Background: Symbioses throughout the animal kingdom are known to extend physiological and ecological capabilities to hosts. Insect-microbe associations are extremely common and are often related to novel niche exploitation, fitness advantages, and even speciation events. These phenomena include expansions in host diet, detoxification of insecticides and toxins, and increased defense against pathogens. However, dissecting the contributions of individual groups of symbionts at the molecular level is often underexplored due to methodological and analytical limitations. Termites are one of the best studied systems for physiological collaborations between host and symbiota; however, most work in lower termites (those with bacterial and protist symbionts) focuses on the eukaryotic members of this symbiotic consortium. Here we present a metatranscriptomic analysis which provides novel insights into bacterial contributions to the holobiont of the eastern subterranean termite, Reticulitermes flavipes, in the presence and absence of a fungal pathogen. Results: Using a customized ribodepletion strategy, a metatranscriptome assembly was obtained representing the host termite as well as bacterial and protist symbiota. Sequence data provide new insights into biosynthesis, catabolism, and transport of major organic molecules and ions by the gut consortium, and corroborate previous findings suggesting that bacteria play direct roles in nitrogen fixation, amino acid biosynthesis, and lignocellulose digestion. With regard to fungal pathogen challenge, a total of 563 differentially expressed candidate host and symbiont contigs were identified (162 up-and 401 downregulated; a/FDR = 0.05) including an upregulated bacterial amidohydrolase. Conclusions: This study presents the most complete bacterial metatranscriptome from a lower termite and provides a framework on which to build a more complete model of termite-symbiont interactions including, but not limited to, digestion and pathogen defense.

abstract: This thesis research focuses on phylogenetic and functional studies of microbial communities in deep-sea water, an untapped reservoir of high metabolic and genetic diversity of microorganisms. The presence of photosynthetic cyanobacteria and diatoms is an interesting and unexpected discovery during a 16S ribosomal rRNA-based community structure analyses for microbial communities in the deep-sea water of the Pacific Ocean. Both RT-PCR and qRT-PCR approaches were employed to detect expression of the genes involved in photosynthesis of photoautotrophic organisms. Positive results were obtained and further proved the functional activity of these detected photosynthetic microbes in the deep-sea. Metagenomic and metatranscriptomic data was obtained, integrated, and analyzed from deep-sea microbial communities, including both prokaryotes and eukaryotes, from four different deep-sea sites ranging from the mesopelagic to the pelagic ocean. The RNA/DNA ratio was employed as an index to show the strength of metabolic activity of deep-sea microbes. These taxonomic and functional analyses of deep-sea microbial communities revealed a `defensive' life style of microbial communities living in the deep-sea water. Pseudoalteromonas sp.WG07 was subjected to transcriptomic analysis by application of RNA-Seq technology through the transcriptomic annotation using the genomes of closely related surface-water strain Pseudoalteromonas haloplanktis TAC125 and sediment strain Pseudoalteromonas sp. SM9913. The transcriptome survey and related functional analysis of WG07 revealed unique features different from TAC125 and SM9913 and provided clues as to how it adapted to its environmental niche. Also, a comparative transcriptomic analysis of WG07 revealed transcriptome changes between its exponential and stationary growing phases. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2013

abstract: This project was completed to understand the evolution of the ability to digest wood in termite symbiotic protists. Lower termites harbor bacterial and protist symbionts which are essential to the termite ability to use wood as a nutritional source, producing glycoside hydrolases to break down the polysaccharides found in lignocellulose. Yet, only a few molecular studies have been done to confirm the protist species responsible for particular enzymes. By mining publicly available and newly generated genomic and transcriptomic data, including three transcriptomes from isolated protist cells, I identify over 200 new glycoside hydrolase sequences and compute the phylogenies of eight glycoside hydrolase families (GHFs) reported to be expressed by termite hindgut protists. Of those families examined, the results are broadly consistent with Todaka et al. 2010, though none of the GHFs found were expressed in both termite-associated protist and non-termite-associated protist transcriptome data. This suggests that, rather than being inherited from their free-living protist ancestors, GHF genes were acquired by termite protists while within the termite gut, potentially via lateral gene transfer (LGT). For example one family, GHF10, implies a single acquisition of a bacterial xylanase into termite protists. The phylogenies from GHF5 and GHF11 each imply two distinct acquisitions in termite protist ancestors, each from bacteria. In eukaryote-dominated GHFs, GHF7 and GHF45, there are three apparent acquisitions by termite protists. Meanwhile, it appears prior reports of GHF62 in the termite gut may have been misidentified GHF43 sequences. GHF43 was the only GHF found to contain sequences from the protists not found in the termite gut. These findings generally all support the possibility termite-associated protists adapted to a lignocellulosic diet after colonization of the termite hindgut. Nonetheless, the poor resolution of GHF phylogeny and limited termite and protist sampling constrain interpretation. / Dissertation/Thesis / Masters Thesis Biological Design 2019

