Les protéines en cours de synthèse subissent des modifications très précoces de leur extrémité N-terminale, dès lors que celle-ci émerge du tunnel de sortie du ribosome. La première modification est l’excision de la méthionine initiatrice, assurée par une méthionine aminopeptidase (MetAP), précédée de sa déformylation par une enzyme peptide déformylase (PDF) chez les bactéries et dans les mitochondries et chloroplastes. Ce processus est ubiquitaire et essentiel, et a été décrit dans tout le règne du vivant. Chez les bactéries, les PDFs de type 1B se fixeraient au ribosome à proximité de l’extrémité du tunnel de sortie du peptide naissant, via son hélice α C-terminale. Or des analyses métagénomiques récentes ont révélé la présence insoupçonnée de gènes codant des PDFs putatives chez des virus marins. De manière inattendue, toutes les PDF virales présentent des séquences C-terminales très courtes et dépourvues de l’hélice α3. L’identification de ces PDFs atypiques soulève alors de nouvelles questions quant à leur possible interaction au ribosome et à leur fonction biologique. L’objectif de ma thèse a donc été de réaliser la caractérisation complète et intégrée de la peptide déformylase du bactériophage Vp16T, dont la séquence est l’une des plus courtes connues à ce jour. J’ai montré que le phage Vp16T code une protéine active, in vivo et in vitro, et qu’elle peut se lier au ribosome malgré l’absence d’hélice α C-terminale. La caractérisation structure-fonction de Vp16PDF a révélé des caractéristiques uniques qui pourraient alors expliquer sa fonction au cours de la réplication du phage. Ainsi j’ai montré que l’expression de Vp16PDF chez E. coli modifie la structure de l’enveloppe, induit l’accumulation d’agrégats et finalement inhibe la croissance bactérienne. De plus, l’étude de souches bactériennes mutantes a montré que Vp16PDF interfère spécifiquement avec le repliement et l’adressage de protéines membranaires. Cette dernière fonction pourrait permettre de déstabiliser la membrane de l’hôte et ainsi favoriser la libération des particules virales. / Being synthesized proteins undergo very early changes in their N-terminal end, since it emerges from the outlet channel of the ribosome. The first modification is the excision of the initiator methionine, provided by a methionine aminopeptidase (MetAP), preceded by its deformylating enzyme peptide deformylase (PDF) in bacteria and in mitochondria and chloroplasts. This process is ubiquitous and essential, and has been described in the kingdom of life. In bacteria, Type 1B PDFs would bind to the ribosome near the end of the outlet tunnel of the nascent peptide via its C-terminal helix α. But recent metagenomic analyzes revealed the unexpected presence of genes encoding putative PDFs in marine viruses. Unexpectedly, all viral PDF have very short C-terminal sequences and lacking the α3 helix. The identification of these atypical PDFs then raises new questions about their possible interaction with ribosome and their biological function. The aim of my thesis was therefore to achieve the complete and integrated characterization of peptide deformylase bacteriophage Vp16T, the sequence is one of the shortest known to date. I showed that the phage Vp16T code an active protein in vivo and in vitro, and can bind to the ribosome despite the absence of the C-terminal helix α. The structure-function characterization Vp16PDF revealed unique features that could then explain its function in the replication of the phage. Thus I have shown that expression in E. coli Vp16PDF modifies the envelope structure, induces accumulation of aggregates and ultimately inhibits bacterial growth. In addition, the study of mutant bacterial strains showed that Vp16PDF specifically interfere with the folding and addressing of membrane proteins. This latter function could help destabilize the membrane of the host and thereby promote release of viral particles.

