Le rôle des protéines avec un motif TerD est depuis toujours insaisissable. La séquence en acides aminés qui correspond au motif TerD est répandue dans les génomes de plusieurs espèces bactériennes. Les recherches effectuées dans le cadre de ce doctorat avaient pour objectif d’identifier le rôle du gène tdd8 (SCO2368) qui code pour une protéine avec un motif TerD chez Streptomyces coelicolor. Sur la base d’une étude comparative du transcriptome de souches présentant une expression différentielle de tdd8, il a été possible de déterminer l’implication de tdd8 dans plusieurs systèmes de régulation. Les résultats obtenus ont permis d'établir que le niveau d’expression de tdd8 peut jouer un rôle dans le mécanisme de la différenciation morphologique et de la sporulation, dans le métabolisme de l’azote et dans l’équilibre redox. La protéine Tdd8 semble avoir un rôle dans divers processus cellulaires de par son implication dans l’homéostasie du calcium intracellulaire qui a été démontrée dans cette étude. Parmi les gènes qui semblent affectés par le taux d’expression de tdd8, ces recherches ont identifié un regroupement de gènes impliqués dans la réponse au stress redox. La plupart de ces gènes sont positionnés sur deux loci et leur expression implique un système de régulation analogue au régulon DosR retrouvé chez Mycobactérium tuberculosis. La croissance de la souche M145 de S. coelicolor en conditions de stress (hypoxie et présence d’oxyde nitrique) a permis de confirmer l’induction de ces gènes et des recherches bioinformatiques ont permis d’identifier un motif de liaison DosR dans les séquences qui précèdes la région codante de plusieurs gènes situés dans les deux loci identifiés. Les recherches ont également permis une meilleure caractérisation du métabolisme de l’azote et notamment une implication de tdd8 dans la régulation de ce métabolisme. Ces travaux s’inscrivent dans un processus de recherche fondamentale qui permet de mieux comprendre le rôle des protéines avec un motif TerD.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A alga marinha vermelha Solieria filiformis, abundante no litoral cearense, biossintetiza, dentre outros polissacarÃdeos, um ficocolÃide denominado carragenana de grande importÃncia comercial devido Ãs suas propriedades espessantes, estabilizantes e gelificantes. No Brasil, espÃcies exÃticas de algas marinhas vÃm sendo cultivadas em escala comercial com o objetivo de suprir a demanda industrial desse ficocolÃide. O objetivo desse trabalho foi analisar a textura de gÃis aquosos e lÃcteos elaborados com ι-carragenana extraÃda da alga S. filiformis, cultivada no litoral cearense atravÃs da tÃcnica de esporulaÃÃo natural, com a finalidade de estabelecer um comparativo entre esses gÃis e aqueles elaborados com carragenanas comerciais (ι e κ carragenanas). As algas foram cultivadas atravÃs de esporulaÃÃo natural, onde mensalmente estruturas de PVC (25 mm) de tamanho 25 x 44 cm, contendo cada uma Ãnica corda de nylon (10 mm) de 23 m de comprimento foram mantidas em profundidades de 1 e 2 m durante um perÃodo de nove meses. Para extraÃÃo da ι-carragenana foram coletadas em ambas profundidades amostras da alga S. filiformis dos bastidores que apresentaram maior e menor biomassa, determinadas para a estaÃÃo chuvosa (Janeiro- 26,2 Kg e Abril- 46,5 Kg) e seca (Outubro- 53,0 Kg e Novembro- 31,7 Kg). Inicialmente, as algas foram secas, trituradas em moinho elÃtrico e submetidas à extraÃÃo aquosa (1,5% m⁄v), sob agitaÃÃo a cada 20 minutos, em banho-maria a 90 ÂC durante 4 h. Os homogenatos foram filtrados em tecido de nylon, os resÃduos descartados. Os filtrados foram submetidos a uma filtraÃÃo à vÃcuo em funil de placa sinterizada, e as carragenanas obtidas foram congeladas, liofilizadas e pesadas. Os maiores rendimentos de ι-carragenana obtidos foram das algas coletadas dos bastidores que acumularam esporos no perÃodo seco (35,6%- Novembro 2M a 45,3%- Outubro 1M), enquanto as algas que acumularam no perÃodo chuvoso apresentaram os menores rendimentos (29,6%- Abril 1M a 33,8%- Abril 2M). Jà os teores de aÃÃcares totais variaram entre 37,6 e 50,5% e os teores de sulfato das carragenanas variaram de 21,6 a 33,3%, de acordo com os teores jà verificados para carragenanas do tipo ι. A textura dos gÃis aquosos na presenÃa de CaCl2 0,1% e lÃcteos preparados com as ι- caragenanas de S. filiformis e caragenanas comerciais nas concentraÃÃes de 0,5 e 1,5%, foram analisados quanto a firmeza, adesividade, coesividade, elasticidade, gomosidade e mastigabilidade. Todas as