Les quatre étapes principales d'infection des bactéries par leurs virus sont (i) la reconnaissance spécifique de la cellule hôte et l'entrée du génome dans le cytoplasme,(ii) la réplication du génome viral, (iii) l'assemblage des particules virales, et (iv) leur relâchement, menant dans la plupart des cas à la lyse de la cellule. Bien que la description des étapes individuelles du cycle viral a été relativement bien établie, les détails de comment d'ADN viral chemine du virion jusqu’au cytoplasme de la bactérie hôte et de comment l'environnement cellulaire participe au processus restent mal compris.La première étape de l’infection est la reconnaissance d’un récepteur à la surface de la bactérie hôte par la machinerie d’adsorption du phage. Les barrières que l’agent infectieux doit franchir par la suite sont la membrane externe de la bactérie Gram-negative, la paroi cellulaire et la membrane cytoplasmique. Ceci implique une dégradation localisée de la paroi et le cheminement de l’ADN à travers un pore dans la membrane. L‘ADN linéaire se circularise normalement dans le cytoplasme et il est répliqué par la suite. On a utilisé le bactériophage SPP1 qui infecte la bactérie Gram-positive Bacillus subtilis comme modèle d’étude pour disséquer ces différentes étapes clés pour le démarrage de l’infection virale. Dans ce travail de thèse les conditions d’infection et d’acquisition de données pour suivre en temps réel la dépolarisation de la membrane cellulaire de B. subtilis lors de l’infection par SPP1 ont été mis au point. Il est montré que le démarrage de l’infection déclenche une dépolarisation très rapide de la membrane cytoplasmique.Le potentiel de membrane n’est plus rétablit pendant toute la durée du cycle d'infection. Ce changement du potentiel de membrane au début de l’infection dépend de la présence du récepteur YueB. L’amplitude de la dépolarisation dépend du nombre de particules virales infectieuses présentes et de la concentration du récepteur YueB à la surface de la bactérie hôte. L’interaction du phage avec le récepteur YueB conduit à l’interaction irréversible et à l'éjection de l’ADN de SPP1. Pour établir si c’est l’interaction avec YueB ou le début de l’entrée de l’ADN qui conduit à la dépolarisation de la membrane on a utilisé des phages SPP1 éclates par EDTA qui adsorbent normalement à B. subtilis mais qui n’avaient plus leur ADN. Les résultats obtenus ont montré que la dépolarisation requiert l’interaction du virus intacte avec le récepteur YueB. Des concentrations sous-millimolaire de Ca2+ sont nécessaires et suffisantes pour SPP1 liaison réversible à l'enveloppe d'hôte et donc de déclencher la dépolarisation.La cinétique d’entrée de l’ADN du bactériophage SPP1 dans la bactérie Bacillus subtilis a été suivie en temps réel par microscopie de fluorescence. On a mis au point une méthode de microscopie pour visualiser des particules virales marquées avec des «quantum dots» ce qui permit de démontrer que ces particules se fixent préférentiellement aux pôles des bacilli. L’immuno-marquage du récepteur de SPP1,la protéine YueB, a montré que celle-ci a une organisation ponctuée à la surface de B.subtilis et se concentre particulièrement aux extrémités de la bactérie. Cette localisation particulière du phage sur la surface de la cellule hôte corrèle avec l’observation que l’ADN viral rentre dans le cytoplasme (<2 min) et se réplique dans des foci situés dans la plupart des cas à proximité des pôles de B. subtilis. L’étude spatio-temporelle de l’interaction de SPP1 avec son hôte Gram-positive montre que le virus cible des régions spécifiques de la bactérie pour son entrée et pour sa réplication. Transfert d'ADN dans le cytoplasme dépend des concentrations millimolaires de Ca2+. Un modèle décrivant les événements précoces de l'infection bactériophage SPP1 est présenté. / The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB – GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.

