Yes / Several bacterial families are known to be highly abundant within the human microbiome, but their ecological roles and evolutionary histories have yet to be investigated in depth. One such family, Lachnospiraceae (phylum Firmicutes, class Clostridia) is abundant in the digestive tracts of many mammals and relatively rare elsewhere. Members of this family have been linked to obesity and protection from colon cancer in humans, mainly due to the association of many species within the group with the production of butyric acid, a substance that is important for both microbial and host epithelial cell growth. We examined the genomes of 30 Lachnospiraceae isolates to better understand the origin of butyric acid capabilities and other ecological adaptations within this group. Butyric acid production-related genes were detected in fewer than half of the examined genomes with the distribution of this function likely arising in part from lateral gene transfer (LGT). An investigation of environment-specific functional signatures indicated that human gut-associated Lachnospiraceae possess genes for endospore formation, whereas other members of this family lack key sporulation-associated genes, an observation supported by analysis of metagenomes from the human gut, oral cavity, and bovine rumen. Our analysis demonstrates that adaptation to an ecological niche and acquisition of defining functional roles within a microbiome can arise through a combination of both habitat-specific gene loss and LGT. / Canadian Institute for Health Research (grant number CMF-108026), Genome Atlantic and the Canada Research Chairs program to R.G.B.

Viruses, and particularly bacteriophages, are key players in many microbial ecosystems and can profoundly influence the human microbiome and its impact on human health. While the bacterial and archaeal fraction of the human microbiome can now be profiled at an unprecedented resolution via cultivation-free metagenomics, viral metagenomics is still extremely challenging. The lack of universal viral genetic markers limits the de-novo discovery of viral entities, and the low number of available viral reference genomes from cultivation studies does not cover well the phage diversity in human microbiome samples. Viral-like particle (VLP) purification has been proposed as a set of experimental tools to concentrate viruses in samples prior to sequencing, but it remains unclear how efficient and reproducible such tools are in practice. In this thesis we aim to address some of these challenges and better exploit the potential of viral metagenomics in the context of the human microbiome. First, we performed and studied the performance of VLP procedures on freshwater and sediment samples. We found that bacteria can still be abundant at the end of the filtration process, thus lowering the efficiency of the enrichment. Analyzing samples with a low enrichment may lead to inconsistent conclusions, as the residual bacterial contamination might misdirect the computational analysis. To better quantify the extent of non-viral contamination in VLP sequencing, we designed ViromeQC, a novel open-source tool able to assess and rank viromes by their viral purity directly from the raw reads. In ViromeQC, rRNA genes and bacterial single-copy proteins are used as a proxy to estimate non-viral contamination. With the ViromeQC, we conducted the largest meta-analysis on the degree of enrichment of thousands of viral metagenomes, and concluded that the vast majority of them are three-fold less enriched than a standard metagenome. ViromeQC was then used to select the human gut viromes that had the highest enrichment as a starting point for a novel reference-free pipeline for the discovery of previously uncharacterized viral entities. The approach included metagenomic assembly of the enriched viromes as well as extensive mining of many thousands of assembled metagenomes, and led to a catalog of 162,876 sequences of highly-trusted viral origin. Most of these predicted viral sequences had no match against any known virus in RefSeq even though some of them showed a prevalence in gut metagenomes of up to 70%. Our analyses and publicly available tools and resources are helping to uncover the still hidden virome diversity and improve the support for current and future investigations of the human virome.