The interaction between figs (Ficus spp.) and their pollinating wasps (fig wasps; Chalcidoidea, Hymenoptera) is a classic example of an ancient and apparently stable mutualism. A striking property of this mutualism is that fig wasps consistently oviposit in the inner flowers of the fig syconium (gall flowers, which develop into galls that house developing larvae), but typically do not use the outer ring of flowers (seed flowers, which are pollinated and develop into seeds). This dissertation explores the potential differences between gall and seed flowers that might influence oviposition choices, and the unknown mechanisms underlying gall formation. To identify the microbial community that could influence oviposition choice, I identified fungi in both flower types across six species of Ficus. I found that whereas fungal communities differed significantly as a function of developmental stages of syconia and lineages of fig trees, communities did not differ significantly between receptive gall and seed flowers. Because secretions from the poison sac that are deposited at oviposition are thought to be important in gall formation by both pollinating fig wasps and non-pollinating, parasitic wasps, I examined poison sac morphology in diverse galling wasps from several species of Ficus in lowland Panama. I found that the size of the poison sac was positively associated with egg number across pollinating and non-pollinating fig wasps. Finally to determine difference in defense and metabolism between gall and seed flowers, and to identify genes involved in galling, I compared gene expression profiles of fig flowers at the time of oviposition choice and early gall development. I found a prominence of flavonoids and defensive genes in both pollinated and receptive gall flowers of Ficus obtusifolia, and revealed detectable differences between gall flowers and seed flowers before oviposition. Several highly expressed genes were also identified that have implications for the mechanism of gall initiation. This dissertation explores previously unstudied aspects of the fig and fig wasp mutualism and provides important molecular tools for future study of this iconic and ecologically important association.

Deep-sea corals have long been regarded as cold-water coral; however a reevaluation of their habitat limitations has been suggested after the discovery of deep-sea coral in the Red Sea where temperatures exceed 20˚C. To gain further insight into the biology of deep-sea corals at these temperatures, the work in this PhD employed a holotranscriptomic approach, looking at coral animal host and bacterial symbiont gene expression in Dendrophyllia sp., Eguchipsammia fistula, and Rhizotrochus sp. sampled from the deep Red Sea. Bacterial community composition was analyzed via amplicon-based 16S surveys and cultured bacterial strains were subjected to bioprospecting in order to gauge the pharmaceutical potential of coralassociated microbes. Coral host transcriptome data suggest that coral can employ mitochondrial hypometabolism, anaerobic glycolysis, and surface cilia to enhance mass transport rates to manage the low oxygen and highly oligotrophic Red Sea waters. In the microbial community associated with these corals, ribokinases and retron-type reverse transcriptases are abundantly expressed. In its first application to deep-sea coral associated microbial communities, 16S-based next-generation sequencing found that a single operational taxonomic unit can comprise the majority of sequence reads and that a large number of low abundance populations are present, which cannot be visualized with first generation sequencing. Bioactivity testing of selected bacterial isolates was surveyed over 100 cytological parameters with high content screening, covering several major organelles and key proteins involved in a variety of signaling cascades. Some of these cytological profiles were similar to those of several reference pharmacologically active compounds, which suggest that the bacteria isolates produce compounds with similar mechanisms of action as the reference compounds. The sum of this work offers several mechanisms by which Red Sea deep-sea corals cope with environmental conditions in which no other deep-sea corals have yet to be reported. These deep-sea coral are associated with rich microbial communities, which produce molecules that induce bioactivity. The aggregate of this work provides direction for future research of Red Sea deep-sea coral and highlights the potential pharmacological benefit of conserving these species and their unique ecosystem.

Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen. It causes blinding trachoma and sexually transmitted disease such as chlamydia, pelvic inflammatory disease and lymphogranuloma venereum. Ct has a unique biphasic development cycle and replicates in an intracellular vacuole called inclusion. Normally it has two forms: the infectious form, elementary body (EB); and the non-infectious form, reticulate body (RB). Ct is not easily amenable to genetic manipulation. Hence, to understand the infection process, it is crucial to study how the metabolic activity of Ct exactly evolves in the host cell and what roles of EB and RB play differentially in Ct metabolism during infection. In addition, Ct was found regularly coinfected with other pathogens in patients who got sexually transmitted diseases (STDs). A lack of powerful methods to culture Ct outside of the host cell makes the detailed molecular mechanisms of coinfection difficult to study. In this work, a genome-scale metabolic model with 321 metabolites and 277 reactions was first reconstructed by me to study Ct metabolic adaptation in the host cell during infection. This model was calculated to yield 84 extreme pathways, and metabolic flux strength was then modelled regarding 20hpi, 40hpi and later based on a published proteomics dataset. Activities of key enzymes involved in target pathways were further validated by RT-qPCR in both HeLa229 and HUVEC cell lines. This study suggests that Ct's major active pathways involve glycolysis, gluconeogenesis, glycerolphospholipid biosynthesis and pentose phosphate pathway, while Ct's incomplete tricarboxylic acid cycle and fatty acid biosynthesis are less active. EB is more activated in almost all these carbohydrate pathways than RB. Result suggests the survival of Ct generally requires a lot of acetyl-CoA from the host. Besides, both EB and RB can utilize folate biosynthesis to generate NAD(P)H but may use different pathways depending on the demands of ATP. When more ATP is available from both host cell and Ct itself, RB is more activated by utilizing energy providing chemicals generated by enzymes associated in the nucleic acid metabolism. The forming of folate also suggests large glutamate consumption, which is supposed to be converted from glutamine by the glutamine-fructose-6-phosphate transaminase (glmS) and CTP synthase (pyrG). Then, RNA sequencing (RNA-seq) data analysis was performed by me in a coinfection study. Metatranscriptome from patient RNA-seq data provides a realistic overview. Thirteen patient samples were collected and sequenced by our collaborators. Six male samples were obtained by urethral swab, and seven female samples were collected by cervicovaginal lavage. All the samples were Neisseria gonorrhoeae (GC) positive, and half of them had coinfection with Ct. HISAT2 and Stringtie were used for transcriptomic mapping and assembly respectively, and differential expression analysis by DESeq2, Ballgown and Cuffdiff2 are parallelly processed for comparison. Although the measured transcripts were not sufficient to assemble Ct's transcriptome, the differential expression of genes in both the host and GC were analyzed by comparing Ct positive group (Ct+) against Ct-uninfected group. The results show that in the Ct+ group, the host MHC class II immune response was highly induced. Ct infection is associated with the regulation of DNA methylation, DNA double-strand damage and ubiquitination. The analysis also shows Ct infection enhances host fatty acid beta oxidation, thereby inducing mROS, and the host responds to reduce ceramide production and glycolysis. The coinfection upregulates GC's own ion transporters and amino acid uptake, while it downregulates GC's restriction and modification systems. Meanwhile, GC has the nitrosative and oxidative stress response and also increases the ability for ferric uptake especially in the Ct+ group compared to Ct-uninfected group. In conclusion, methods in bioinformatics were used here in analyzing the metabolism of Ct itself, and the responses of the host and GC respectively in