amostras com ι-carragenanas de S. filiformis dos bastidores obtiveram valores de firmeza, gomosidade e mastigabilidade superiores aos encontrados para ι-carragenana comercial. Os bastidores de novembro das amostras com ι-carragenanas de S. filiformis obtiveram os maiores valores no parÃmetro de firmeza para os gÃis aquosos e lÃcteos quando comparados aos gÃis comerciais. Adesividade, elasticidade e coesividade mostraram valores prÃximos aos encontrados para ι-carragenana comercial. Os resultados obtidos para firmeza e mastigabilidade da κ-carragenana foram superiores quando comparados a ι-carragenana de S. filiformis e ι-carragenana comercial. Os gÃis formulados com leite reconstituÃdo obtiveram resultados de firmeza superiores aos formulados com soluÃÃo de cloreto de cÃlcio 0,1%, devido Ãs interaÃÃes entre as proteÃnas do leite e a ι-carragenana. Com os dados obtidos pode-se concluir que a utilizaÃÃo da ι-carragenana de S. filiformis oriunda de cultivo por esporulaÃÃo natural à capaz de formar gÃis mais firmes quando comparada a ι- carragenana comercial, podendo gerar economia atravÃs da utilizaÃÃo de concentraÃÃes de carragenana inferiores a comercial, para se obter a firmeza desejada para um produto. / The red seaweed Solieria filiformis, abundant in CearÃ, biossintetiza, among other polysaccharides, phycoloide called carrageenan of great commercial importance due to its properties thickeners, stabilizers and gelling agents. In Brazil, exotic species of marine algae are being grown on a commercial scale in order to meet the demand of industrial phycoloide. The aim of this study was to analyze the texture of aqueous gels made with milk and carrageenan extracted from seaweed S. filiformis, cultivated in Cearà through sporulation natural technique, in order to establish a comparison between these gels and those prepared with commercial carrageenans (iota and kappa carrageenan). The algae were grown through sporulation natural, where monthly PVC structures (25 mm) in size 25 x 44 cm, each containing a single nylon cord (10 mm) of 23 m length were kept at depths of 1 and 2 m over a period of nine months. For extraction of carrageenan were collected at both depths seaweed samples S. filiformis backstage with higher and lower biomass, some for the rainy season (January and April) and dry (October and November). Initially, the algae were dried, crushed in electric grinder and subjected to aqueous extraction (1.5% m / v), stirring every 20 minutes in a water bath at 90 ÂC for 4 h. The homogenates were filtered through nylon cloth, the waste discarded. The filtrates were subjected to filtration on a vacuum funnel sintered plate, and carrageenans obtained were frozen, lyophilized and weighed. The highest yields were obtained from carrageenan seaweed collected from the scenes that have accumulated during the dry spores (35.6% - November 2M to 45.3% - October 1M), while the algae that accumulated during the rainy season had the lowest incomes (29 6% - April 1M to 33.8% - April 2M). In contrast, levels of total sugars ranged between 37.6 and 50.5% and the content of carrageenan sulfate ranged from 21.6 to 33.3% according to the levels already checked to carrageenans of the iota type. The texture of aqueous gels in the presence of CaCl2 and 0.1% milk prepared with carrageenan S. filiformis and commercial concentrations of 0.5 and 1.5%, were analyzed for firmness, adhesiveness, cohesiveness, springiness, gumminess and chewiness. All samples with carrageenan derived from S. filiformis backstage obtained values of firmness, gumminess and chewiness than those found for ι-carrageenan commercial. Backstage November had the highest values in the parameter of firmness for aqueous gels and compared to ι-carrageenan commercial dairy. Adhesiveness, elasticity and cohesiveness showed values close to those found for ι-carrageenan commercial. The results for firmness and chewiness of κ-carrageenan were superior when compared to commercial car ι-carrageenan and S. filiformis. The gels formulated with milk obtained results strongly higher than those formulated with a solution of calcium chloride 0.1% due to interactions between the proteins and the milk carrageenan. With the data obtained it can be concluded that the use of carrageenan S. filiformis derived by sporulation natural cultivation is capable of forming gels when compared to stronger ι-carrageenan commercial could generate savings through the use of lower concentrations of the carrageenan commercial, to obtain the desired firmness of a product.