The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its ability to form lysogens. Based on published analyses of other lambdoid phages, a clear plaque phenotype is commonly due to a mutation in one of 5 phage genes: cI, cII, cIII, int, xis or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the cI and cIII genes and assayed their activities. The HK75 cI gene clone prevented super-infection by HK75. This result demonstrated repressor functionality and thus the clear plaque phenotype cannot be due to a mutation in the HK75 cI gene. Several amino acid differences were noted between the HK022 and HK75 CIII proteins. To determine if the clear plaque phenotype was due to mutations in the HK75 cIII gene, we cloned it into an expression vector. Only under conditions of cIII gene overexpression were lysogens of HK75 recovered. The phage CIII protein normally protects CII from proteolysis. Stabilization of CII by mutations in specific host proteases has been shown to suppress a clear plaque phenotype caused by mutations in the cIII gene. When HK75 was plated on a protease deficient strain of E. coli, turbid plaques were formed and lysogens were recovered. These results support the idea that the clear plaque phenotype of HK75 is due to a defect in the expression of the phage cIII gene.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

<p>  </p> <p>There is an increasing global awareness of the threat posed by antimicrobial resistance. Measures are being taken by non-government organizations, nations and individual entities to address this intensely pressing issue which ultimately threatens human lives. The One Health initiative provides a framework which may advance public understanding of and willingness to address antimicrobial resistance. One Health seeks to identify alternative solutions to problems through an understanding of the human-animal-ecological interconnection. There are several alternatives to antibiotics that have been proposed in livestock (and specifically poultry) production systems. This work focused specifically on the prospect of bacteriophages as a tool for microbial control. A sample of 1,497 respondents targeted to be representative of the population of Pakistan completed a survey providing data about knowledge of antibiotics, the threats of antimicrobial resistance, and their food shopping behaviors. A hypothetical discrete choice experiment was used to elicit survey respondent’s choices amongst various chicken products which varied according to purchase location (supermarket versus wet market) and were labeled with regard to the use of antibiotics in production. Respondents were randomly assigned into one or two groups. One group saw in-depth information about antibiotics and bacteriophage technology alongside basic information about poultry prices, purchase location, and product labeling. The other group saw only basic information about purchase location, pricing, and product labeling, but were not provided the additional information about antibiotics or bacteriophage technology and its potential effectiveness for microbial control. In addition to the estimation of consumer willingness to pay for poultry production processes, respondent’s food shopping behavior, familiarity with antibiotic use, and familiarity with bacteriophages or phages was assessed. A random parameters logit model was used to estimate Pakistan poultry consumer’s willingness to pay for bacteriophage technology as an alternative to antibiotics in poultry production. </p>

Les exopolysaccharides (EPS) sont des biopolymères pouvant être synthétisés par les Eucaryotes, lesArchées et les Procaryotes. Au niveau bactérien les EPS peuvent être impliqués dans la constitution du biofilm (phénomène de biofouling) lors de la colonisation de nouveaux milieux. Ces biopolymères ont des propriétés physico-chimiques et biologiques spécifiques et innovantes à haut potentiel biotechnologique (agroalimentaire, santé, cosmétique, ingénierie environnementale ...). A l'opposé, leurs rôles écologiques lors de l'établissement de biofilms de souches potentiellement pathogènes peuvent rendre leur éradication compliquée.Les processus de dépolymérisation par voie enzymatique sont nécessaires pour réaliser l'élucidation structurale fine des EPS complexes, pour la production de dérivés bio-actifs calibrés à faible poids moléculaire ou pour empêcher la formation de biofilm. La mise en évidence de ces phénomènes enzymatiques sur des microorganismes modèles peut également permettre de mieux cerner les flux de matière au sein de certains compartiments biologiques en particulier en milieu marin. Néanmoins la complexité et grande diversité de structures des EPS rendent la recherche d’enzymes de dépolymérisation spécifiques difficile.Deux stratégies ont été employées pour trouver des sources d'enzymes.1. La voie bactérienne via l’utilisation de bactéries marines productrices d’EPS.2. La voie virale par la recherche de polysaccharidases de bactériophages marins. En plus d’EPS marins déjà connus, de nouveaux substrats (EPS) originaux ont été produits et caractérisés à partir de batéries marines d’intérêts biotechnologiques et/ou écologiques pour les besoins du projet. Un criblage enzymatique sur 11 souches bactériennes du genre Alteromonas a permis de mettre en évidence que 7 d’entre elles présentaient une activité de dépolymérisation