a coinfection study with and without Ct. These methods provide metabolic and metatranscriptomic details to study Ct metabolism during infection and Ct associated coinfection in the human microbiota. / Chlamydia trachomatis (Ct) ist ein obligater intrazellulärer Pathogen des Menschen. Er verursacht Trachoma und sexuell übertragbare Krankheiten, wie Chlamydiose, Unterleibsentzündung und Lymphogranuloma venereum. Ct besitzt einen biphasischen Entwicklungszyklus und vermehrt sich in intrazellulären Vakuolen, sogenannten Einschlusskörperchen. Normalerweise können zwei Formen beobachtete werden: Die infektiöse Form, Elementarkörperchen (EK); und die nicht-infektiöse Form, Retikularkörperchen (RK). Ct ist nicht einfach genetisch zu manipulieren. Um den Infektionsablauf besser zu verstehen, ist es wichtig, zu untersuchen, wie sich genau die metabolische Aktivität von Ct in der Wirtszelle entwickelt und welche Rolle EK und RK im Metabolismus von Ct während der Infektion spielen. Zusätzlich wurde Ct häufig bei Patienten mit sexuell übertragbaren Krankheiten (STD) in Co-Infektion mit anderen Erregern gefunden. Ein Mangel an leistungsfähigen Methoden zur Kultivierung von Ct außerhalb der Wirtszelle macht es schwierig die genauen molekularen Mechanismen von Co-Infektionen zu untersuchen. In dieser Arbeit wurde erstmals ein genomweites metabolisches Model mit 321 Metaboliten und 277 Reaktionen aufgebaut, um die metabolische Adaption von Ct in der Wirtzelle während der Infektion zu untersuchen. Dieses Model wurde erstellt und umfasst 84 „extreme pathways“ (Grenz-Stoffwechselwege). Darauf aufbauend wurde die metabolische Fluss-Stärke berechnet. Die Zeitpunkte 20 hpi (20 Stunden nach der Infektion), 40 hpi und die anschließende Infektionsphase wurden durch Nutzung von Proteom-Daten modelliert. Die Aktivitäten von Schlüsselenzymen, welche in wichtigen Stoffwechselwegen involviert sind, wurden zusätzlich durch RT-qPCR überprüft. Dabei wurden die Ergebnisse sowohl für HeLA229- als auch HUVEC-Zellen nachgemessen. Diese Untersuchungen zeigten, dass Ct’s wichtigste aktive Stoffwechselwege die Glykolyse, die Gluconeogenese und der Pentosephosphatweg sind, während der unvollständige Zitronensäurezyklus und die Fettsäuresynthese weniger aktiv sind. Gegenüber RK sind bei EK fast alle diese Kohlenhydratwege stärker aktiviert. Im Allgemeinen benötigt Ct eine größere Menge an Acetyl-CoA. Außerdem können sowohl EK, als auch RK die Folsäurebiosynthese nutzen, um NAD(P)H zu generieren. Dabei werden möglicherweise unterschiedliche Pathways genutzt, abhängig vom Bedarf an ATP. Sobald mehr ATP sowohl durch die Wirtszellen als auch von der Ct-Zelle selbst zur Verfügung steht, wird die Nutzung von Energieträgern, produziert durch Enzyme des Nukleinsäurestoffwechsels, in RK stärker aktiviert. Die Bildung von Folsäure lässt den Schluss zu, dass große Mengen von Glutamat umgesetzt werden, welches vermutlich aus der Umwandlung von Glutamin durch die Glutamine-fructose-6-phosphate-transaminase (glmS) und CTP-Syntase (pyrG) stammt. Anschließend wurde eine Analyse von RNA-Sequenzierungsdaten (RNA-seq) aus einer Co-Infektions-Studie (Chlamydien und andere Keime, insbesondere Gonokokken (GC)) durchgeführt. Dafür wurden Proben von dreizehn Patienten gesammelt und von Kollaborationspartnern sequenziert. Sechs Proben männlicher Patienten wurden durch Abstrich der Harnröhre und sieben Proben weiblicher Patientinnen durch cervicovaginale Lavage gewonnen. Alle Proben waren Neisseria gonorrhoeae (GC) positiv, wobei die Hälfte eine Co-Infektion mit Ct aufwies. Die Programme HISAT2 and Stringtie wurden zum Abbilden der transgenomischen Reads beziehungsweise zur Assemblierung des Genoms verwendet, und eine Analyse der differentiellen Expression wurde jeweils mit DESeq2, Ballgown und Cuffdiff2 durchgeführt und die Ergebnisse verglichen. Obwohl nicht ausreichend viele Transkripte von Ct gewonnen werden konnten, um das Transkriptom komplett assemblieren zu können, wurde die differentielle Expression der Gene sowohl von Wirt als auch von GC durch den Vergleich zwischen der Gruppe