Molecular profiling is exploiting the unprecedented power of next generation DNA sequencing to illuminate the microbial diversity of the natural world. The composition of microbiomes has been implicated as an important factor in human health and the function of ecosystems. It is thus of great importance that measurements of microbiomes are accurate and reliable, and moreover it is essential that the accuracy and reliability of such measurements are well understood. This project sought to provide assessments of the accuracy and precision of measurements made by 16S rDNA amplicon sequencing and whole genome shotgun sequencing, as well as investigate the impact of different experimental and bioinformatics choices on quantitative measurements. To address these aims next generation sequencing data from a well quantified metagenomic control material was utilized. Good precision and accuracy were recorded for 16S primer pairs which were perfectly complementary to the target organisms. Where primers were not perfectly complementary to an organism, its abundance was underestimated. Whole genome shotgun sequencing demonstrated very high levels of precision, with a mean coefficient of variation of 2%, and showed good agreement with the 16S rDNA amplicon sequencing using primer pairs optimized specifically for the target species. Small changes in relative species abundance (less than three fold) should be treated with caution as this thesis demonstrated that sequencing results for species can vary by this amount from digital polymerase chain reaction results. Issues with publically available 16S rDNA sequence databases contribute to a lack of taxonomic resolution; taxa measured at low abundance are also likely to be artifacts of the analysis. In addition to the established sequencing platforms, this thesis also investigated the performance of a promising new experimental DNA sequencing platform developed by Oxford Nanopore Technologies (ONT). The ONT MinION, has an error rate of greater than 40% and, while it produces exceptionally long reads, it is not yet suitable for quantitative metagenomics. This thesis also demonstrated that the use of control materials in molecular profiling is important to verify findings and to understand the impact different experimental and bioinformatics choices have on measurements of the microbiome.

La métagénomique virale est une approche prometteuse pour la détection et l’identification sans a priori de potentiels nouveaux pathogènes.Cependant, son utilisation reste encore marginale en raison de l’importante contamination des viromes par les séquences nucléiques de l’hôte.L'objectif de cette thèse était d’améliorer l’approche de métagénomique pour le diagnostic clinique de maladies infectieuses virales en augmentant le ratio de séquences pathogène/hôte par déplétion des acides nucléiques humains.Le premier chapitre consiste en une synthèse bibliographique des approches de métagénomique virale en recherche clinique et des challenges à relever dans ce domaine. Elle inclut également une revue sur les approches de capture/séquençage ciblées de certains pathogènes dans le domaine des maladies infectieuses humaines.Le deuxième chapitre propose une mise au point méthodologique permettant d’enrichir les métagénomes en séquences non-humaines basée sur l’hybridation et la capture de l’ensemble des acides nucléiques de l’hôte après hybridation avec des sondes ARN humaines biotinylées.Le troisième chapitre est divisé en deux sous-chapitres qui proposent l’application de ce protocole à la détection d’agents potentiellement impliqués (1) dans un cas fatal d’encéphalite et (2) dans un cas énigmatique d’endocardite infectieuse à hémoculture négative.Dans un quatrième chapitre, l’approche méthodologique que nous avons développée est discutée et les résultats sont replacés dans un contexte élargi d’émergence des maladies infectieuses et de lien de causalité entre l’agent détecté et la pathologie observée. / Viral metagenomics, which is based on the random shotgun sequencing of all viral genomes present in a sample, is a promising approach for blind detection and identification of potential new pathogens. Its use is however still marginal because of the large proportion of human nucleic sequences. In this context, this thesis work aims at improving the metagenomic approach for the clinical diagnosis of viral infectious diseases by increasing the ratio of pathogen-to-host sequences trough depletion of human nucleic acids from the samples. The first chapter of this thesis consists in a bibliographic synthesis of viral metagenomic approaches in clinical research and the challenges we faced in this field. This bibliographic overview also includes a review article on targeted-enrichment sequencing approaches for pathogen detection in the field of human infectious diseases. The second chapter proposes a methodological development allowing the enrichment of non-human sequences from metagenomes through hybridization and capture of human nucleic acids with biotinylated human RNA probes. The third chapter is divided into two sub-chapters that propose the application of this protocol to the detection of putative pathogens in (1) a fatal case of encephalitis and (2) an enigmatic case of blood-culture negative infectious endocarditis. The methodological approach developed during this work is finally discussed in a fourth chapter, which also replaces the results obtained in the broader context of emerging infectious diseases and validation of the causal link between the agent detected and the observed pathology.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

Metabolic models were constructed from the metagenome of a methanogenic benzene-degrading community to understand the metabolite interactions among the key microbes in the culture. The metagenomic sequences were assembled, and it was found that assembling the short DNA fragments before they were combined with longer reads can contribute to the overall lengths of the resulting sequences. The metagenome was then taxonomically classified into the domain of archaea and bacteria, and domain-specific models were built. A mathematical framework to fill metabolic gaps at the community level was then developed and applied to the benzene-degrading community model to study how metabolic gaps can be filled by via interspecies metabolite transfer, and it suggested that among other metabolites, acetate, hydrogen, formate, coenzyme A and histidine produced by the bacteria population could potentially contribute to the growth of the methanogens. The computational framework demonstrated its ability to generate testable hypotheses about microbial interactions.

