Few genomic and metagenomic studies have focused on freshwater systems in the last years. Most of the studies carried out on these particular environments so far rely on microscopy, physiology, phenotypic observations, individual genes and 16S rRNA sequencing. Here, we shed light on microbial communities from oligotrophic and mesotrophic freshwater systems using high-throughput deep sequencing metagenomics and genome-resolved metagenomics. We have focused on the study of ubiquitous and cosmopolitan microbial groups from two temperate Spanish reservoirs (Tous, Amadorio). Among these, we studied freshwater picocyanobacteria from Synechococcus and Cyanobium genera, which so far have not been well characterized at the genomic level, compared to the marine representatives. In particular, we were able to isolate two of the most abundant picocyanobacteria from Tous reservoir, which were previously studied via metagenomics. These picocyanobacteria are not only abundant in this reservoir but are widely distributed in different freshwater and brackish systems. In this work we also shed light on some of the first freshwater representatives of the phylum Verrucomicrobia, that are ecologically uncharacterized in freshwater systems about which relatively little is known. We discovered a wide range of metabolisms in these microbes, ranging from nitrogen fixation and photoheterotrophy via rhodopsin pumps to important contributions in the degradation of recalcitrant matter and polysaccharides. We also include the first metagenomic study of the microbial communities under the ice waters of the largest (by volume) ultraoligotrophic lake in the world, Lake Baikal. This study has provided a first glimpse and a particular microbial composition on the sub-ice, having found an unusual fraction of Verrucomicrobia and new microbial lineages from many typical freshwater phyla, including the first freshwater representative of the groups I/II of SAR11 lineage and novel genomes of Proteobacteria, Thaumarchaeaota, Gemmatimonadetes, Cyanobacteria, Planctomycetes, Bacteroidetes, Acidobacteria, Nitrospirae, Verrucomicrobia or Actinobacteria.

Tetrodotoxin (TTX) is a lethal neurotoxin isolated mainly from the organs of wild puffer fishes. Although the neurotoxicity mechanisms of TTX are well known, the TTX origin and the biosynthetic mechanisms inside its hosts remain unresolved. In recent decades, the numerous reports of TTX-producing bacteria strongly suggested its bacterial origin. However, this origin is currently being challenged by the low and inconsistent TTX productions in vitro by the previously reported TTX-producing bacteria. Culturable TTX-producing bacteria were frequently isolated and reported from the guts of TTX-bearing animals including puffer fishes, however, these bacteria were estimated to account for 0.1% of the total gut bacteria. Moreover, the identification and functions of the non-culturable gut bacteria participating in TTX biosynthesis have never been reported. I hypothesize that the puffer fish gut bacteria and the entire gut environment serve as a functional integrality responsible for TTX biosynthesis. In this study, 16S rRNA amplicon metagenomics pipeline was established to profile the entire gut bacterial structures of both toxic and non-toxic puffer fishes respectively. UniFrac based principal coordinate analysis showed that bacterial diversities were significantly different (P-value < 0.001) between the gut environments of toxic puffer fishes and the non-toxics. Vibrio and Cyanobacteria were identified as centralities of gut bacteria co-occurrence network in toxic puffer fishes, implying their key roles in TTX biosynthesis. The results of metagenome prediction and gene set enrichment indicated that arginine biosynthesis was significant enriched (P-value < 0.05) in the toxic group. To further investigate the roles of key bacteria and arginine biosynthesis in producing TTX, metabolomics pipeline was established along with 16S rRNA amplicon metagenomics to monitor the dynamics of metabolites and bacterial compositions in guts of toxic puffer fishes during their detoxification process. The average TTX concentrations in the liver after a 60-day culture (6.41 ± 3.00 µg/g) was found significantly lower (P-value < 0.01) than that of the same species from the wild (31.86 ± 22.20 µg/g). The relative abundance of Vibrio was found positively correlated with the liver TTX concentrations. With the increase of culture periods, the relative abundance of Vibrio and Cyanobacteria decreased. In addition, both the metabolites and functional genes in arginine biosynthesis metabolic pathway were found significantly down-regulated (P-value < 0.05). These results indicated that both Vibrio and Cyanobacteria bacterial symbionts participated in TTX biosynthesis using arginine as a potential precursor in the gut environment of toxic puffer fishes.