With respect to a multienzyme complex of deoxyribonucleoside triphosphate (dNTP) synthesis somehow juxtaposed with DNA replication sites, our laboratory has demonstrated the existence of a multienzyme complex in T4-infected E. coli, named the T4 dNTP synthetase complex, but the idea of direct linkage of dNTP synthesis to DNA replication and organization of the complex has not been well established. This study had two objectives. The first objective was to test the specific hypothesis that gp32, the single-stranded DNA binding protein encoded by gene 32, plays a role in recruiting enzymes of dNTP synthesis to the replisome and in organizing the dNTP synthetase complex. By use of two newly created gene 32 mutants along with several experimental approaches, DNA-cellulose chromatography, coimmunoprecipitation, and glutathione-S-transferase pull downs, interactions of gp32 with thymidylate synthase (gptd), ribonucleotide reductase (gpnrdA/B), and E. coli NDP kinase have been identified. These results support the hypothesis that gp32 helps to recruit enzymes of dNTP synthesis to DNA replication sites. As the second objective, I investigated contributions of two host proteins, E. coli nueleoside diphosphate kinase (NDP kinase) and adenylate kinase (Adk), to the organization of the complex. As an important step to understand roles of E. coli NDP kinase in the complex, I identified direct interactions of E. coli NDP kinase with gpnrdA/B, dCMP hydroxymethylase (gp42), and dihydrofolate reductase (gpfrd) by means of coimmunoprecipitation and glutathione-S-transferase pull-down experiments. Interestingly, these interactions were influenced by the presence of substrate nucleotides or an analog for E. coli NDP kinase, suggesting that metabolite flux may affect the preference of E. coli NDP kinase binding to enzymes in the complex in vivo. Meanwhile, Adk involvement in DNA precursor synthesis has been suggested, particularly in phage T4-infected E. coli, from observations of increased thermostability of temperature-sensitive Adk in situ. The involvement of E. coil Adk in the complex was demonstrated by identifying some proteins of the T4 dNTP synthetase complexgp42, dNMP kinase (gpl), gpfrd, and E. coli NDP kinasedirectly interacting with Adk, implying that E. coil Adk would be properly located in the complex to efficiently carry out the conversion of dNDPs to dNTPs. This implication was supported by measurements of T4 DNA synthesis. Taken together, this research strongly supports the idea of connection of dNTP synthesis to DNA replication and allows us to take a step toward understanding the organization of the complex at DNA replication sites. / Graduation date: 2005

Many T=3 plant and insect viruses use a molecular switch in form of order/disorder of a segment of the polypeptide chain to regulate the quasi-equivalent contacts. The structure of a mutant of the T=3 capsid of bacteriophage fr confirms that this virus and other members of the Leviviridae family lack a switch mechanism. The geometric principles underlying the construction of spherical virus capsid do not allow more than 60 protein monomers to from a capsid while maintaining an identical chemical environment. Most virus capsid, however, contain many more protein subunits. Quasi-equivalence explains how the capsid proteins can have slightly different interactions in the virus shell. Quasi-equivalence requires the capsids to be constructed from multiples of 60 subunits, where the T number denotes the multiplicity. The structure of the T=4 Nudaurelia capensis ω Virus shows a molecular switch in form of a C-terminal helix inserted in some contacts between protein dimers. This virus is very similar in structure to the T=3 nodaviruses. In the nodaviruses a five-membered helix bundle, formed by cleaved peptides around the five-fold axes on the inside of the shell, are suggested to aid in membrane translocation of the genomic RNA. In Nudaurelia capensis ω Virus the helix bundle is formed by 10 helices, of which 5 are still covalently attached to the capsid proteins. Bacteriophage HK97 has T=7 quasi-symmetry. A domain that is degraded during maturation and is not present in the structure of the mature virion controls the quasi-equivalence. During maturation covalent bonds are formed between the protein subunits, producing a set of interlocking covalently bound rings, resembling chainmail. Structural studies of complexes between the bacteriophage MS2 and variants of its translational operator are also included in this work. A dimer of the MS2 coat protein binds with sequence specificity to an operator in its genomic RNA, and causes translational repression. Structures of multiple RNA segments with altered sequence at some positions which are required for binding to the capsid protein, has been determined.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