With the environmental problems caused by man-induced pollution by persistent toxic compounds, the importance of finding remediation solutions is immense. As an emerging field, microbial environmental biotechnology may provide the tools to achieve novel solutions. Microbial communities in the environment have biodegradation capacities which could be, and historically have been, exploited for bioremediation. The novelty lies in being able to access the capacity of the uncultured majority of the microbial community. Every day, more and more knowledge is gained in the field and thanks to new approaches such as metagenomics, along with the access to databases and archives where scientists share information and data, the quest becomes considerably facilitated. Microorganisms are highly diverse in metabolic pathways and some have become highly developed during evolution; detoxification and biotransformation of naturally occurring toxic compounds are therefore not novel concepts. The environmental problem occurs when synthetically manufactured compounds are less efficiently biodegraded. However, improved knowledge about the degradation potential in nature and the involved enzymes may help in developing bioremediation procedures. For this reason, an enzyme involved in catabolic pathways of chlorinated aromatic compounds, dienelactone hydrolase, which has been less well studied, was selected as a target. This study investigated the biogeographical distribution of the dienelactone hydrolase gene identified in metagenomes sampled from different environments globally in order to detect potential environmental patterns. Results may cast light on its significance for degradation of chlorinated aromatic compounds in nature. The results indicate a broad biogeographical distribution of dienelactone hydrolase in varying microbial habitats in the environment. The enzyme was found in environments ranging from water and soil habitats to hypersaline-, dechlorinating-, hot-spring- and other extremophillic habitats, in which the gene sequences shared high similarity within each group. A broad environmental distribution suggests that dienlactone hydrolase could be useful in bioremediation.

Metabolic models were constructed from the metagenome of a methanogenic benzene-degrading community to understand the metabolite interactions among the key microbes in the culture. The metagenomic sequences were assembled, and it was found that assembling the short DNA fragments before they were combined with longer reads can contribute to the overall lengths of the resulting sequences. The metagenome was then taxonomically classified into the domain of archaea and bacteria, and domain-specific models were built. A mathematical framework to fill metabolic gaps at the community level was then developed and applied to the benzene-degrading community model to study how metabolic gaps can be filled by via interspecies metabolite transfer, and it suggested that among other metabolites, acetate, hydrogen, formate, coenzyme A and histidine produced by the bacteria population could potentially contribute to the growth of the methanogens. The computational framework demonstrated its ability to generate testable hypotheses about microbial interactions.

