie présente la première étude de métagénomique virale dans le désert du Sahara. Dans la troisième partie de ma thèse, je présente de viromes associés a l'Homme: i) le premier métagénome viral issu d'un coprolithe humain du Moyen Âge; ii) la première étude de métagénomique virale sur de liquides péricardiques provenant de patients atteints d’une péricardite infectieuse d'origine inconnue; iii) une analyse fonctionnelle de métagénomes viraux précédemment publiés associés aux expectorations de patients atteints de mucoviscidose qui décrit les gènes de résistance aux antibiotiques portés par les bactériophages dans ces patients.Ce travail présente ainsi des données inédites sur certaines communautés virales peu étudiées et confirme le potentiel de la métagénomique virale pour étudier la diversité virale, révéler la présence de virus inattendus ou inconnus et comprendre leur rôle dans leur écosystème d’origine. / Viruses are the most abundant and diverse organisms but little is known about their diversity. Recently, viral metagenomics has allowed performing broad unselective exploration of uncultivated viral communities, bypassing the limits of classical viral detection tools. However, most viral metagenomes are generated from temperate regions (for environmental studies) or from modern stool samples, sera/blood and naso-/oro- pharyngeal samples (for human-associated studies). Therefore, the purpose of my thesis is to study viral communities in the least investigated environments or human samples, using viral metagenomics.The first part of my thesis is a review of the main computational tools for the analysis of viral metagenomes. The second part of my thesis presents the first viromes generated from the Sahara desert. In the third part, I investigate human-associated viral communities: i) the first virome from a human coprolite; ii) the first viromes generated from human pericardial fluids, in idiopathic pericarditis cases; ii) a functional-level investigation of previously described viral metagenomes from cystic fibrosis patient sputa that focuses on antimicrobial resistance genes carried by bacteriophages to better understand the emergence of multidrug-resistance bacteria in the airways of cystic fibrosis patients.This thesis work provides original data on unexplored viral communities and shows the potential of viral metagenomics to give insights on viral diversity, reveal the presence of expected and unexpected viruses and decipher their role in the ecosystem.

Les glucides sont très rependus dans la nature et sont impliqués dans une multitude de phénomènes biologiques. Sous forme de saccharides et de glycoconjugués, ils constituent une partie substantielle de la biomasse produite sur terre et représentent une source potentielle d’énergie renouvelable de première importance. La diversité des glucides complexes est créée et contrôlée par un panel d’activités enzymatiques qui interviennent dans leur assemblage, dégradation et modification. L’étude structurale et fonctionnelle des enzymes actives sur les glucides (CAZymes) est à la base de multiples efforts de recherche appliquée en biotechnologie. L’industrie recherche actuellement des enzymes avec des activités et des spécificités encore plus performantes. L’activité de recherche de ces nouvelles enzymes est grandement facilitée par l’accumulation de séquences biologiques dans les bases de données, provenant notamment des études génomiques.Mon sujet de recherche s’inscrit dans un objectif de développement d’outils pour la classification et l’identification de nouvelles enzymes impliqués dans la conversion de la biomasse. Tous ces travaux sont en lien direct avec la mise en place d’une nouvelle infrastructure de la base de données CAZy et l’analyse de données génomiques, métagénomiques et biochimiques. La refonte complète de la structure de la base de données préexistantes et de son interface a été ainsi réalisée. Cet effort a été validé par l’analyse des familles de polysaccharide lyases et la création de sous-familles, dont l’homogénéité fonctionnelle a été révélée. De plus, la détection systématique de protéines modulaires portant des modules d’adhésion aux composants de la paroi végétale a permis l’identification de nouvelles protéines potentiellement impliquées dans la dégradation de la biomasse végétale. Enfin, j’ai implémenté des approches automatisées capables d’analyser de grands volumes de données (méta)génomiques pour en extraire le contenu en CAZymes. / Carbohydrates are widely distributed in nature, where they are involved in a multitude of important biological events. Saccharides and glycoconjugates constitute the main component of the biomass produced on earth, therefore they represent a plentiful source of renewable energy. The diversity of complex carbohydrates is created and controlled by a panel of enzyme activities involved in their assembly, degradation and modification. The structural and functional study of Carbohydrate Active enZymes on (CAZymes) has been the basis for many applied research efforts in biotechnology. For exemple, the biotechnology industry is currently searching enzymes with enhanced activities and specificities. The identification of new enzymes is potentially facilitated by the large-scale accumulation of gene sequences, particularly from current genomic studies.This thesis aimed at developing tools for the classification and identification of new enzymes involved in biomass degradation. To this end, a new structure of the CAZy database was developed and applied to mining genomic, metagenomic and biochemical data. A complete reorganisation of the structure of the existing database and its interface has been achieved. In this effort the analysis of all known families of polysaccharide lyases has been validated and subfamilies were created, which revealed functional homogeneity. In addition, the systematic identification of modular proteins containing plant cell wallbinding modules allowed the identification of new proteins potentially targeting plant biomass. Finally, I show that it is indeed possible to analyze large volumes of (meta)genomic data by automated methods in order to understand their CAZyme contents.