Tetrodotoxin (TTX) is a lethal neurotoxin isolated mainly from the organs of wild puffer fishes. Although the neurotoxicity mechanisms of TTX are well known, the TTX origin and the biosynthetic mechanisms inside its hosts remain unresolved. In recent decades, the numerous reports of TTX-producing bacteria strongly suggested its bacterial origin. However, this origin is currently being challenged by the low and inconsistent TTX productions in vitro by the previously reported TTX-producing bacteria. Culturable TTX-producing bacteria were frequently isolated and reported from the guts of TTX-bearing animals including puffer fishes, however, these bacteria were estimated to account for 0.1% of the total gut bacteria. Moreover, the identification and functions of the non-culturable gut bacteria participating in TTX biosynthesis have never been reported. I hypothesize that the puffer fish gut bacteria and the entire gut environment serve as a functional integrality responsible for TTX biosynthesis. In this study, 16S rRNA amplicon metagenomics pipeline was established to profile the entire gut bacterial structures of both toxic and non-toxic puffer fishes respectively. UniFrac based principal coordinate analysis showed that bacterial diversities were significantly different (P-value < 0.001) between the gut environments of toxic puffer fishes and the non-toxics. Vibrio and Cyanobacteria were identified as centralities of gut bacteria co-occurrence network in toxic puffer fishes, implying their key roles in TTX biosynthesis. The results of metagenome prediction and gene set enrichment indicated that arginine biosynthesis was significant enriched (P-value < 0.05) in the toxic group. To further investigate the roles of key bacteria and arginine biosynthesis in producing TTX, metabolomics pipeline was established along with 16S rRNA amplicon metagenomics to monitor the dynamics of metabolites and bacterial compositions in guts of toxic puffer fishes during their detoxification process. The average TTX concentrations in the liver after a 60-day culture (6.41 ± 3.00 µg/g) was found significantly lower (P-value < 0.01) than that of the same species from the wild (31.86 ± 22.20 µg/g). The relative abundance of Vibrio was found positively correlated with the liver TTX concentrations. With the increase of culture periods, the relative abundance of Vibrio and Cyanobacteria decreased. In addition, both the metabolites and functional genes in arginine biosynthesis metabolic pathway were found significantly down-regulated (P-value < 0.05). These results indicated that both Vibrio and Cyanobacteria bacterial symbionts participated in TTX biosynthesis using arginine as a potential precursor in the gut environment of toxic puffer fishes.