The pirarucu (Arapaima gigas) from Amazon basin is currently the largest farmed fish species and its production is increasing rapidly in Brazil. However, there is a concern about bacterial disease outbreaks and resulting mortalities in pirarucu farms. The use of probiotic bacteria as prophylactic method is recognized as beneficial practice to enhance fish production. The aim of this thesis was to isolate autochthonous probiotic strains from the pirarucu intestine, characterize their potential probiotic characteristics in vitro, and perform in vivo colonization and growth experiments. To this end, the intestinal microbial community of A. gigas was assessed at two different growth stages using high-throughput sequencing (HTS) analysis. Proteobacteria, Fusobacteria and Firmicutes were the most abundant phyla. At genus level Bradyrhizobium and Cetobacterium were the most abundant in adult and juvenile fish, respectively. In a further trial two isolated lactic acid bacteria (LAB): Lactococcus lactis subsp. lactis and Enterococcus faecium, showed in vitro suitability as probiotics. Two potential pathogens: Citrobacter freundii and Pseudomonas sp., were also isolated and their pathogenicity assessed. Subsequently, an in vivo experiment was performed to assess the potencial of LAB strains to colonise and modulate the gut microbiome of pirarucu after 21 days of feeding. The results showed a high abundance of Cetobacterium in all treatments. Additionally, both probiotic treatments decreased the levels of Clostridiales in pirarucu intestine and showed adherence to the fish mucosal tissue. Finally, a growth experiment was performed to assess the ability of to the candidate probiotics to improve growth parameters after 42 days of feeding. HTS confirmed that Cetobacterium was the most abundant genus in all treatments. Fish fed with L. lactis subsp. lactis presented higher percentage of increase (%I) of weight, specific growth rate (SGR), and monocytes in blood. The strain E. faecium interacted with the microbial gut community and was able to populate the mucosal tissue. In conclusion, both LAB strains presented probiotic characteristics and should be considered as probiotics in A. gigas farming. These probiotics could contribute to a reduction in antibiotics use in pirarucu farms, thus, adding value to the species as a sustainable aquaculture product.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-23Bitstream added on 2014-06-13T19:35:21Z : No. of bitstreams: 1 gomes_es_me_jabo.pdf: 1563110 bytes, checksum: d05b3a2a3af4b9eac347a74aebd3029e (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As PKSs tipo II são complexos enzimáticos envolvidos na produção de policetídeos. Estes são metabólitos secundários que possuem papel-chave no desencadeamento das respostas bioquímicas que levam à diversificação fisiológica frente a fatores adversos, fazem parte da composição de pigmentos de esporos microbianos, além de comporem a estrutura química de importantes antibióticos explorados pela indústria farmacêutica. Foi feita a prospecção de novos “clusters” gênicos para PKSs tipo II em uma biblioteca metagenômica de 9.320 clones, através de amplificação por PCR com “primers” degenerados para uma região gênica conservada. Dentre três clones encontrados, um foi sequenciado pela estratégia de shotgun, resultando em um consensus de 22.988 pares de bases. Foi feita a predição funcional de ORFs por homologia obtida com a ferramenta Blast (NCBI) e também a identificação de domínios enzimáticos pelo PRODOM e PFAM. Dentre os resultados da predição por homologia de domínios enzimáticos foi possível identificar ORFs homologas à: reguladores gênicos (LacI e LuxR); carboidrases (Lacases, β-glucosidases, Quitinases/Celulases); transferases (O-metil-transferases, Acíl-transferases); pks mínima; ciclases, aromatases, hidroxilases, mono-oxigenases e transportadores ABC. Alguns “clusters” enzimáticos podem ainda atuar em degradação de celulose e lignina. As enzimas candidatas ao core biossíntético do “cluster” (pks mínima) foram submetidas à modelagem de estrutura tridimensional in silico. Os modelos 3D foram avaliados quanto a acurácia, utilizando as ferramentas: Verify3D, Qmean, Procheck, Rampage e Swiss-Model. Uma vez validados, as KSA e CLF foram submetidas a simulações de “docking” enzimático / The type II PKSs are enzyme complexes involved in the production of polyketides. These are secondary metabolites that have key role in triggering the biochemical responses that lead to physiological diversification against adverse factors are part of the pigment composition of microbial spores, and