Metagenomic sequencing has provided scientists with the ability to investigate microbial populations, termed microbiomes, in environmental and clinical settings. Current approaches to metagenomic research involve the use of next-generation sequencing (NGS) to generate short, precise reads for characterization of microbial compositions. While highly accurate, short reads possess several limitations that restrict their application in metagenomic research. Third generation, long-read sequencing technologies may offer several advantages for metagenomic research. Here, we used simulated datasets, as well as experimental data from murine fecal samples, to compare the relative performance of short and long reads for metagenomic research, and their impact on assessing microbial composition. Long-read data demonstrated increased precision for identification of microbiome constituents and assessing abundance without sacrificing sensitivity. Hierarchical clustering of microbiome similarity from paired short- and long-read datasets obtained from murine fecal samples revealed clustering was driven by read type as opposed to sample type, underscoring the importance of sequence type. These findings led us to use long-read sequencing for elucidating the effects of propionic acid (PPA) on the murine gut microbiome. PPA has been shown to induce physiological changes like those observed in autism spectrum disorder (ASD). Individuals with ASD may suffer from gastrointestinal comorbidities, suggesting an association with the gut microbiome. Murine offspring fed a PPA-rich diet were assessed for microbiota perturbations. Our results demonstrated that a PPA-rich diet alters the gut microbiome of progeny mice, selecting for several bacterial species that have previously been found in greater abundance among people iv with ASD. In our study, changes to microbial abundance were also associated with significant variation in bacterial metabolic pathways related to steroid hormone biosynthesis, amino sugar, and nucleotide sugar metabolism. Taken together, our findings provide empirical evidence supporting the use of long-read sequencing in metagenomic research by elucidating links between PPA exposure and gut microbiome composition.

Next Generation Sequencing (NGS) is a new technology that has revolutionized the way we study living organisms. Where previously only a few genes could be studied at a time through targeted direct probing, NGS offers the possibility to perform measurements for a whole genome at once. The drawback is that the amount of data generated in the process is large and extracting useful information from it requires new methods to process and analyze it. The main contribution of this thesis is the development of a novel experimental method coined tagRNA-seq, combining 5’tagRACE, a previously developed technique, with RNA-sequencing technology. Briefly, tagRNA-seq makes it possible to identify the 5’ ends of RNAs in bacteria and directly probe for their type, primary or processed, by ligating short RNA sequences, the tags, to the beginnings of RNA molecules. We used the method to directly probe for transcription start and processing sites in two bacterial species, Escherichiacoli and Enterococcus faecalis. It was also used to study polyadenylation in E. coli, where the ability to identify processed RNA molecules proved to be useful to separate direct and indirect regulatory effects of this mechanism. We also demonstrate how data from tagRNA-seq experiments can be used to increase confidence on the discovery of anti-sense transcripts in bacteria. Analyses of RNA-seq data obtained in the context of these experiments revealed subtle artifacts in the coverage signal towards gene ends, that we were able to explain and quantify based Kolmogorov’s broken stick model. We also discovered evidences for circularization of a few RNA transcripts, both in our own data sets and publicly available data. Designing the tags used in tagRNA-seq led us to the problem of words absent from a text. We focus on a particular subset of these, the minimal absent words (MAWs), and develop a theory providing a complete description of their size distribution in random text. We also show that MAWs in genomes from viruses and living organisms almost always exhibit a behavior different from random texts in the tail of the distribution, and that MAWs from this tail are closely related to sequences present in the genome that preferentially appear in regions with important regulatory functions. Finally, and independently from tagRNA-seq, we propose a new approach to the problem of bacterial community reconstruction in metagenomic, based on techniques from compressed sensing. We provide a novel algorithm competing with state-of-the-art techniques in the field. / <p>QC 20150930</p>