Signal transduction pathways allow organisms to sense and respond appropriately to a complex bouquet of environmental cues. The molecular determinants of specificity are constrained by the demands of signaling fidelity, yet flexible enough to allow pathway remodeling to meet novel environmental challenges. A detailed picture of how these forces shape bacterial two-component signaling systems has emerged over the last decade. However, the tension between constraint and flexibility in more complex architectures has not been well-studied. In this thesis, I combine comparative genomics and in vitro phosphotransfer experiments to investigate pathway remodeling using the Firmicutes sporulation initiation (Spo0) pathway as a model. The present-day Spo0 pathways in Bacilli and Clostridia share common ancestry, but possess different architectures. In Clostridia, a sensor kinase phosphorylates Spo0A, the master regulator of the sporulation, directly. In Bacilli, Spo0 is phosphorylated/activated indirectly via a four-protein phosphorelay. The presence in sister lineages of signaling pathways that activate the same response regulator and control analogous phenotypes, yet possess with different architectures, suggests a common ancestral pathway that evolved through interaction remodeling. The prevailing theory is that the ancestral pathway was a simpler, direct phosphorylation architecture; the more complex phosphorelay emerged within the Bacillar lineage. In contrast to this prevailing view, my analysis of 84 representative genomes supports a novel hypothesis for the evolution of Spo0 architectures, wherein the two protein, direct phosphorylation architecture is a derived state, which arose from an ancestral Spo0 phosphorelay. The combination of my bioinformatic analysis and the first experimental characterization of a Clostridial phosphorelay provide evidence for the presence of functional phosphorelays in both classes Bacilli and Clostridia. Further, a cross-species complementation assay between phosphorelays from each class suggests that interaction specificity has been conserved since the divergence of this phylum, 2.7 BYA. My results reveal a patchy phylogenetic distribution of both Spo0 pathway architectures, consistent with repeated remodeling events, in which a phosphorelay was replaced with a two protein, direct phosphorylation pathway. This remodeling likely occurred via acquisition of a sensor kinase with direct specificity for Spo0A. Further, my analysis suggests that the unusual architectures of the Spo0 pathway and its striking tendency to gain and lose interactions may be due to the juxtaposition of three key properties: the maintenance of interaction specificity through molecular recognition; the ecological role of endosporulation; and the degeneracy of interaction space that permits the ongoing recruitment of kinases to recognize novel environmental signals.

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to European indigenous crayfish species, e.g., the noble crayfish (Astacus astacus). The only way to protect susceptible crayfish species from the disease is to prevent the dispersion of the pathogen to their populations. One of the most important sources of the crayfish plague pathogen in Central Europe is the spiny-cheek crayfish (Orconectes limosus), a species of North American origin, which can carry the parasite in its cuticle for years. Some literature sources claimed that the pathogen dispersion from the American vectors is restricted to periods of moulting or to the time before and after the crayfish death. However, experimental evidence for such hypotheses was lacking. The main aim of my thesis was to test these predictions, and the alternative scenario that the crayfish plague pathogen can be transmitted from the infected spiny-cheek crayfish also in other periods. For this purpose, experiments were set up to investigate A. astaci transmission from infected spiny-cheek crayfish to non-infected spiny-cheek or noble crayfish. As expected, the pathogen was transmitted to noble crayfish much more easily than to the uninfected American host. Nevertheless, we succeeded in the pathogen transmission also among spiny-cheek...

