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Caractérisation moléculaire des protéines OrfM et OrfL dans l’initiation de la conjugaison chez ICESt3 de Streptococcus thermophilus
No full textÉtude des mécanismes moléculaires et cellulaires impliqués dans la conjugaison de ICESt3 chez Streptococcus thermophilus
No full textThèse confidentielle jusqu'au 12 décembre 2027.Conjugation is one of the main mechanisms of horizontal gene transfer, and plays a major role in the spread of antibiotic resistance among bacteria. The transfer of conjugative plasmids and integrative and conjugative elements (ICEs) is mediated by a multiprotein complex localized in the cell envelope of the donor cell and is called the type IV secretion system (T4SS). Little is known about the architecture and the function of T4SSs in Gram-positive bacteria compared with their counterparts in Gram-negative bacteria. The aim of my project is to identify proteins involved in the assembly and function of T4SSs in Gram-positive bacteria. Our model is ICESt3, a mobile genetic element found in Streptococcus thermophilus shown to propagate efficiently by conjugation to various Gram-positive bacteria. ICESt3 comprises a conjugation module which encodes a set of proteins involved in T4SS assembly. The approach we used for this study involved techniques in protein biochemistry, structural biology, immunology, genetics, molecular biology and also fluorescence microscopy. Many of these techniques have required the establishment of several collaborations between our laboratory and several other laboratories at national and international level. In this project, we demonstrated that OrfG, the VirB8-like of ICESt3, functions as a trimeric subunit. Structure determination revealed that its assembly is shared among VirB8-like proteins within β-class, which is widespread in Firmicutes. We also identified the interaction of OrfG with DNA and other T4SS components, in particular the predicted peptidoglycan hydrolase OrfA, the VirB1-like of the system. On the basis of these data, we propose a model for T4SS assembly in Gram-positive bacteria, consisting of two complexes: the cell wall complex formed by the VirB8-like and VirB1-like subunits, and the translocon formed by the other T4SS proteins. We reinforced this model by studying OrfD, the VirB4-like of the system, where we demonstrated its multimerisation, its interaction with OrfG, as well as with DNA. Our study thus provides new insights into the conjugation process and more specifically into T4SS assembly.La conjugaison est l'un des principaux mécanismes du transfert horizontal de gènes, notamment les gènes de résistances aux antibiotiques. Elle permet l'échange des éléments génétiques mobiles, incluant les plasmides conjugatifs et les éléments conjugatifs intégratifs (ICE), entre bactéries de manière contact dépendant. Le transfert de ces éléments est médié par un complexe multiprotéique localisé dans l'enveloppe cellulaire de la cellule donneuse et appelé système de sécrétion de type IV (T4SS). On sait peu de choses sur l'architecture et la fonction des T4SS chez les bactéries Gram-positives par rapport à leurs homologues chez les bactéries Gram-négatives. Le but de mon projet de thèse a été d'étudier l'assemblage et l'architecture du T4SS chez les bactéries Gram-positives. Notre modèle d'étude est le T4SS codé par ICESt3, un élément génétique mobile présent chez Streptococcus thermophilus, capable de se transférer par conjugaison entre des microorganismes de la même espèce ou d'espèces différentes. Afin de répondre aux questionnements scientifiques de mon projet doctoral, nous avons utilisé plusieurs approches complémentaires notamment la biochimie des protéines, la biologie structurale, l'immunologie, la génétique, la biologie moléculaire ainsi que la microscopie. Plusieurs de ces techniques ont nécessité la mise en place de nombreuses collaborations entre notre laboratoire et d'autres laboratoires au niveau nationale et international. Nous avons démontré que OrfG, la VirB8-like de ICESt3, fonctionne sous forme de sous-unité trimétrique. La détermination de sa structure nous a révélé que son assemblage est conservé chez la famille des protéines VirB8 de la classe β, très répondu chez les Firmicutes. Nous avons également identifié une interaction de OrfG avec l'ADN et d'autres composants du T4SS ; en particulier la prédite hydrolase du peptidoglycane OrfA. Grace à ces données, nous proposons un modèle d'assemblage du T4SS chez les bactéries Gram-positives qui serait composé de deux complexes : le complexe de la paroi cellulaire formé par les sous-unités VirB8-like et VirB1-like puis le translocon formé par les autres protéines du T4SS. Nous renforçons ce modèle par l'étude de OrfD, la VirB4-like du système où nous avons mis en évidence sa multimérisation et son interaction avec OrfG mais aussi avec l'ADN. Notre étude apporte ainsi des nouvelles perspectives sur le processus de conjugaison et plus spécifiquement dans l'assemblage du T4SS
Study of the molecular and cellular mechanism involved on the conjugative transfer of ICESt3 from Streptococcus thermophilus