Orientadores: Valéria Maia Merzel, Itamar Soares de Melo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T12:19:42Z (GMT). No. of bitstreams: 1 Domingos_DanielaFerreira_D.pdf: 4459680 bytes, checksum: c21b44008997f453425f3732e5729029 (MD5) Previous issue date: 2014 / Resumo: O manguezal é um ambiente rico em diversidade microbiana, entretanto existem poucos estudos sobre esse tema no Brasil, tornando imperativo o conhecimento e a exploração de novos micro-organismos e seus metabólitos neste ecossistema. A prospecção da diversidade microbiana em ambientes pouco explorados, como os manguezais, potencializa as chances de sucesso na busca por novas moléculas bioativas, contribuindo para o desenvolvimento econômico e ambiental mais sustentável. Entretanto, sabemos que embora as técnicas de cultivo tenham sido aprimoradas e tenham permitido a recuperação in vitro de um número crescente de micro-organismos ainda não cultivados, nosso conhecimento sobre sua ecologia permanece insuficiente para cultivar a maioria deles. Neste contexto, as bibliotecas metagenômicas surgem como uma ferramenta poderosa para acessar de maneira mais abrangente a diversidade microbiana total em um dado ambiente, permitindo a análise e exploração de genes funcionais de membros da microbiota, principalmente de micro-organismos não-cultivados, e a descoberta de novos compostos bioativos. O objetivo geral desse trabalho foi a prospecção de biossurfactantes a partir de microbiota de manguezal utilizando uma abordagem polifásica. Para a abordagem independente-de-cultivo, foi construída uma biblioteca metagenômica de alto peso molecular a partir de sedimento de manguezal contaminado com petróleo. Os clones obtidos foram submetidos à triagem funcional e molecular para compostos com atividade biossurfactante. Três clones potencialmente produtores foram selecionados e submetidos ao sequenciamento fosmidial para a caracterização gênica dos insertos. Embora os clones apresentassem uma redução na tensão superficial, não foram identificados genes que pudessem estar envolvidos na síntese de algum biossurfactante. Os resultados obtidos revelaram que o uso da metagenômica funcional para a exploração metabólica de uma comunidade microbiana pode oferecer grandes limitações quando se trata de prospecção de biossurfactantes, cujos operons são muitas vezes maiores que 30-40 kb e podem conter elementos de regulação esparsos no genoma da bactéria selvagem. Na abordagem dependente-de-cultivo, foram selecionadas duas linhagens produtoras de biossurfactantes, Bacillus safensis CCMA-560 e Gordonia sp. CCMA-559, isoladas e testadas em estudo prévio. Através de planejamento experimental do tipo Plackett-Burman e Delineamento Composto Central Rotacional foi otimizada a produção do biossurfactante por essas linhagens. A pumolicidina produzida pelo B. safensis CCMA-560 foi caracterizada quimicamente, bem como a via metabólica responsável por sua produção. Este foi o primeiro trabalho a reportar a análise fisiológica, genética e química da produção de biossurfactante por representantes da espécie B. safensis / Abstract: Mangrove is an environment rich in microbial diversity, but little has been reported about it in Brazil, making the knowledge and exploitation of new microorganisms in mangroves an imperative issue. The prospection of microbial diversity in underexplored environments, such as mangroves, increses possibility of success in looking for new bioactive molecules, contributing to a more sustentable economy and environment. Although the cultivation techniques have been improved, allowing an increased number of yet uncultivated microorganisms to be able recuperated in vitro, our knowledge about their ecology is still insufficient to cultivate the majority of them. Therefore, the metagenomic library have emerged as a powerful tool to more widely access the overall microbial diversity in an environment, enabling the functional analyses of genes and the discovery of new bioactive compounds, mainly uncultured-microorganisms. The aim of the current work was the prospection of biosurfactant from mangrove microbiota through a polifasica approach. For the independent-cultivation approach, a metagenomic library of high molecular weight was constructed from mangrove sediment contaminated with oil. The clones were subjected to functional and molecular screening to identify biosurfactant activity. Three clones with the potential to procuce biosurfactant were selected, and the fosmid sequencing for genetic chatacterization of the insert was carried out. Although the clones showed the ability to reduce the surface tension, genes that may involved in biosurfactant synthesis were not identified. The results showed that using the functional metagenomic to explore metabolic pathways of microbial communities can have great limitation when in the prospection of biosurctants when the operon are larger then 30-40 kb and the genome of wild bacteria contains sparse regulation elements. For the dependent-cultivation approach, two strains that produce biosurfactant were selected: Bacillus safensis CCMA-560 and Gordonia sp. CCMA-559. They were isolated and tested in previous study. The biosurfactant production was opmized through the Placktt-Burman design and the Central Composite Design. The pumilacidin produced by B. safensis CCMA-560 was chemically characterized, and the metabolic pathway that is responsible for its production was genetically characterized. This work was the first report the physiologic, genetic, and chemical analysis on the biosurfactant production by the B. safensis strain / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.