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Made available in DSpace on 2016-12-08T16:24:07Z (GMT). No. of bitstreams: 1 PGCA12MA056.pdf: 526904 bytes, checksum: 7a4c82b6cb16c016c29f0182cfbbaa51 (MD5) Previous issue date: 2012-02-27 / The analysis methods for Salmonella spp. detection are of great importance in the system of animal products processing industries, because they reflect directly on the microbiological quality of food, impacting the storage time and therefore the release of the product for sale. The present study verified the efficiency of a new ELFA ELISA test using a recombinant protein from bacteriophage (VIDAS UP®) to detect the presence of Salmonella spp. in products of animal origin. Two hundred fifteen samples (15 samples artificially contaminated and 200 samples from agribusiness routine), from different products of animal origin were analyzed. The samples were tested by five methods of diagnosis: ELFA ELISA, conventional methodology in accordance with ISO 6579, polymerase chain reaction - PCR, semi-solid Rappaport-Vassiliadis Modified - MSRV and MSRV medium + supplement. Eleven out of 215 (5.12%) samples were positive in at least one test, which four samples were from agribusiness routine and seven were from artificially contaminated samples. The sensitivity and specificity of the ELFA ELISA when compared with the conventional method was 90.0% and 99.51% respectively. The ELFA ELISA was equivalent to the other methodologies, showing to be effective and rapid to detect Salmonella spp. in different products of animal origin / Os métodos de análise para detecção de Salmonella spp. são de grande importância no processo da agroindústria de produtos de origem animal, pois refletem diretamente na qualidade microbiológica dos alimentos, impactando no tempo de estocagem e por conseguinte na liberação do produto para venda. O presente trabalho verificou a eficiência de um novo teste ELFA ELISA utilizando proteína recombinante oriunda de bacteriófago (VIDAS UP®) para detectar a presença de Salmonella spp. em produtos de origem animal. Foram analisadas 215 amostras (15 amostras artificilamente contaminadas e 200 amostras de rotina da agroindústria), de diferentes produtos de origem animal. As amostras foram submetidas a cinco métodos de diagnóstico: ELFA ELISA, metodologia convencional de acordo com ISO 6579, reação em cadeia da polimerase - PCR, meio semi-sólido Rappaport-Vassiliadis Modificado - MSRV e meio MSRV + suplemento. Onze amostras das 215 (5,12%) foram positivas em pelo menos um dos testes, sendo quatro amostras de rotina da agroindústria e sete amostras artificialmente contaminadas. A sensibilidade e especificidade do teste ELFA ELISA com a metodologia convencional foi de 90,0% e 99,51%, respectivamente. O teste ELFA ELISA apresentou equivalência com as demais metodologias, mostrando ser eficaz e rápido na detecção de Salmonella spp. em diferentes produtos de origem animal

O uso de diferentes tipos de medicação intracanal para o controle do processo infeccioso, principalmente nos casos em que há presença de microrganismos resistentes às manobras de desinfecção, tem sido alvo de muitas pesquisas. A proposta deste estudo foi avaliar, in vitro, o efeito antimicrobiano de bacteriófagos específicos diante de cinco cepas de Enterococcus faecalis e a ação de um lisado híbrido polivalente na eliminação da infecção causada por essas cinco cepas da mesma espécie. Foram utilizados 37 dentes unirradiculares humanos, recentemente extraídos e de proporções aproximadas. As coroas foram removidas e os canais instrumentados até a lima tipo K de número 45. Os espécimes foram, então, esterilizados e utilizados em dois experimentos