Les approches de métagénomique basées sur l’utilisation des techniques de séquençage haut débit ont ouvert une nouvelle ère pour la découverte non biaisée et la caractérisation génomique des virus. Comme pour les autres virus, de telles études montrent que la diversité des virus phytopathogènes a jusqu’à tout récemment été fortement sous-estimée. Ces virus constituant une composante potentiellement importante des écosystèmes naturels ou des agrosystèmes anthropisés, il est important d’explorer la diversité des virus associés aux populations végétales et de comprendre les forces structurant cette diversité dans le temps et dans l’espace. Dans le même temps, le développement de telles études reste confronté à des questions d’ordre méthodologique concernant, par exemple, le choix des populations d’acides nucléiques à séquencer, la reproductibilité des analyses ou la disponibilité d’une stratégie permettant de comparer de façon fiable la richesse virale dans différents environnements. Dans le présent travail, le virome associé à des populations végétales échantillonnées dans différents écosystèmes, avec un focus sur les adventices et les plantes sauvages, a été caractérisé par des approches de métagénomique par séquençage haut débit. Dans ces travaux, l’analyse bioinformatique de la richesse du virome a été conduite par deux approches, l’une classique basée sur l’annotation Blast pour l’identification des familles virales présentes dans un échantillon, et l’autre, décrite et validée ici, qui permet de classifier les séquences virales métagénomiques en unités taxonomiques opérationnelles (operational taxonomic units, OTUs) utilisées comme proxy des espèces virales. Toujours dans une perspective méthodologique, les résultats obtenus avec des pools complexes de plantes représentatifs de la diversité végétale au site d’échantillonnage (approche « tondeuse à gazon ») ont permis de comparer les performances des deux techniques actuellement utilisées pour enrichir les séquences virales, la purification d’ARN bicaténaires (double-stranded RNA, dsRNA) ou d’acides nucléiques associés aux virions (virion-associated nucleic acids, VANA). Les résultats obtenus par les deux approches ont mis en évidence des viromes riches mais montrent que l’approche dsRNA devrait être préférée pour l’analyse de tels pools complexes car elle permet de façon reproductible une description plus complète du phytovirome, à l’exception des virus ADN. Les viromes caractérisés montrent, pour les populations végétales de milieux cultivés ou non gérés tempérés échantillonnées, une forte incidence virale (jusqu’à 86.5% dans 126 pools monospécifiques collectés sur une période de deux ans) et confirment la prédominance des virus dsRNA qui représentent plus de 70% des OTU identifiés. Alors qu’une proportion significative des virus ssRNA détectés sont déjà connus, plus de 90% des virus dsRNA détectés sont nouveaux et n’avaient pas été caractérisés auparavant. Un effort important en culturomique visant à comparer le phytovirome avec le mycovirome de cultures fongiques obtenues à partir des mêmes échantillons végétaux a révélé un nombre remarquablement faible d’OTUs partagés, renforçant le questionnement sur la nature, phytovirus ou mycovirus, des virus dsRNA identifiés dans les viromes des plantes. La composition en OTU des viromes analysés s’est révélée variable entre sites d’échantillonnage mais aussi très dynamique dans le temps, avec seulement une très faible fraction des OTUs ré-échantillonnés de façon stable dans la même population végétale sur une période de deux ans. Pris dans leur ensemble, ces travaux exploratoires permettent de mieux raisonner les choix méthodologiques pour l’étude des viromes associés aux plantes et étendent notre connaissance de la diversité des phytovirus, en particulier dans des espèces végétales sauvages largement négligées, apportant des points de référence importants pour de nouveaux travaux en écologie et en évolution virale. / Metagenomics based on high throughput sequencing (HTS) has opened a new era of unbiased discovery and genomic characterization of viruses. As for other viruses, such metagenomic studies indicate that the diversity of plant viruses was until recently far underestimated. As potentially important components of unmanaged and cultivated ecosystems, there is a need to explore the diversity of the viruses associated with plant populations and to understand the drivers shaping their diversity in space and time. At the same time, the development of such studies is still faced by methodological questions concerning, for example, the choice of target nucleic acids populations, the reproducibility of the analyses or the implementation of a strategy to accurately compare virus richness in different environments. In the present thesis the phytovirome associated with plant populations sampled in various ecosystems, with an emphasis on wild plant or weed species was characterized using HTS-based metagenomics. In these experiments, the bioinformatic analysis of the virome complexity was performed using two strategies, a classical one based on Blast-based contigs annotation for the identification of the viral families present in a sample and a novel one, described and validated here, and which allows to classify the metagenomic viral sequences into operational taxonomic units (OTUs) as a proxy to viral species. Also from the methodological perspective, the results obtained using complex plant pools such as those used in the “lawn-mower” sampling strategy allowed to compare the performance of the two currently used viral enrichment methods, double-stranded RNA (dsRNA) or Virion-associated nucleic acids (VANA) purification. The results indicate both of approaches uncovered rich viromes and suggest that the dsRNA approach should be preferred when analyzing complex plant pools since it consistently provided a more comprehensive description of the analysed phytoviromes, with the exception of the DNA viruses. The virome characterization results obtained showed, for the temperate plant populations from unmanaged and cultivated sampling sites, a high virus incidence (up to 86.5% in 126 single species pools collected over a two-year period) and confirmed the predominance of dsRNA viruses with greater than 70% of the phytovirome OTUs. While a significant proportion of detected single-stranded RNA (ssRNA) viruses are already known agents, more than 90% of the dsRNA viruses are novel and had not previously been characterized. A large scale culturomics effort to contrast the phytovirome with the mycovirome of fungal cultures obtained from the same plant samples revealed an extremely low number of shared OTUs, further deepening the debate about the phytovirus or mycovirus nature of the dsRNA viruses identified in plant viromes. The OTU composition of the analyzed phytoviromes varied significantly between sampling sites but was also shown to be highly dynamic over time, with a very low proportion of OTUs consistently re-sampled in the same plant population over a 2 years period. Taken together, these exploratory studies allow a more reasoned choice of methodology for the study of plant-associated viromes and expand our knowledge of plant virus diversity especially in neglected wild plant populations, providing important references for the further viral ecology and evolution studies.