In den vergangenen Jahren rückten Pflanzen-assoziierte Bakterien auf Grund ihrer Bedeutung für die Pflanzengesundheit und das ökologische Gleichgewicht zunehmend in den Fokus aktueller Forschungen. Trotz der stetig steigenden Zahl wissenschaftlicher Studien ist der Einfluss von Bewirtschaftungsmaßnahmen auf die Diversität dieser Bakteriengemeinschaften in Grünlandökosystemen ver-gleichsweise wenig untersucht. In dieser Studie haben wir neue und interessante Erkenntnisse über die Diversität von Pflanzen-assoziierten Bakterien in Grünlandökosystemen gewonnen. Sämtliche Untersuchungen dieser Arbeit wurden auf der GrassMan-Fläche in den Mittelgebirgslagen des Solling in Deutschland durchgeführt. Das GrassMan-Experiment wurde 2008 in einer Matrix von Wiesenplots schachbrett-artig auf historisch altem Grünland errichtet. Die Bewirtschaftungsintensität unterschied sich bezüglich der Häufigkeiten (einmal jährlich im Juli oder dreimal jährlich im Mai, Juli und September) und der Düngung (keine Düngung bzw. Düngung mit NPK). Außerdem wurde durch gezielten Herbizid-Einsatz gegen Monokotylen oder gegen Dikotylen ein Gradient in der Anzahl der Pflanzenarten erzeugt. Die Arbeit umfasst drei Hauptthemen. Erstens wurde der Einfluss verschiedener Bewirtschaftungsmaßnahmen auf die bakterielle Endophyten-gemeinschaft in den drei Grasarten Festuca rubra, Lolium perenne und Dactylis glomerata untersucht. Hierfür wurden im September 2010 und im April, Juli und September 2011 Pflanzenproben auf den Dikotylen-reduzierten Plots gesammelt. Die Umwelt-DNS wurde aus den Proben extrahiert und als Template für 16S PCRs eingesetzt. Die Struktur der bakteriellen Endophyten-Gemeinschaft wurde mittels DGGE-Analyse der erhaltenen PCR-Produkte untersucht. Wir konnten Unterschiede der Endophyten-Gemeinschaftsstrukturen hinsichtlich der verschiedenen Bewirtschaftungsintensitäten feststellen. Während die Düngung einen starken Effekt auf die bakterielle endophytische Diversität sowohl in F. rubra als auch in L. perenne hatte, wurden die bakteriellen Endo-phyten in D. glomerata nicht dadurch beeinflusst. Die Proben von L. perenne, die von den ungedüngten Plots stammten, bildeten zudem eindeutige Gruppen bei der Analyse der DGGE-Banden bezüglich der zwei Schnitthäufigkeiten. Somit beeinflusste auch die Mahd die bakterielle Endophyten-Gemeinschaft in den Pflanzen. Weiterhin konnten wir einen starken saisonalen Effekt auf die Struktur der endophytischen Gemeinschaft nachweisen. Da saisonale Veränderungen und die Pflanzenart die Zusammensetzung der endophytischen Bakteriengemeinschaft beeinflussten, können sich die Auswirkungen unterschiedlicher Bewirtschaf-tungsintensitäten mit der Zeit und der untersuchten Pflanzenart verändern. Dieses Ergebnis sollte bei zukünftigen Studien berücksichtigt werden. Das zweite Ziel dieser Arbeit war die Beantwortung der Frage, wie sich oberirdische Herbivorie auf die Bakteriengemeinschaft in der Rhizosphäre auswirkt. Hierfür wurde im Herbst 2010 ein Lysimeter-Experiment auf der GrassMan-Fläche errichtet. Nach einer zweiwöchigen Herbivorie durch Grashüpfer und Schnecken im Sommer 2011 wurden Bodenproben von jedem Lysimeter genommen. Um Einblicke in die Zusammensetzung der bakteriellen Gemeinschaft in der Rhizosphäre zu erhalten, wurde die Gesamt-DNS aus den Bodenproben extrahiert und als Template in 16S rDNS PCRs eingesetzt. Die Gemeinschaftsstruktur wurde mittels DGGE-Analyse bzw. Pyrosequenzierung der erhaltenen PCR Produkte untersucht. Die Herbivorie hatte keinen Einfluss auf die Anzahl der Bakterien (richness), während leichte Änderungen in der relativen Abundanz von einigen Bakteriengruppen festgestellt wurden. So war zum Beispiel die relative Abundanz einer unkultivierten Acidobacterium-Art in den Herbivorie-Lysimetern erhöht. Bestandteil des Lysimeter-Experiments war zudem die Untersuchung des Einflusses der Pflanzenartenanzahl und der verschiedenen Bewirtschaftungs-maßnahmen auf die bakterielle Gemeinschaft in der Rhizosphäre. Der Einsatz von Herbiziden und eine niedrigere Schnittfrequenz reduzierten die Artenanzahl (richness) der Bakterien in der Rhizosphäre. Die Düngung hatte keinen Einfluss auf die Anzahl der Arten. Weitere Analysen zeigten, dass eine Vielzahl von verschiedenen bakteriellen Taxa in der Rhizosphäre durch die untersuchten Maßnahmen beeinflusst wurde. So war die Abundanz der Acidobacteria in den gedüngten Plots signifikant geringer. Das Gegenteil trat bei den Actinobacteria auf. Abschließend lässt sich sagen, dass Pflanzen-assoziierte Bakterien sowohl in der Endosphäre und Rhizosphäre durch Bewirtschaftungsmaßnahmen beeinflusst werden. Die Untersuchung der Wirkung von verschiedenen Bewirtschaftungsintensitäten im Grünland und von oberirdischer Herbivorie auf Pflanzen-assoziierte Bakterien kann zu einem besseren Verständnis der multitrophischen Interaktionen zwischen Pflanzenart, Bakteriengemeinschaft und oberirdischen Herbivoren führen. Außerdem können uns die Ergebnisse dieser Arbeit helfen, die Effekte unterschiedlicher Bewirtschaftungsmaßnahmen auf Pflanzen-assoziierte Bakteriengemeinschaften und damit zusammenhängende Effekte auf das Bodenökosystem vorherzusagen.