distintos. O primeiro experimento utilizou 25 raízes divididas em cinco grupos de cinco espécimes. Três espécimes de cada grupo foram inoculados com uma das culturas bacterianas e seus fagos correspondentes na proporção 1:1, por um período de três horas a 37 °C, enquanto os outros dois, receberam a cultura de microrganismos ou somente meio de cultura (controle positivo e negativo, respectivamente). No segundo experimento, 11 espécimes receberam um inóculo formado pelas cinco cepas por um período de 10 dias de incubação a 37 °C, com o propósito de manter condições apropriadas para a penetração das bactérias no interior dos túbulos dentinários, e um outro espécime recebeu apenas meio de cultura (controle negativo). Essa penetração foi confirmada empregando-se microscopia ótica e eletrônica realizada em dois espécimes. Após o período de incubação, o lisado polivalente, preparado com os cinco fagos, foi aplicado por 24 horas a 37 °C em 8 espécimes, e os demais preenchidos com meio de cultura (controle positivo e negativo). Alíquotas do interior de todos os canais foram colhidas antes e depois do contato com os fagos e no segundo experimento, também 24 e 48 horas depois, para semeadura e contagem de unidades formadoras de colônia. Os resultados do primeiro experimento mostraram 100% de redução do crescimento bacteriano nos espécimes que receberam a suspensão de fagos específicos, em comparação a seus respectivos controles positivos, em todos os grupos. No segundo experimento, foi comparado o crescimento obtido após os 10 dias de infecção com aquele posterior a aplicação dos fagos, redução que variou entre 50% e 100%. Diante desses resultados, conclui-se que os bacteriófagos foram eficazes na diminuição dos microrganismos presentes no interior de canais radiculares e nos túbulos dentinários de dentes humanos. / Many studies have investigated different intracanal medications to control infection processes, especially in cases of microbial resistance to disinfection procedures. The purpose of this study was to evaluate the in vitro antimicrobial effect of specific bacteriophages on five isolates of Enterococcus faecalis, as well as the activity of a lysate cocktail in eliminating the infection caused by these bacteria. Thirty-seven recently extracted human teeth of approximately equal size and with single roots were used. The crowns were removed and each canal was prepared using K files,up to # 45, and sterile physiological saline. Specimens were then sterilized and used in two separate studies. The first study utilized 25 individual roots divided into five groups of five specimens each. Three specimens of each group were inoculated with one of the bacterial cultures and the corresponding bacteriophage in a proportion of 1:1, and incubated for three hours at 37°C; the other two specimens were inoculated with only the bacterial culture or only the culture medium (positive and negative controls, respectively). In the second study, 11 specimens were inoculated with all five strains and incubated for ten days at 37°C in order to allow bacteria to penetrate the interior of the dental tubules, and another one, received just the culture medium (negative control). Penetration into the tubules was confirmed by optical and electron microscopy of two specimens. Following incubation, the lysate cocktail prepared using all five bacteriophages was applied to the other 8 specimens for 24 hours at 37°C, and 2 specimens were filled with the culture medium (positive and negative controls). In the first study, samples were taken from the lumen of all canals before and after contact with bacteriophages; in the second, aliquots were also taken 24 and 48 hours after the bacteria were exposed to the phages. All samples were diluted and plated and the number of colony forming units was counted. In the first study, there was a 100% reduction in bacterial growth in specimens that received the specific bacteriophage suspension compared to the positive controls within each group. In the second study, after ten days the number of bacteria was reduced by 50% to 100% following the bacteriophage application. These results suggest that bacteriophages are effective in reducing the number of bacteria inside the root canal and in the dental tubules of human teeth.