endogène vis-à-vis de leur propre EPS. Une bioprospection a été réalisée afin de constituer une virothèque à partir d’hôtes bactériens producteurs d’EPS dans le but de fournir une source de Cazymes virales potentielles. Sur 33 bactériophages, 10 ont été sélectionnés pour leur capacité à rester infectieux lorsque leurs hôtes synthétisent des EPS. Finalement un système hôte/virus a été sélectionné.Les 5 virus (appelés Carin-1 à 5) infectant Cobetia marina DSMZ 4741 ont été étudiés au niveau de leurs traits de vie. Les capacités de dépolymérisation de Carin-1 et Carin-5 sur l'EPS L6 ont été explorés plus en détail. En parallèle, la structure chimique de l'EPS L6 a été intégralement élucidée. / Exopolysaccharides (EPSs) are a class of biopolymer synthesized by Eukarya, Archea and Procarya.Bacterial EPSs are involved in biofilm establishment and biofouling phenomenon. These polymers have physicochemical and biological properties suitable with biotechnological valorization. At the opposite, their involvment in biofouling of pathogenic strains can be problematic.Enzymatic depolymerization process are necessary for EPSs structural elucidation, Bioactive oligosaccharides production or to disrupt polysaccharidic biofilms. The highlight of enzymatic phenomenon can help to understand biogeochimical process in the ocean. Nevertheless the important structural diversity as well as their complexity make the sourcing of specific enzymes difficult.Two strategies were used to find enzymes.1. The bacterial way by using EPS-producing marine strains2. The viral way, with marine bacteriophages.For the need of the study, several EPS-substrates were produced and characterized. The majority of them were totally new. An enzymatic screening on 11 marine Alteromonas strains shown that 6 were able to depolymerize their EPS in an endogenous way. A bioprospection was realized to isolates marine bacteriophages with potential viral Cazymes. 10 out of 33 phages were selectionned for their ability to be infectious with their hosts in EPS production induced. Finally, a host/virus system was chosen. The bacteriophages infecting Cobetia marina DSMZ 4741 (named Carin-1 to 5) were studied. The polysaccharidase activities of Carin-1 and Carin-5 on the L6 EPS were studied more deeply. In parallel, the complete structural elucidation of the L6 EPS was realized.

Les chercheurs en biologie de synthèse programment l’ADN pour construire des systèmes biologiques capables de répondre à certaines conditions de manière prédéfinie. Cette capacité pourrait avoir un impact sur plusieurs domaines, de la médecine à la fermentation industrielle. Le traitement de signal par des circuits biologiques synthétiques est en train d’être démontré à large échelle, mais hélas la variété des signaux d’entrée capables de contrôler ces circuits est pour l’instant limitée. Ce manque de diversité est un obstacle majeur au développement de nouvelles applications car en général chaque application requiert une réponse à des signaux de nature particulière qui lui sont spécifiques. Cette thèse cherche à apporter des solutions au manque de signaux d’entrée appropriés contrôlant les circuits biologiques en développant deux nouvelles stratégies d’induction. La première stratégie vise à étendre la diversité chimique des signaux d’entrée. A l’inverse des approches existantes, qui reposent sur la modification des systèmes de détections naturels tels que les riboswitchs ou les facteurs de transcription allostériques, j’ai cherché ici à modifier directement des molécules préalablement non-détectables afin de les rendre détectables par les systèmes de détection actuels. Pour ce faire, la transformation chimique des molécules cibles est réalisée in situ grâce à l’expression de voies métaboliques synthétiques dans la cellule. Afin de pouvoir utiliser cette stratégie de manière systématique, j’ai employé la conception assistée par ordinateur et puisé dans l’ensemble des réactions biochimiques connues afin de prédire des voies de détections pour de nouvelles molécules. J’ai ensuite implémenté in vivo plusieurs prédictions qui ont permis à E. coli de détecter de nouveaux composés. Au-delà de l’intérêt de cette méthode en biotechnologie, cela montre que le métabolisme peut jouer un rôle dans le transfert d’information, en plus de son rôle dans le transfert de matière et d’énergie, ce qui soulève la question de l’utilisation potentielle de cette stratégie de détection par la nature. Un second axe présente une façon d’épargner l’utilisation d’inducteurs chimiques pour les programmes biologiques simples, et propose d’utiliser des inducteurs biologiques à la place. Lorsqu’une seule étape d’induction ou de répression de gènes est nécessaire, comme c’est le cas en fermentation industrielle, je propose de remplacer la coûteuse étape d’induction chimique par l’infection simultanée de toutes les cellules d’une population par des particules virales capables d’injecter en temps réel l’ensemble des informations nécessaires pour déclencher l’activité biologique recherchée. A des fins de fermentation, j’ai développé des particules virales modifiées qui reprogramment dynamiquement le métabolisme d’une large population de bactérie au moment opportun et les forcent à produire des molécules à haute valeur ajoutée. / Synthetic biologists program DNA with the aim of building biological systems that react under