der Ct-positiven (Ct+) der Gruppe der Ct-unifizierten Patienten analysiert. Die Ergebnisse zeigten, dass in der (Ct+)-Gruppe die auf der MHC-Klasse-II basierte Immunantwort stark induziert war. Die Infektion von Ct ist mit der Regulation der DNA-Methylierung, DNA-Doppel-Strang-Schädigung und Ubiquitinierung verbunden. Die Analyse zeigte zusätzlich, dass die Infektion mit Ct die Fettsäure β-Oxidation des Wirts steigert, dadurch mROS induziert, und sowohl die Ceramid-Produktion als auch die Glycolyse reduziert. Die Co-Infektion reguliert GC’s eigene Eisentransporter und Aminosäureaufnahme hoch, während Restriktions- und Modifikationssysteme herunterreguliert werden. Gleichzeitig zeigt GC sowohl eine stickstoffsensitve Stress Antwort als auch eine oxidative. Dies verstärkt zusätzlich die Fähigkeit für die Aufnahme von Eisen, insbesondere in der (Ct+)-Gruppe. Zusammenfassend wurden Methoden der Bioinformatik genutzt, um den Metabolismus von Ct selbst, und die Antwort des Wirtes respektive GC‘s in einer Co-Infektionsstudie mit und ohne Ct zu analysieren. Diese Methoden lieferten wichtige metabolische und metatranskriptomische Details, um den Metabolismus von Ct während der Infektion, aber auch das Mikrobiom während einer Ct assoziierter Co-Infektion zu untersuchen.

No description available.

Les sols pollués par des métaux lourds sont colonisés par des communautés de microorganismes qui ont développé différentes adaptations leur permettant de résister à ces contaminants. Afin d'analyser au niveau moléculaire la diversité de ces adaptations, une approche expérimentale innovante basée sur l'étude du méta transcriptome eucaryote des sols a été utilisée pour comparer les fonctions exprimées au sein d'une communauté de microorganismes eucaryotes colonisant un sol contaminé, anciennement contaminé et non contaminé par des métaux lourds. Les banques d’ADNc eucaryotes construites à partir des ARNm extraits directement de ces sols ont été criblées par séquençage aléatoire de leurs inserts et par complémentation fonctionnelle de mutants de levures sensibles au cadmium.Cette étude a permis d’identifier de nouveaux gènes et de nouveaux mécanismes impliqués dans la résistance au cadmium ainsi qu’un nombre important de nouvelles protéines hypothétiques. Ceci démontre l’intérêt appliqué de cette approche pour la recherche de nouveaux bio catalyseurs et molécules bio actives.En parallèle, la diversité microbienne eucaryote a été révélée et comparée entre les sols par le clonage-séquençage du gène codant la petite sous-unité ribosomique 18S. Cette étude a mis en évidence une diversité inattendue de microorganismes eucaryotes dans ces sols et une analyse phylogénétique a permis de découvrir un nouveau clade de protistes (Rhizaria,Cercozoa). / Heavy metal-polluted soils are colonised by microbial communities which have developed different adaptations that allow them to resist to these pollutants. The objectives of this study were to reveal at the molecular level the diversity of these adaptations. To this aim,we implemented an innovative approach based upon the analysis of soil eukaryotic metatranscriptome to compare resistance mechanisms expressed by eukaryotic microorganisms living in heavy metal contaminated and control non contaminated or formerly-contaminated soils. Eukaryotic cDNA libraries were prepared using mRNA directly extracted from these soils and screened by either systematic sequencing of their inserts or functional complementation of yeast mutants sensitive to cadmium. This study allowed us to characterise novel genes and mechanisms implicated in Cd resistance as well as numerousnovel hypothetical proteins. This study also demonstrates the potential of this experimental approach to look for novel biocatalysts as well as novel bio-active molecules.In addition, eukaryotic molecular diversity was studied by the cloning-sequencing of the gene encoding the 18S ribosomal RNA. This study revealed an unexpected diversity of eukaryotic diversity in the studied soils and allowed us to discover a novel clade of protists(Rhizaria, Cercozoa).