Bacteriophage genomes show pervasive mosaicism, indicating that horizontal gene exchange plays a crucial role in their evolution. Phage genomes represent unique combinations of modules, each of them with a different phylogenetic history. Thus, a web-like, rather than a hierarchical scheme is needed for an appropriate representation of phage evolutionary relationships. Part of the virology community has long recognized this fact and calls for changing the traditional taxonomy that classifies tailed phages according to the type of genetic materials and phage tail and head/capsid morphologies. Moreover, based on morphological features, the current system depends on inspection of phage virions under the electron microscope and cannot directly classify prophages. With the genomic era, many phages have been sequenced that are not classified, calling for development of an automatic classification procedure that can cope with the sequencing pace. The ACLAME database provides a classification of phage proteins into families and assigns the families with at least 3 members to one or several functions.<p>In the first contribution of this work, the relative contribution of those different protein families to the similarities between the phages is assessed using pair-wise similarity matrices. The modular character of phage genomes is readily visualized using heatmaps, which differ depending on the function of the proteins used to measure the similarity. <p>Next, I propose a framework that allows for a reticulate classification of phages based on gene content (with statistical assessment of the significance of number of shared genes). Starting from gene/protein families, we built a weighted graph, where nodes represent phages and edges represent phage-phage similarities in terms of shared families. The topology of the network shows that most dsDNA phages form an interconnected group, confirming that dsDNA phages share a common gene pool, as proposed earlier. Differences are observed between temperate and virulent phages in the values of several centrality measures, which may correlate with different constraints to rampant recombination dictated by the phage lifestyle, and thus with a distinct evolutionary role in the phage population. <p>To this graph I applied a two-step clustering method to generate a fuzzy classification of phages. Using this methodology, each phage is associated with a membership vector, which quantitatively characterizes the membership of the phage to the clusters. Alternatively, genes were clustered based on their ‘phylogenetic profiles’ to define ‘evolutionary cohesive modules’. Phages can then be described as composite of a set of modules from the collection of modules of the whole phage population. The relationships between phages define a network based on module sharing. Unlike the first network built from statistical significant number of shared genes, this second network allows for a direct exploration of the nature of the functions shared between the connected phages. This functionality of the module-based network runs at the expense of missing links due to genes that are not part of modules, but which are encoded in the first network. <p>These approaches can easily focus on pre-defined modules for tracing one or several traits across the population. They provide an automatic and dynamic way to study relationships within the phage population. Moreover, they can be extended to the representation of populations of other mobile genetic elements or even to the entire mobilome.<p>Finally, to enrich the phage sequence space, which in turn allows for a better assessment of phage diversity and evolution, I devise a prophage prediction tool. With this methodology, approximately 800 prophages are predicted in 266 among 800 replicons screened. The comparison of a subset of these predictions with a manually annotated set shows a sensitivity of 79% and a positive predictive value of 91%, this later value suggesting that the procedure makes few false predictions. The preliminary analysis of the predicted prophages indicates that many may constitute novel phage types.<p>This work allows tracing guidelines for the classification and analysis of other mobile genetic elements. One can foresee that a pool of putative mobile genetic elements sequences can be extracted from the prokaryotic genomes and be further broken down in groups of related elements and evolutionary conserved modules. This would allow widening the picture of the evolutionary and functional relationships between these elements.