No full textLa conjugaison est l'un des principaux mécanismes du transfert horizontal de gènes, notamment les gènes de résistances aux antibiotiques. Elle permet l'échange des éléments génétiques mobiles, incluant les plasmides conjugatifs et les éléments conjugatifs intégratifs (ICE), entre bactéries de manière contact dépendant. Le transfert de ces éléments est médié par un complexe multiprotéique localisé dans l'enveloppe cellulaire de la cellule donneuse et appelé système de sécrétion de type IV (T4SS). On sait peu de choses sur l'architecture et la fonction des T4SS chez les bactéries Gram-positives par rapport à leurs homologues chez les bactéries Gram-négatives. Le but de mon projet de thèse a été d'étudier l'assemblage et l'architecture du T4SS chez les bactéries Gram-positives. Notre modèle d'étude est le T4SS codé par ICESt3, un élément génétique mobile présent chez Streptococcus thermophilus, capable de se transférer par conjugaison entre des microorganismes de la même espèce ou d'espèces différentes. Afin de répondre aux questionnements scientifiques de mon projet doctoral, nous avons utilisé plusieurs approches complémentaires notamment la biochimie des protéines, la biologie structurale, l'immunologie, la génétique, la biologie moléculaire ainsi que la microscopie. Plusieurs de ces techniques ont nécessité la mise en place de nombreuses collaborations entre notre laboratoire et d'autres laboratoires au niveau nationale et international. Nous avons démontré que OrfG, la VirB8-like de ICESt3, fonctionne sous forme de sous-unité trimétrique. La détermination de sa structure nous a révélé que son assemblage est conservé chez la famille des protéines VirB8 de la classe β, très répondu chez les Firmicutes. Nous avons également identifié une interaction de OrfG avec l'ADN et d'autres composants du T4SS ; en particulier la prédite hydrolase du peptidoglycane OrfA. Grace à ces données, nous proposons un modèle d'assemblage du T4SS chez les bactéries Gram-positives qui serait composé de deux complexes : le complexe de la paroi cellulaire formé par les sous-unités VirB8-like et VirB1-like puis le translocon formé par les autres protéines du T4SS. Nous renforçons ce modèle par l'étude de OrfD, la VirB4-like du système où nous avons mis en évidence sa multimérisation et son interaction avec OrfG mais aussi avec l'ADN. Notre étude apporte ainsi des nouvelles perspectives sur le processus de conjugaison et plus spécifiquement dans l'assemblage du T4SS.Conjugation is one of the main mechanisms of horizontal gene transfer, and plays a major role in the spread of antibiotic resistance among bacteria. The transfer of conjugative plasmids and integrative and conjugative elements (ICEs) is mediated by a multiprotein complex localized in the cell envelope of the donor cell and is called the type IV secretion system (T4SS). Little is known about the architecture and the function of T4SSs in Gram-positive bacteria compared with their counterparts in Gram-negative bacteria. The aim of my project is to identify proteins involved in the assembly and function of T4SSs in Gram-positive bacteria. Our model is ICESt3, a mobile genetic element found in Streptococcus thermophilus shown to propagate efficiently by conjugation to various Gram-positive bacteria. ICESt3 comprises a conjugation module which encodes a set of proteins involved in T4SS assembly. The approach we used for this study involved techniques in protein biochemistry, structural biology, immunology, genetics, molecular biology and also fluorescence microscopy. Many of these techniques have required the establishment of several collaborations between our laboratory and several other laboratories at national and international level. In this project, we demonstrated that OrfG, the VirB8-like of ICESt3, functions as a trimeric subunit. Structure determination revealed that its assembly is shared among VirB8-like proteins within β-class, which is widespread in Firmicutes. We also identified the interaction of OrfG with DNA and other T4SS components, in particular the predicted peptidoglycan hydrolase OrfA, the VirB1-like of the system. On the basis of these data, we propose a model for T4SS assembly in Gram-positive bacteria, consisting of two complexes: the cell wall complex formed by the VirB8-like and VirB1-like subunits, and the translocon formed by the other T4SS proteins. We reinforced this model by studying OrfD, the VirB4-like of the system, where we demonstrated its multimerisation, its interaction with OrfG, as well as with DNA. Our study thus provides new insights into the conjugation process and more specifically into T4SS assembly
A new twin expands the VirB8-like protein family
No full textInternational audienceConjugative transfer is mediated by specialized type IV secretion systems (T4SSs). However, their architecture and mode of function remain poorly defined in Gram-positives. In this issue of Structure, Jager et al. reveal an exclusive assembly of PrgL and illustrate the importance of its structural organization in pCF10 conjugative transfer
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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