No description available.

Les tourbières couvrent 3 % des surfaces continentales et jouent un rôle important dans le cycle du carbone en stockant le tiers du carbone des sols. L'accumulation de tourbe est liée au déséquilibre production primaire/décomposition du à une activité microbienne limitée par les conditions environnementales. L'infection et la lyse virale ont un impact sur la diversité et l'activité des communautés microbiennes, et influent sur le cycle du carbone. Cependant, le fonctionnement de ce compartiment viral n'avait jamais été pris en compte dans les études de fonctionnement des tourbières. Le but de ce travail de thèse était d'analyser et comprendre la dynamique spatiale et temporelle de l'abondance et de la diversité virale des tourbières à Sphagnum. L'analyse de l'abondance virale et procaryote et de 12 metaviromes en lien avec la physico-chimie d'une tourbière tempérée en France montre une forte variation saisonnière des communautés virales. Cette variation semble très liée aux conditions environnementales générées par la fluctuation de la nappe d'eau. Dans cette même tourbière, l'analyse de la diversité taxonomique et fonctionnelle des communautés de microorganismes présents (métagénomes) et métaboliquement actifs (métatranscriptomes) indique que la structure taxonomique est différente entre les des deux principaux stades, le fen et le bog, mais que ces communautés présentent une diversité fonctionnelle similaire, dont l'expression est liée aux changements des conditions environnementales avec la profondeur. L'abondance des particules virales étudiées dans 5 tourbières à Sphagnum réparties en Finlande, au Canada, en France, et sur l'île subantarctique d'Amsterdam varie fortement avec les sites. L'analyse de la diversité virale de la matrice et de l'eau de tourbe du Canada et de Finlande montre que la diversité virale est structurée par le site, puis le stade dynamique, puis la profondeur, avec un rôle important de la saturation en eau au niveau du site. Ces résultats valident le fonctionnement proposé du compartiment viral et de la communauté d'hôtes procaryotes. Ces connaissances ont été utilisées pour analyser le fonctionnement du compartiment microbien de tourbières à Sphagnum soumises à des perturbations d'origine anthropique. Les 31 métaviromes produits pour cette thèse constituent l'une des plus grandes bases de données sur la diversité virale des écosystèmes alors que la diversité virale des sols n'avait presque jamais été étudiée auparavant. / Peatlands cover 3 % of the continental surfaces but represent up to a third of the soil carbon stock. Peat accumulation results from the imbalance between primary production and decomposition due to the limitation of the prokaryote activity caused by the environmental conditions. Viral infection and lysis impact the diversity and the activity of the microbial communities and influence the carbon cycle. However, the functioning of the viral compartment had never been taken into account in peatlands. The aim of this thesis was to gain knowledge about the spatio-temporal dynamic of viral abundance and diversity in Sphagnum-dominated peatlands. Spatio-temporal analysis of viral and prokaryote abundance and of 12 metaviromes (viral diversity) in relation to the physico-chemical features in a temperate Sphagnum-dominated peatland in France revealed the high seasonal variability of the viral communities. This dynamic appeared mainly related to the environmental conditions shaped by the fluctuation of the water-table level. In the same peatland the taxonomic diversity of the present microorganisms (metagenomes) differed between the fen and the bog, but these communities present a similar functional diversity, which expression in selected in the same way in the two dynamic stages, in relation to depth-related environmental conditions. Viral abundance analyzed in 5 Sphagnum-dominated peatlands from Finland, Canada, France and subantarctic Amsterdam Isle presented a high geographical variability. Investigation of the diversity of the viral communities from the peat matrix and the pore-water in Finland and Canada emphasized the structuration of the viral communities by the site, then the dynamic stage, and finally depth. These results confirm the first hypotheses about the functioning of the viral compartment depending on environmental conditions and prokaryote activity. Effects of human-derived disturbances on viral ecology in peatlands were investigated based on this knowledge. While soil viral diversity was poorly documented at the start of this thesis, the collection of 31 metaviromes from Sphagnum-dominated peatlands produced for this project represents the second largest dataset representing the viral diversity from environmental samples.