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Récemment, le système de sécrétion de type VI (SST6) a été identifié comme un nouvel acteur clé dans la compétition inter-bactérienne parmi le large arsenal dont dispose les bactéries. L’une des particularités du SST6 est de cibler à la fois des cellules eucaryotes et procaryotes. Le T6SS est un complexe protéique formé par l’assemblage de deux ‘sous-complexes’. Le premier sert à l’ancrage de la machinerie au sein de l’enveloppe bactérienne et le second agit comme une arbalète moléculaire. Le mécanisme d’action du SST6 est très similaire à celui d’autres machineries contractiles telles que celui des bactériophages : la contraction d’un fourreau propulse une flèche, composée d’un tube avec une aiguille à son extrémité, directement dans la cellule cible afin de délivrer les différentes toxines. Mon projet de thèse consiste à comprendre quelles sont la structure et la biogénèse des deux différents complexes et de comprendre comment ils sont assemblés. Nous utilisons comme modèle la bactérie pathogène à Gram négatif Escherichia coli entéroagrégative. J’ai pu démontrer que le complexe membranaire est assemblé en premier, avec l’adressage de la lipoprotéine de membrane externe TssJ, puis le recrutement séquentiel de TssM et TssL, deux protéines de membrane interne. Le complexe membranaire recrute ensuite une plateforme d’assemblage, appelée ‘baseplate’. Nous avons identifié et caractérisé les composants de cette ‘baseplate’ qui sert de plateforme d’assemblage pour le recrutement du reste de la machinerie (fourreau et flèche). Enfin, nous avons identifié et déterminé le rôle de la protéine TssA, une protéine qui coordonne la polymérisation du fourreau et de la flèche. / Among the broad weaponry of bacteria, the recently identified type VI secretion system (T6SS) emerges as one of the key player in bacterial competition. T6SS is a versatile machinery that targets both eukaryotic and prokaryotic cells. This molecular weapon assembles two evolutionarily different sub-assemblies. One complex anchors the machinery to the cell envelope while the second acts as a molecular crossbow. The mechanism of action of the T6SS is similar to other known contractile machineries such as bacteriophages: the contraction of a sheath propels an arrow, constituted of a tail tube capped by a cell-puncturing device, directly into the prey cell to deliver effector toxins. My Ph.D project was to provide mechanistic details on the structure and biogenesis of the two T6SS sub-complexes and to understand how they are connected, using entero-aggregative Escherichia coli as model bacterium. I have demonstrated that the membrane complex is assembled first and starts with the positioning of the outer membrane TssJ lipoprotein and proceeds inward, from the outer to the inner membrane, through the sequential recruitment of the TssM and TssL subunits. After assembly, the membrane complex recruits an assembly platform called the baseplate. We identified and characterized the components of this baseplate, which serves as assembly platform for the tail. We further demonstrated that the functional and physical interaction between the T6SS membrane complex and the baseplate is mediated by multiple contacts. Finally, we identified and deciphered the role of TssA, a protein that coordinates the polymerizations of the tail tube and sheath.

Les bactériophages neutralisent les systèmes de défense et détournent les fonctions vitales de leur hôte pour favoriser leur multiplication. Les gènes de phages qui gouvernent cette prise de contrôle de l’hôte restent mal connus, pourtant leur caractérisation présente un intérêt majeur pour mettre à jour des fonctions bactériennes spécifiquement ciblées par les phages et pour concevoir de nouveaux agents antibactériens.Le phage T5 injecte son ADN dans la bactérie Escherichia coli en deux étapes. Seuls les gènes précoces codés par 8% du génome entrent dans la cellule et le transfert s’arrête. Leur expression induit la dégradation du chromosome de l’hôte et l’inactivation de ses systèmes de restriction et de réparation de l’ADN. Après quelques minutes, le reste de la molécule d’ADN est injecté, ce qui permet la production de nouveaux phages. Deux gènes précoces A1 et A2 ont été identifiés comme essentiels pour la reprise du transfert de l’ADN et A1 est également nécessaire pour induire la dégradation de l’ADN de l’hôte. A1 et A2 sont les deux seuls gènes connus pour être impliqués dans la régulation de ce système original d’infection, mais leur fonction n’a jamais été identifiée.Ma thèse porte sur la caractérisation fonctionnelle et structurale des protéines A1 et A2. J’ai purifié A1 et démontré in vitro qu’elle avait une activité DNase dépendante du manganèse. Sa structure atomique a été résolue par cryomicroscopie électronique à 3.01 Å de résolution, montrant une organisation octamérique de symétrie D4 inédite pour une DNase. Chaque monomère (61kDa) contient un domaine exonuclease dont le site actif lie deux ions Mn2+ et qui s’apparente au site catalytique des domaines exonucléases de la DNA polymerase II et des DNAses associées aux systèmes de recombinaison homologue