No description available.

No description available.

In arid and semi-arid ecosystems, dominant tree species create dramatic mosaics of plant islands of fertility and relatively barren plant interspaces that exert immense pressure on ecosystem processes and offers an ideal opportunity to explore the impact of bacterial communities. We evaluated potential links between soil respiration and N mineralization, and community co-occurrence networks and predicted gene function across three tree island microsites (i.e., beneath tree canopies, at the canopy edge, and in interspaces) in a replicated field experiment in thirty-eight woodlands sites in the Great Basin Desert in UT, USA. Additionally, we potentially intensified the effects of tree islands by creating a treatment where whole trees were shredded and the resulting fine woody debris (FWD) was deposited onto the soil surface and measured a suite of characteristics relating to the metabolic functional state of communities (i.e., microbial efficiency as the microbial quotient, C substrate quality, biomass, and dissolved organic C) to improve our interpretation of potential links between function and structure. We found that tree islands were the predominant driver, creating highly complex and connected assemblies of bacterial populations and easily discernable differences in abundance and composition of predicted functional genes. Specifically, communities directly beneath Juniperus and Pinus canopies were comprised of at least 5.2-times more connections between bacterial taxa than present in networks from interspace and edge. Using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) to predict the gene expression, differences in the functional potential mirrored shifts in network complexity. Tree island communities expressed 236 genes with many related to the degradation of polyaromatic or polycyclic compounds, while interspace communities expressed only 66 genes associated with the decomposition of more labile C substrates. We observed a robust tree island microsite effect on all ecosystem processes, with soil respiration rates increasing 12% and N mineralization decreasing 29% in canopy than interspace soils demonstrating that a more recalcitrant substrate from a sole C source provided high amounts of low quality of DOC and lead to a decrease in metabolic efficiency, but ultimately selected for a specific community assembly. Alternatively, communities at the edge of canopies, experiencing both tree island and interspace soil conditions generated similar levels of soil respiration as canopy soils regardless of not selecting for a highly connected community and/or specific genes suggesting that a diverse composition of labile and recalcitrant C substrates from multiple sources (e.g., trees, perennial grasses, annual grasses, and forbs) potentially elevates function by promoting the activity of a wide range of taxa. Our results identify that tree islands exert enough pressure to create distinct interactions between bacteria and alter gene expression resulting in changes in ecosystem function, but the link between structure and function is mediated through the diversity and quality of C substrates.