Viruses are the most abundant biological entity on Earth and outnumber their hosts ten-to-one. Ocean viruses (phages) impact bacterial-driven global biogeochemical cycles through lysis, manipulating host metabolism, and horizontal gene transfer. However, knowledge of virus-host interactions and viral roles in ecosystems remains limited due to few cultured marine phage genomes and non-quantitative culture-independent metagenomes. Here, I develop and apply novel and well-tested bioinformatic techniques to explore Pacific Ocean viral communities using quantitative datasets derived from rigorously-tested preparation methods. To evaluate concentration and purification methods, I examined triplicate metagenomes from a single ocean sample using four protocols. Concentration protocols showed statistical differences in taxonomy whereas purification protocols did not. Specifically, TFF-concentrated metagenomes contained trace bacterial contamination and had fewer abundant taxa as compared to FeCl₃-precipitated metagenomes. K-mer analysis using the complete dataset revealed polymerase choice defined access to "rare" sequences.To explore unknown viral sequences, I organized known and unknown sequence space into 27K high-confidence protein clusters (PCs) from 32 diverse Pacific Ocean Virus (POV) metagenomes, which doubled available PCs and included the first pelagic deep-sea viral metagenomes. Using PCs as a whole-viral-community diversity metric revealed decreases from coastal to open ocean, winter to summer, and deep to surface, that correlate with data from microbial genetic diversity markers (no parallel viral markers exist).Biologically, POV metagenomes showed that viruses likely reprogram central metabolic pathways in microbial communities far beyond the "photosynthesis viruses" paradigm. Gene distribution patterns from 35 viral gene families (31 new) revealed niche-specific (photic vs aphotic zone) altered pathway carbon flux presumably optimized to best locally generate energy and drive viral replication. Further, these PCs define the first "core" (180 genes) and "flexible" (423K genes total) viral community genome. Functionally, core genes again suggest niche-differentation with extensive Fe-S cluster-related genes for electron transport and metabolic enzyme catalysis in photic samples, and manipulation of host pressure-sensitive genes in aphotic samples. Taxonomically, these data deconstruct the culture-based paradigm that tailed viruses dominate in the wild - instead they appear ubiquitous, but not abundant.

No description available.

Aquatic bacteria respond to changing environmental conditions through a variety of mechanisms including changes in abundance, shifts in community composition and variable activity states. In the tidal-freshwater James River, variation in bacterial abundance was linked to nutrient availability and autochthonous production with highest bacterial densities associated with low-nutrient, high-chlorophyll a conditions. Laboratory experiments revealed that bacterial growth rates were nutrient limited at the low-nutrient site, while co-limitation (nutrients, glucose, light) was apparent at the high nutrient site. Despite large differences in abundance, community composition was similar based on TRFLP and 16S rDNA pyrosequencing. Community similarity was lower among rRNA libraries suggesting that variable activity states are prevalent in natural communities. Rare taxa were more likely to be metabolically active and were capable of dramatic growth under microcosm conditions.