et de réparation de l’ADN comme Mre11. En construisant différents mutants de A1, j’ai identifié certains acides aminés essentiels pour l’activité catalytique et, par des expériences de complémentation fonctionnelle, j’ai montré que cette activité était indispensable pour l’infection. L’ensemble de ces résultats suggèrent que A1 est la DNase, jusqu’ici inconnue, responsable de la dégradation massive du génome de l’hôte au tout début de l’infection. Enfin, j’ai observé que la production de A1 pendant l’infection induit une forte activité recombinase. De nombreux autres bactériophages qui n’appartiennent pas à la famille des T5virus produisent également une protéine similaire à A1 dont la fonction n’a jamais été identifiée. Ce travail est un premier pas vers la compréhension de son rôle dans le mécanisme général d‘infection par les phages. Une deuxième partie de cette thèse porte sur la caractérisation structurale de A2. Des recherches de similarité indiquent la présence d’un domaine Helix-Turn-Helix typique des régulateurs transcriptionnels. J’ai purifié A2 et montré que cette protéine de 14 kDa est un dimère en solution. La caractérisation des propriétés biochimiques de A2 a permis de débuter l’étude de sa structure par RMN.Les résultats de ma thèse ont révélé la structure originale d’une DNase de bactériophage qui contrôle la dégradation du génome bactérien et la régulation du transport de l’ADN viral au début du cycle infectieux. Ces résultats soulèvent des questions intrigantes : comment l’ADN de T5 est-il protégé de l’activité DNase de A1 ? Comment A1 et A2 interagissent-elles lors des étapes de prise de contrôle de l’hôte ? / Bacteriophages defeat bacterial defences and hijack host cell machineries to establish a favourable environment for their multiplication. Early-expressed viral genes that govern host takeover are highly diverse from one phage to another and most of them have no assigned function. They thus represent a pool of novel genes whose products potentially subvert bacterial cell vital functions and could help in designing new antibacterial strategies.T5 phage uses a unique 2-step mechanism to deliver its DNA into its host Escherichia coli. At the onset of the infection, only 8 % of the genome enter the cell before the transfer temporarily stops. Expression of the genes encoded by this DNA portion leads to host chromosome degradation and inactivation of host restriction and DNA mending systems. After a few minutes, T5 DNA transfer resumes, allowing further phage multiplication. A1 and A2 are early genes required for DNA transfer completion and A1 is also necessary to trigger host DNA degradation. A1 and A2 are the only two genes known to be involved in the regulation of this original infection system, but their function yet remains to be characterized.The objectives of this work were to characterize the function and structure of A1 and A2 proteins. I have purified the A1 protein and shown that it has a manganese-dependent DNase activity in vitro. Cryo Electron Microscopy at 3.01 Å resolution unravelled its structure, showing an octameric organization with a D4 symmetry, which is unprecedented for a DNase. Each monomer (61 kDa) carries an exonuclease domain harbouring an active site with two Mn2+ ions. This site is similar to those from the exonuclease domain of the DNA polymerase II and from DNases involved in DNA mending and recombination events like Mre11. I identified essential catalytic residues for the DNase activity and demonstrated that this activity is crucial for infection by engineering A1 mutant proteins and by doing functional complementation assays. Taken together, my results suggest that A1 could then be the elusive DNase responsible for the massive host genome degradation observed during T5 phage infection. Eventually, I uncovered a recombinase activity associated to A1 production during infection. Similar proteins to A1 with unknown functions are produced in several other bacteriophages outside of the T5virus family. This work is a first step towards understanding the role of this protein in the general mechanism of infection by bacteriophages. In a second part, I worked on the structural characterisation of A2 protein. Similarity searches revealed a helix-turn-helix domain typically found in transcriptional regulators. I purified and demonstrated the dimeric organisation of this 14-kDa protein in solution. This initial characterization of A2 has opened avenues for further NMR studies.During my Ph.D., I uncovered the structure of an original bacteriophage DNase that controls bacterial genome degradation and that regulates viral DNA transport at the beginning of the infectious cycle. These results open the intriguing question about the mechanism for T5 DNA protection from A1 DNase activity as well as about the interplay between A1 and A2 during the host takeover.