certain conditions in a predefined way. This ability could have impact in several fields, from medicine to industrial fermentation. While the scalability of synthetic biological circuits in terms of signal processing in now almost demonstrated, the variety of input signals for these circuits is limited. Because each application typically requires a circuit to react to case-specific molecules, the lack of input diversity is a major obstacle to the development of new applications. Two axis are developed over the course of this thesis to try to address input-related problems. The main axis consists in a new strategy aiming at systematically and immediately increasing the chemical diversity of inputs for synthetic circuits. Current approaches to expand the number of potential inputs focus on re-engineering sensing systems such as riboswitches or allosteric transcription factors to make them react to previously non-detectable molecules. On the contrary, here we developed a method to transform the non-detectable molecules themselves into molecules for which sensing systems already exist. These chemical transformations are realized in situ by expressing synthetic metabolic pathways in the cell. In order to systematize this strategy, we leveraged computer-aided design to predict ways of detecting new molecules by digging into all known biochemical reactions. We then implemented several predictions in vivo that successfully enabled E. coli to detect new chemicals. Aside from the interest of the method for biotechnological applications, this shows that in addition to transferring matter and energy, metabolism can also play a role in transferring information, raising the question of potential occurrences of this sensing strategy in nature. A second axis introduce a way to exempt simple programs from the need for a chemical input, and explore the use of a biological input instead. In situations where a single timely induction or repression of multiple genes is required, such as in industrial fermentation processes, we propose to replace expensive chemical induction by simultaneous infection of all the members of a growing population of cells with viral particles inputting in real-time all the necessary information for the task at hand. In the context of fermentation, we developed engineered viral particles that can dynamically reprogram the metabolism of a large population of bacteria at the optimal stage of growth and force them to produce value-added chemicals.

Les systèmes CRISPR-Cas confèrent aux bactéries une immunité adaptative contre les éléments génétiques mobiles jouant ainsi un rôle important dans l’évolution bactérienne. Cependant, moins de la moitié des génomes bactériens encodent des systèmes CRISPR-Cas ; cela, malgré la protection qu’ils confèrent et leur haut taux de transfert horizontal. Des hypothèses telles que le coût des phénomènes d’auto-immunité ou de posséder des défenses adaptatives plutôt qu’innées ont été mises en avant pour expliquer ce paradoxe. Je propose une nouvelle hypothèse complémentaire : le contexte génétique jouerait un rôle important dans la fixation d’un système CRISPR-Cas après son transfert. Plus précisément, j’ai étudié comment les interactions entre les systèmes de réparation de l’ADN et les CRISPR-Cas influencent la distribution de ces derniers. Pour cela, j’ai d’abord examiné finement la distribution des systèmes CRISPR-Cas dans les génomes bactériens. J’ai ensuite analysé les co-occurences des systèmes de réparation de l’ADN et des CRISPR-Cas et démontré l’existence d’associations positives et négatives entre eux. Enfin, je me suis concentrée sur une des associations négatives découvertes pour valider mes prédictions expérimentalement et comprendre les mécanismes moléculaires sous-jacents. Mes travaux permettent de mieux comprendre les interactions complexes entre systèmes de réparation de l’ADN et CRISPR-Cas et démontrent la nécessite d’accommodation des CRISPR-Cas à un contexte génétique pour être sélectionnés et maintenus dans les génomes bactériens. / CRISPR-Cas systems confer bacteria and archea an adaptative immunity against phages and other invading genetic elements playing an important role in bacterial evolution. Only 47% of bacterial genomes harbor a CRISPR-Cas system despite their high rate of horizontal transfer. Hypothesis such as the cost of autoimmu- nity or the trade off between a constitutive or an inducible defense system have been put forward to explain this paradox. I propose that the genetic background plays an important role in the process of maintaining a CRISPR-Cas system af- ter its transfer. More precisely I hypothesized that CRISPR-Cas systems interact with DNA repair pathways. To test this idea, we detected DNA repair pathways and CRISPR-Cas systems in bacterial genomes and studied their co-occurences. We report both positive and negative associations that we interpret as poten- tial antagonistic or synergistic interactions. We then focused on one interaction to validate our result experimentally and explored molecular mechanisms behind those interactions. My findings give insights on the complex interactions between CRISPR-Cas systems and DNA repair mechanisms in bacteria and provide a first example on the necessity of accommodation of CRISPR-Cas systems to a specific genetic context to be selected and maintained in bacterial genomes.