Les microorganismes, par leurs fascinantes capacités d’adaptation liées à l’extraordinaire diversité de leurs capacités métaboliques, jouent un rôle fondamental dans les tous les processus biologiques. Ils interviennent notamment au niveau des changements globaux, comme le réchauffement climatique, en partie occasionné par les émissions croissantes de méthane dans l’atmosphère, mais également par les pollutions résultant de la dispersion de molécules comme les Hydrocarbures Aromatiques Polycycliques. Ainsi, les communautés microbiennes vont participer à réduire ou à augmenter les effets délétères de l’anthropisation des écosystèmes. La régulation des changements globaux passe donc par une meilleure connaissance de ces communautés qui doivent être explorées dans leur globalité au sein des environnements. Néanmoins en raison de leur forte complexité, une telle exploration n’est possible qu’en utilisant des outils d’analyse haut-débit. Cependant, l’emploi d’outils moléculaires à haut-débit comme les biopuces à ADN passe par la détermination de sondes combinant à la fois une forte sensibilité, une très bonne spécificité et un caractère exploratoire. Pour concevoir de telles sondes un nouveau logiciel KASpOD a donc été développé. De même, en utilisant des sondes présentant les mêmes caractéristiques, le développement d’une nouvelle approche innovante en écologie microbienne de capture de gènes en solution été entrepris. Cette nouvelle méthode d’enrichissement de gènes d’intérêt couplée à du séquençage haut-débit a été appliquée pour l’exploration des communautés méthanogènes du lac Pavin. Les résultats obtenus montrent la pertinence de l’approche qui assure une meilleure évaluation de diversité de l’écosystème avec notamment l’identification de populations appartenant à la biosphère rare. L’autre ajout majeur de cette approche est qu’elle autorise l’identification de grandes régions d’ADN génomique exploitable pour caractériser de nouveaux gènes ou de nouveaux processus adaptatifs. / Microorganisms play a crucial role in all biological processes related to their huge metabolic potentialities. They are involved in global changes such as global warming partially caused by the growing methane emissions in the atmosphere, but also by the release of pollutants such as Polycyclic Aromatic Hydrocarbons. Thus, microbial communities will contribute to reduce or increase the negative effects of human impacts on ecosystems. The regulation of global changes needs a better knowledge of the microbial communities involved in complex environments functioning. Nevertheless, a complete exploration of such environments requires the use of high-throughput tools, due to the extraordinary diversity of microorganisms within the ecosystems. The use of DNA microarrays requires a probe design step allowing the selection of highly sensitive, specific and explorative oligonucleotides. For this purpose, we have developed KASpOD, a new software, allowing the generation of efficient probes dedicated to environmental applications. Using high quality probe sets, an innovative in solution-based gene capture method combined with Next Generation Sequencing, was developed and applied for the exploration of the methanogen communities in lake Pavin, Results showed the relevance of this approach that allows a better evaluation of the methanogen diversity with an efficient detection of populations belonging to the rare biosphere. The other main advantage of this approach is the identification of large regions of genomic DNA, useful for the characterization of new genes or adaptive processes.