Arbuscular mycorrhizal (AM) fungi are key soil symbiotic microorganisms, intensively studied for their roles in improving plant fitness and their ubiquity in terrestrial ecosystems. Research on AM fungi is difficult because their obligate biotrophic nature makes it impossible to culture them in the absence of a host. Over the last three decades, Ri T-DNA transformed roots have been the gold standard to study AM fungi under in vitro conditions. However, only two host plant species (Daucus carota and Cichorium intybus) have been routinely used to in vitro propagate less than 5% of the known AM fungal species. There is much evidence that host identity can significantly affect AM symbioses, therefore, we investigated any potential host-specific effects of two novel Ri T-DNA transformed root species, Medicago truncatula and Nicotiana benthamiana, by associating them with seven AM fungal species selected based on their contrasting behaviors when grown with Ri T-DNA transformed D. carota roots. To evaluate the performance of new Ri T-DNA transformed roots to host and propagate AM fungal species, a factorial set-up was used to generate nine unique pairs of hosts (M. truncatula, N. benthamiana, D. carota) and AM fungi (Rhizophagus irregularis, R. clarus, Glomus sp.). Using statistical modeling, all pairs of hosts and AM fungi were compared by their symbiosis development (SD) and sporulation patterns in the hyphal compartments (HCs) of two-compartment Petri dishes. Our results show that 1) most of the variation between host and AM fungus pairs relating to SD or HC sporulation was explained by an interaction between host and AM fungal identity, i.e., host identity alone was not sufficient to explain AM fungal behaviour, 2) AM symbioses involving different combinations of symbiont identities trigger heterogenous fungal behaviours. This work provides a robust framework to develop and evaluate new Ri T-DNA roots for the in vitro propagation of AM fungi, an important asset for germplasm collections and biodiversity preservation.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

In this work, dRNA-seq (differential RNA sequencing) and RNAtag-seq were applied to first define the global transcriptome architecture of C. difficile, followed by Hfq RIP-seq (RNA immunoprecipitation followed by RNA-seq) and RIL-seq (RNA interaction by ligation and sequencing) to characterize the Hfq-mediated sRNA interactome on a transcriptome-wide scale. These approaches resulted in the annotation of > 60 novel sRNAs. Notably, it not only revealed 50 Hfq-bound sRNAs, but also > 1000 mRNA-sRNA interactions, confirming Hfq as a global RNA matchmaker in C. difficile. Similar to its function in Gram-negative species, deletion of Hfq resulted in decreased sRNA half-lives, providing evidence that Hfq affects sRNA stability in C. difficile. Finally, several sRNAs and their function in various infection relevant conditions were characterized. The sRNA nc085 directly interacts with the two-component response regulator eutV, resulting in regulation of ethanolamine utilization, an abundant intestinal carbon and nitrogen source known to impact C. difficile pathogenicity. Meanwhile, SpoY and SpoX regulate translation of the master regulator of sporulation spo0A in vivo, thereby affecting sporulation initiation. Furthermore, SpoY and SpoX deletion significantly impacts C. difficile gut colonization and spore burden in a mouse model of C. difficile infection. / Der anaerobe Gram-positive humanpathogene Erreger Clostridioides difficile (C. difficile) gilt als Hauptursache für nosokomiale Antibiotika-assoziierte Diarrhöe. Verschiedene Virulenzfaktoren und -eigenschaften beeinflussen das Fortschreiten und den Schweregrad der Krankheit, darunter Toxinexpression und Sporenbildung. Kleine regulatorische RNAs (sRNAs) sind bekannte post- transkriptionelle Regulatoren von Virulenz- und Stress-assoziierten Stoffwechselwegen in vielen pathogenen Bakterien. In Gram-negativen Arten wird sRNA-abhängige post-transkriptionelle Regulierung häufig durch das RNA-Chaperon Hfq vermittelt, welches die sRNA-mRNA- Basenpaarung erleichtert. Trotz ihrer Bedeutung in Gram-negativen Bakterien ist vergleichsweise wenig über die verschiedenen Aspekte der post-transkriptionellen Regulation in Gram-positiven Arten bekannt. Erste Daten deuten auf eine wichtige Funktion von Hfq bei der Regulierung verschiedener infektionsassoziierter Signalwege in C. difficile hin, sowie auf die Existenz eines umfangreichen post-transkriptionellen Netzwerks. Eine globale Identifizierung von Hfq- assoziierten RNAs und deren Einfluss auf die Virulenz von und Kolonisierung durch C. difficile ist jedoch bisher noch nicht erfolgt. In dieser Arbeit wurde dRNA-seq (differentielle RNA-Sequenzierung) und RNAtag-seq angewandt, um zunächst die globale Transkriptom-Architektur von C. difficile zu definieren. Anschließend wurde Hfq RIP-seq (RNA-Immunpräzipitation gefolgt von RNA-seq) und RIL-seq (RNA-Interaktion durch Ligation und Sequenzierung) durchgeführt, um das Hfq-vermittelte sRNA-Interaktom auf globaler Ebene zu charakterisieren. Diese Ansätze führten zur Annotation von > 60 neuen sRNAs. Darüber hinaus wurden 50 Hfq-gebundene sRNAs, sowie > 1000 mRNA- sRNA-Interaktionen identifiziert, wodurch Hfq als globaler RNA-Matchmaker in C. difficile bestätigt wurde. Analog zu seiner Funktion in Gram-negativen Arten, führte die Deletion von Hfq zu verringerten sRNA-Halbwertszeiten, was darauf hindeutet, dass Hfq die sRNA-Stabilität in C. difficile beeinflusst. Schließlich wurden mehrere sRNAs und ihre Funktion unter verschiedenen infektionsrelevanten Bedingungen charakterisiert. Die sRNA nc085 interagiert direkt mit dem Zweikomponenten-Regulator eutV, was zu einer Regulierung der Ethanolaminverwertung führt. Als häufig vorkommenden Kohlenstoff- und Stickstoffquelle im Darm, kann Ethanolamin die Pathogenität von C. difficile beeinflussen. SpoY und SpoX regulieren dagegen die Translation des Hauptregulators der Sporulation spo0A in vivo und damit die Sporulationsinitiation. Darüber hinaus hat die Deletion von SpoY und SpoX signifikante Auswirkungen auf die Besiedlung des Darms mit C. difficile sowie die Sporenbelastung in einem Mausmodell der C. difficile-Infektion. Insgesamt liefert diese Arbeit Beweise für eine umfassende Hfq-abhängige post-transkriptionelle Regulierung, die die Physiologie und Virulenz eines Gram-positiven Erregers beeinflusst. Auch wenn mit dieser Arbeit die Charakterisierung der sRNA-vermittelten Regulation in C. difficile gerade erst begonnen hat, können die RIL-seq-Daten als Grundlage für zukünftige mechanistische Studien der RNA-basierten Genregulation in C. difficile herangezogen werden.

Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. The streptomycetes divisome is however, similar to that of other prokaryotes. We hypothesized chemical inhibitors of sporulation in model streptomycetes might interfere with cell division in rod shaped bacteria. To test this, we investigated 196 compounds that inhibit sporulation in Streptomyces coelicolor. We show that 19 of these compounds cause filamentous growth in Bacillus subtilis, consistent with impaired cell division. One of the compounds is a DNA damaging agent and inhibits cell division by activating the SOS response. The remaining 18 act independently of known stress responses and may therefore act on the divisome or on divisome positioning and stability. Three of the compounds (Fil-1, 2 and 3) confer distinct cell division defects on B. subtilis. They also block B. subtilis sporulation, which is mechanistically unrelated to the sporulation pathway of streptomycetes but which is also dependent on the divisome. We discuss ways in which these differing phenotypes can be used in screens for cell division inhibitors. In addition to the molecules affecting the divisome, DNA and cell wall damage also affects the process indirectly by temporarily halting the cell division. To further explore the cell division regulation in stressful conditions, we carried the complete transcriptomic analysis of S. venezuelae after the DNA damage. The observed changes in the gene expression as a result of the DNA damage paves the way for identification of the DNAdamage induced cell division inhibitor in streptomycetes. / Thesis / Doctor of Philosophy (PhD)

<p> Deletion in the chromosome of Bacillus subtilis strain Sp-H12-3 was demonstrated by Hg-Cs(2)SO(4) density gradient centrifugation. The base composition of the deleted DNA segments and transcription of m-RNA from these DNA segments were investigated. Physiological and biochemical studies of the mutant Sp-H12-3 yielded information on uridine derivatives which may be intimately associated with the process of sporulation. </p> / Thesis / Doctor of Philosophy (PhD)

<p>The chaplin proteins are functional amyloids that are produced by filamentous <em>Streptomyces</em> bacteria. The chaplins are essential for the morphological development of <em>S. coelicolor</em>, and are important for altering the surface ultrastructure of aerial hyphae and spores. Although it is well established that the chaplins play an important role in <em>S. coelicolor </em>aerial development, there is still much that remains unknown regarding their activity; in particular, how each of the chaplins contribute to promoting aerial development, and the importance that chaplin amyloidogenesis has in this process.</p> <p>Previous work has revealed that only three of the eight chaplins (ChpE, ChpC, and ChpH) are necessary for promoting aerial development, and that ChpH plays a significant role in this process. For this reason, ChpH was used as the ‘model chaplin’ to examine the primary sequence determinants governing chaplin amyloidogenesis, and to explore the relationship between ChpH amyloid fiber formation and ChpH-dependent aerial development. This analysis revealed that ChpH contains two amyloidogenic regions, at the N- and C-termini, both of which are necessary for promoting aerial development, while the N-terminal domain is dispensable for surface fiber assembly.</p> <p>A separate study focused on the role of the short chaplin ChpE. One of the surprising findings of this work is that, unlike the other chaplins, ChpE is essential for maintaining cell viability. The relationship between ChpE cell surface localization and the presence of the long chaplins was also examined. This work showed that the long chaplins are not required for the surface attachment of ChpE (nor the other short chaplins), but do function to enhance the activity of the short chaplins in promoting aerial development, in addition to being necessary for the organization and assembly of surface fibers.</p> / Doctor of Philosophy (PhD)