Bacteriophages (viruses that infect bacteria) are the most abundant biological life-forms on Earth. However, very little is known regarding the structure of phage-bacteria infections. In a recent study we showed that phage-bacteria infection assay datasets are statistically nested in small scale communities while modularity is not statistically present. We predicted that at large macroevolutionary scales, phage-bacteria infection assay datasets should be typified by a modular structure, even if there is nested structure at smaller scales. We evaluate and confirm this hypothesis using the largest study of the kind to date. The study in question represents a phage-bacteria infection assay dataset in the Atlantic Ocean region between the European continental shelf and the Sargasso Sea. We present here a digitized version of this study that consist of a bipartite network with 286 bacteria and 215 phages including 1332 positive interactions, together with an exhaustive structural analysis of this network. We evaluated the modularity and nestedness of the network and its communities using a variety of algorithms including BRIM (Bipartite, Recursively Induced Modules), NTC (Nestedness Temperature Calculator) and NODF (Nestedness Metric based on Overlap and Decreasing Filling). We also developed extensions of these standard methods to identify multi-scale structure in large phage-bacteria interaction datasets. In addition, we performed an analysis of the degree of geographical diversity and specialization among all the hosts and phages. We find that the largest-scale ocean dataset study, as anticipated by Flores et al. 2013, is highly modular and not significantly nested (computed in comparison to null models). More importantly is the fact that some of the communities extracted from Moebus and Nattkemper dataset were found to be nested. We examine the role of geography in driving these modular patterns and find evidence that phage-bacteria interactions can exhibit strong similarity despite large distances between sites. We discuss how models can help determine how coevolutionary dynamics between strains, within a site and across sites, drives the emergence of nested, modular and other complex phage-bacteria interaction networks. Finally, we releases a computational library (BiMAT)to help to help the ecology research community to perform bipartite network analysis of the same nature I did during my PhD.

The purpose of this study was to investigate the feasibility of using bacteriophages - viruses that can lyse bacteria - to control infections caused by uropathogenic Escherichia coli (UPEC) in dogs and cats. Prior to phage experiments, UPEC were subjected to virulence factor genotyping by multiplex polymerase chain reaction assay and phylogenetic 'fingerprinting' by Pulsed-Field Gel Electrophoresis (PFGE). Twenty-five of 30 assessed virulence factor gene (VFG) markers were detected at least once in 31 UPEC isolated from 20 UK cats and 89 UPEC isolated from dogs (56), cats (22) and people (11) living in New Zealand (NZ). The PFGE banding patterns of UPEC isolates from different individuals were markedly dissimilar unless isolates had been collected at the same hospital within one month of each other. In contrast, ≥2 UPEC strains isolated from each of 3 UK cats diagnosed with multiple UTIs were indistinguishable by PFGE. Antibiograms inaccurtely predicted UPEC clonality and, of clinical importance, underestimated the number of relapsing or persistent infections in these cats. A comparison of VFG profiles and PFGE banding patterns of UPEC isolated from NZ and UK cats demonstrated a geographically uneven distribution of pathotypic and phylogenetic traits and indicated that, among other factors, the source of UPEC must be considered when comparing UPEC from different host species. When comparing UPEC isolates from NZ dogs, cats and people, strains with similar VFG profiles were found among the different host species. Other strains, with VFG profiles that differed according to the host species of origin were also detected. The latter finding, which is in contrast to the results of previous studies, may be of interest to researchers aiming to predict the potential zoonotic risk posed by particular UPEC strains sourced from dogs and cats. Forty bacteriophages (phages for short) were isolated from sewage waters and propagated on UPEC strains. The ability of these phages to cause bacterial lysis was tested on 31 canine UPEC, 22 feline UPEC and 7 faecal E. coli. In contrast to faecal E. coli, UPEC strains were highly susceptible to phages. Ten phages with a particularly broad host range each lysed ≥27/53 (≥51%) UPEC strains. Used in combination, these 10 phages were predicted to be able to lyse 49/53 (92%) of the UPEC strains in the collection. Morphological and genotypic studies on 5 of these 10 phages demonstrated that 4 of them belonged to the lytic T4-like genus, while one phage showed similarity to the temperate phage P2. Overall, results of this project indicate that the majority of canine and feline UPEC - with very diverse PFGE banding patterns and VFG profiles - are susceptible to lysis by naturally occurring phages. Hence, phages show promise as therapeutic agents for treatment of canine and feline UTI and, perhaps, for other infections caused by UPEC.

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Microbiology and Immunology . Bibliography: leaves 130 - 137 . Also available online via the Internet.