Hantavirus is a dangerous zoonotic viral pathogen that is found across Asia, Europe, and the Americas. This virus causes a range of symptoms from flu-like malaise to heart failure and death. It is normally transmitted to humans via the aerosolized feces or urine of infected rodents. Currently, there are no known treatments for the disease, and it continues to threaten human health in endemic areas. In order to identify possible future therapeutic targets, we ran a meta-analysis of existing transcriptomic data collected from infected human tissue. Several genes and cellular pathways were identified, in addition to several potential therapeutics that warrant additional testing as potential future therapeutics for hantavirus infection. Such genes include, but are not limited to SLC27A3, NOG, AMIGO1, NUSAP1, and CDC25C which have not been previously associated with hantavirus infection. In addition, we identified that RIG-I and MDA5-associated anti-viral response genes are downregulated, while downstream elements of these pathways are upregulated, indicative of immune activation via alternate pathways. Finally, among the potential therapeutics we identified are dinaciclib, alvodicib, and ruxolitinib, which limit cellular replication, as well as ruxolitinib, baricitinib, and tofacitinib, which target other human intracellular pathways that may aid in successful viral infection. Crohn's disease is an autoimmune disorder that affects the digestive system of more than six million people worldwide, with most cases found in North America and Europe. Although the disease can occur throughout the entire digestive tract, the classical sign of disease progression is inflammation of the intestine. There are a number of factors that have been associated with the onset and progression of the disease including diet, antibiotics, stress, and bacterial infections, but no putative cause has been found. As diet and the gut biome play a significant role in disease progression, we aimed to find commonalities in the gut microbiomes of Crohn's patients, even when located in different geographical areas.

Our ability to sense small molecules with high specificity, over a broad range of concentrations, is limited and difficult to accomplish in a way that is inexpensive and continuous. The most commercially successful biosensor is the enzyme-based blood glucose electrochemical biosensor, yet for nearly all other biomolecules, detection and monitoring require specialized equipment, trained personnel, and long lead times, and are not amenable to continuous monitoring. Industries in need of enzyme-based small-molecule biosensors, including medical diagnostics, industrial production, environmental monitoring, food safety analysis, and international security, would benefit greatly from the development of new devices capable of measuring biomolecules of interest. Environmental microbes have been gaining attention because of the vast array of biomolecules that they are capable of sensing and degrading. These microbes do so, in part, through redox enzymes with diverse substrate specificities that represent an immense resource for developing electrochemical biosensors. However, the development of new enzyme biosensors has largely been limited by the lack of a general high-throughput method to identify these redox enzymes, making discovery slow, laborious, and ad hoc. To address this need, a high-throughput functional screening approach has been developed to isolate microbial oxidase enzymes from complex metagenomic DNA libraries based solely on the enzyme-mediated degradation of any target analyte. The approach can be applied to DNA isolated from any complex microbial sample, including unidentified or unculturable bacteria. In this research, I first describe the development of a general assay to capture the activity of oxidase enzymes expressed in E. coli cells. I then demonstrate how the assay can be used to screen for the nicotine degrading oxidase NicA2 from a genomic DNA library generated from the microbe P. putida. Lastly, I describe the use of this screen to identify a new hydrocortisone-responsive oxidase from a pooled genomic DNA library of eight microbes, representing over 43 Mb of DNA sequence space. This hydrocortisone oxidase represents the first of many new enzymes that can be discovered leveraging our screening platform, which is poised to revolutionize the electrochemical biosensing field and substantially broaden the number of molecules these electrochemical biosensors can detect continuously and quantitatively. / 2023-02-17T00:00:00Z

Bromus tectorum (cheatgrass) is an invasive annual grass that has colonized large portions of the Intermountain west. Cheatgrass stand failures have been observed throughout the invaded region, the cause of which may be related to the presence of several species of pathogenic fungi in the soil or surface litter. In this study, metagenomics was used to better understand and compare the fungal communities between sites that have and have not experienced stand failure. Samples were taken from the soil and surface litter in Winnemucca, Nevada and Skull Valley, Utah. Results show distinct fungal communities between Winnemucca and Skull Valley, as well as between soil and surface litter. In both the Winnemucca and Skull Valley surface litter, there was an elevated abundance of the endophyte Ramimonilia apicalis in samples that had experienced a stand failure. Winnemucca surface litter stand failure samples had increased abundance of the potential pathogen in the genus Comoclathris while the soils had increased abundance of the known cheatgrass pathogen Epicoccum nigrum. Skull Valley surface litter stand failure samples had increased abundance of the known cheatgrass pathogen Clarireedia capillus-albis while the soils had increased abundance of potential pathogens in the genera Olpidium and Monosporascus.