compose the chemical structure of most antibiotics exploited by the pharmaceutical industry. Some clusters could act in enzymatic degradation of cellulose and lignin. It was made a prospection for new clusters gene for type II PKSs in a metagenomic library of 9320 clones by PCR amplification with degenerate primers for a conserved gene region. Among three clones found, one was sequenced by subcloning and shotgun strategy, resulting in a consensus of 22,988 base pairs. It was made the prediction of functional homology ORFs by Blast (NCBI) and also identifying areas for enzymatic Prodom and PFAM. Among them were found homologous to: ABC transporters; minimal PKS, cyclasea; Aromatasea; Hydroxylases; Mono-oxygenases; O-methyl-transferases; Multicopper oxidase/laccase, β-glucosidase, chitinase/ Cellulases; Peptidases; Acyl-transferases, in addition of gene regulators similar to LacI and LuxR. The biosynthetic enzymes candidates for the core of the cluster (minimal pks) underwent three-dimensional structure modeling in silico. The 3D models were evaluated for accurate, using the tools of 'Assessing Verify3D, Qmean, Procheck, Rampage and Swiss-Model. Once validated, the KSA and CLF were subjected to simulated docking enzyme between them

Orientador: Eliana Gertrudes de Macedo Lemos / Banca: Gabriel Padilla Maldonado / Banca: Jesus Aparecido Ferro / Resumo: As PKSs tipo II são complexos enzimáticos envolvidos na produção de policetídeos. Estes são metabólitos secundários que possuem papel-chave no desencadeamento das respostas bioquímicas que levam à diversificação fisiológica frente a fatores adversos, fazem parte da composição de pigmentos de esporos microbianos, além de comporem a estrutura química de importantes antibióticos explorados pela indústria farmacêutica. Foi feita a prospecção de novos "clusters" gênicos para PKSs tipo II em uma biblioteca metagenômica de 9.320 clones, através de amplificação por PCR com "primers" degenerados para uma região gênica conservada. Dentre três clones encontrados, um foi sequenciado pela estratégia de shotgun, resultando em um consensus de 22.988 pares de bases. Foi feita a predição funcional de ORFs por homologia obtida com a ferramenta Blast (NCBI) e também a identificação de domínios enzimáticos pelo PRODOM e PFAM. Dentre os resultados da predição por homologia de domínios enzimáticos foi possível identificar ORFs homologas à: reguladores gênicos (LacI e LuxR); carboidrases (Lacases, β-glucosidases, Quitinases/Celulases); transferases (O-metil-transferases, Acíl-transferases); pks mínima; ciclases, aromatases, hidroxilases, mono-oxigenases e transportadores ABC. Alguns "clusters" enzimáticos podem ainda atuar em degradação de celulose e lignina. As enzimas candidatas ao core biossíntético do "cluster" (pks mínima) foram submetidas à modelagem de estrutura tridimensional in silico. Os modelos 3D foram avaliados quanto a acurácia, utilizando as ferramentas: Verify3D, Qmean, Procheck, Rampage e Swiss-Model. Uma vez validados, as KSA e CLF foram submetidas a simulações de "docking" enzimático / Abstract: The type II PKSs are enzyme complexes involved in the production of polyketides. These are secondary metabolites that have key role in triggering the biochemical responses that lead to physiological diversification against adverse factors are part of the pigment composition of microbial spores, and compose the chemical structure of most antibiotics exploited by the pharmaceutical industry. Some clusters could act in enzymatic degradation of cellulose and lignin. It was made a prospection for new clusters gene for type II PKSs in a metagenomic library of 9320 clones by PCR amplification with degenerate primers for a conserved gene region. Among three clones found, one was sequenced by subcloning and shotgun strategy, resulting in a consensus of 22,988 base pairs. It was made the prediction of functional homology ORFs by Blast (NCBI) and also identifying areas for enzymatic Prodom and PFAM. Among them were found homologous to: ABC transporters; minimal PKS, cyclasea; Aromatasea; Hydroxylases; Mono-oxygenases; O-methyl-transferases; Multicopper oxidase/laccase, β-glucosidase, chitinase/ Cellulases; Peptidases; Acyl-transferases, in addition of gene regulators similar to LacI and LuxR. The biosynthetic enzymes candidates for the core of the cluster (minimal pks) underwent three-dimensional structure modeling in silico. The 3D models were evaluated for accurate, using the tools of 'Assessing "Verify3D, Qmean, Procheck, Rampage and Swiss-Model. Once validated, the KSA and CLF were subjected to simulated docking enzyme between them / Mestre