Through complex metabolic interactions aquatic microbial life is essential as a driver of ecosystem functions and hence a prerequisite for sustaining plant and animal life in the sea and on Earth. Despite its ecological importance, infor­mation on the complexity of microbial functions and how these are related to environmental conditions is limited. Due to climate change and eutrophication, marine areas facing oxygen depletion are increasing and predicted to continue to do so in the future. Vertically steep oxygen gradients are particularly pronoun­ced in the Baltic Sea. In this thesis, therefore, the ecosystem functions of micro­bial communities were investigated, using metagenomics, to understand how they were distributed along the steep oxygen gradient at the Landsort Deep, the deepest point of the Baltic Sea. Furthermore, microbial communities from the Lands­ort Deep transect were compared to microbial communities of other marine environments to establish whether the environment at this site resulted in a characteristic community. To reveal what microbial community functions and taxa were active in the anoxic sediment a metatranscriptomic approach was used. Results showed a marked effect of the coupled environmental parameters dissolved oxygen, salinity and temperature on distribution of taxa and par­ti­cularly community functions. Microbial communities showed functional capa­cities consistent with a copiotrophic life-style dependent on organic ma­terial sinking through the water column. The eutrophic condition with high organic load was further reflected in the metatranscriptome of the anoxic sedi­ment com­munity, which indicated active carbon mineralisation through ana­erobic hetero­trophic-autotrophic community synergism. New putative linkages between nitro­gen and- sulphur metabolisms were identified at anoxic depths. Further­more, viable Cyanobacteria in the anoxic sediment was evident from the tran­script analyses as another reflection of marine snow. High abundance and expres­­sion of integron integrases were identified as a charac­teristic feature of the Lands­ort Deep communities, and may provide these communities with a mech­an­ism for short-term-adaptation to environmental change. In summary, this thesis clearly documents what impact eutrophication and oxygen depletion have on microbial community functions. Furthermore, it specifically advances the mechanistic insight into microbial processes in anoxic deep-water sediment at both genomic and transcriptional level. Given the predicted progress of oxygen depletion in marine and brackish environments, this work advances information necessary to estimate effects on marine and in particular brackish ecosystem functions where anoxic conditions prevail. / Mikroorganismer är essentiella för fungerande ekosystemfunktioner i akvatiska miljöer och därmed en förutsättning för övrigt växt- och djurliv på vår planet. Trots deras ekologiska nyckelroll är kunskapen om mikroorganismernas funk­tion och komplexitet samt hur dessa är relaterade till miljön begränsad. På grund av eutrofiering och klimatförändringar har marina områden som lider av syrebrist ökat och en ytterligare utbredning av marina och bräckta områden med syrebrist är predicerad i framtiden. Stora områden av Östersjön kännetecknas av vertikala syregradienter med syresatt ytvatten och anoxiskt bottenvatten. I denna avhandling undersöktes därför med metagenomik hur mikrobiella ekosystems funktioner var utbredda längs den vertikala syregradienten i Östersjöns djupaste del, Landsortsdjupet. Dessutom jämfördes de mikrobiella samhällena från Lands­­­ortsdjupet med mikrobiella samhällen från andra marina miljöer för att utröna om den karakteristiska miljön i Landsortsdjupet återspeglade de mikro­biella samhällen som lever där. För att undersöka vilka mikroorganismer samt vilka mikrobiella ekosystemfunktioner som var aktiva i det anoxiska sedimentet i Lands­ortsdjupet användes metatranskriptomik. Resultaten visade en stark kor­re­lation mellan miljöparametrarna syrehalt, salinitet och temperatur och för­del­ningen av mikrobiell taxa och i synnerhet mikrobiell funktion längs Lands­orts­djupets transekt. De mikrobiella samhällena uppvisade en funktionell kapa­citet förenlig med en livsstrategi beroende av organiskt material som sjunker genom vattenkolonnen som en konsekvens av eutrofiering. Eutrofa förhållanden med hög halt av organiskt material var även återspeglad i metatranskriptomet från det anoxiska sedimentet, som indikerade aktiv mineralisering av organiskt kol genom anaerob heterotrof-autotrof synergism. Nya möjliga kopplingar mellan kväve- och svavelmetabolism identifierades i det anoxiska vattnet. Vidare visade resultat från metatranskriptom-analys att livsdugliga cyanobakterier var abun­danta i det mörka och anoxiska sedimentet, vilket även detta kan vara en konse­kvens av sjunkande organiskt material. Hög abundans och hög transkribering av integrongener kunde identifieras som ett karakteristiskt kännetecken hos de mikro­biella samhällena i Landsortsdjupet vilket skulle kunna förse dem med en me­kanism för anpassning till miljöförändringar. Sammanfattningsvis dokumen­terar denna avhandling tydligt vilken påverkan eutrofiering och syrebrist har på mikrobiella funktioner. Dessutom för den specifikt kunskapen om mikrobiella processer i anoxiska djupvattensediment framåt på både genom- och transkrip­tions­nivå. Mot bakgrund av en predicerad ökning av syrebristen i marina mil­jöer, bidrar denna avhandling med information som är viktig för att kunna förutse vilka effekter anoxiska förhållanden kan komma att få på ekosystemfunktioner i marina miljöer och i brackvattenmiljöer i synnerhet.