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Étude des mécanismes moléculaires d'initiation du transfert conjugatif d'ICESt3, médiée par une relaxase MOBT chez la bactérie Gram+ Streptococcus thermophilus
No full textBacterial genomes evolve mainly through horizontal gene transfer. Bacterial conjugation is one of the major mechanisms for these transfers. Conjugation is mediated by integrative and conjugative elements (ICE). In addition to their transfer function, ICEs encode other functions that may provide an adaptive advantage to their host, such as resistance to antibiotics whose dissemination is a major public health issue. It is therefore necessary to understand how ICEs are transferred in order to limit their dissemination.The transfer of an ICE from a donor cell to a recipient cell requires its excision from the chromosome, its transfer from one cell to the other and then its integration into the genomes of the two partner cells. According to the literature, the initiation of ICE transfer is mediated by a nucleoprotein complex called relaxosome, whose key protein is the relaxase, a transesterase encoded by the element. The role of the relaxase is to perform a single-stranded cleavage on the DNA of the ICE at a conserved site, called nic. This cleavage releases a free 3'OH end, used as a primer to initiate rolling circle replication. The DNA-relaxase complex is then driven to the conjugation pore.During my PhD thesis, I studied ICESt3 from Streptococcus thermophilus which belongs to the ICESt3/Tn916/ICEBs1 superfamily, widespread among Firmicutes. These ICEs encode a non-canonical relaxase belonging to the MOBT family, which is related to the rolling circle replication initiators of the Rep_trans family. The general objective of my thesis was to elucidate the function of the RelSt3 relaxase in order to decipher the molecular mechanisms of initiation of conjugative transfer mediated by a MOBT relaxase.My work led to the identification of the RelSt3 binding site on ICESt3 origin of transfer (oriT). This site, called bind, is peculiar in that it is distant from the nic site, which is not the case for other relaxase families. RelSt3 possesses an HTH domain at its N-terminus. I have shown that this domain is required for the binding of RelSt3 to its bind site, and that it is important for its catalytic activity. Conjugation assays demonstrated that this HTH domain is crucial for the conjugative transfer of ICESt3. Structural predictions of the HTH domain in complex with DNA led to the identification of the interaction interface with the bind site, confirmed by mutagenesis. I also demonstrated that RelSt3 exhibits a nicking-closing activity and that it covalently binds to the 5' end of the cleaved strand, demonstrating that this enzyme participates in both initial and final steps of conjugation.In the literature, it has been shown that relaxases interact frequently with other accessory proteins, encoded by the ICE or by the host bacteria, participating in relaxosome formation. The second objective of my thesis was to identify RelSt3 partners. Comparisons with available data on ICEBs1 from Bacillus subtilis allowed to identify two candidate proteins, OrfL and OrfM, that may belong to the relaxosome of ICESt3, as well as a cellular helicase, PcrA , probably involved in the rolling circle replication. A characterization of these proteins was performed using biochemical and biophysical approaches. The interaction network between all of these proteins was established using in vitro approaches, as well as with the in vivo two-hybrid approach. These data provide a first insight into the components of the ICESt3 relaxasome. I also showed that OrfL and OrfM stimulate the catalytic activity of RelSt3 in vitro, and that they are both essential for ICESt3 conjugation.This work lead to a better understanding of the molecular mechanisms required during the conjugation of an ICE driven by a MOBT family relaxase.Les génomes bactériens évoluent principalement grâce au transfert horizontal de gènes. La conjugaison bactérienne en est un des mécanismes majeurs. Elle est notamment médiée par les éléments intégratifs et conjugatifs (ICE). En plus de leur transfert, les ICE codent d'autres fonctions conférant à leur hôte un avantage adaptatif, comme par exemple des résistances aux antibiotiques dont la diffusion est un enjeu majeur en santé publique. Il est donc nécessaire de comprendre comment les ICE se transfèrent si l'on veut limiter leur dissémination. Le transfert d'un ICE d'une cellule donatrice vers une cellule réceptrice implique son excision du chromosome, son transfert puis son intégration dans les génomes des deux cellules partenaires. Les données de la littérature révèlent que l'initiation de ce transfert est médiée par un complexe nucléoprotéique appelé relaxosome, dont la protéine clé est la relaxase, une transestérase codée par l'élément. Le rôle de la relaxase est d'effectuer une coupure simple brin sur l'ADN de l'ICE au niveau d'un site conservé, appelé nic. Ce clivage libère une extrémité 3'OH libre, servant d'amorce pour initier la réplication en cercle roulant. Le complexe ADN-relaxase est alors dirigé vers le pore de conjugaison. Au cours de ma thèse j'ai étudié un ICE modèle, ICESt3 de Streptococcus thermophilus qui appartient à la superfamille ICESt3/Tn916/ICEBs1, très répandue chez les Firmicutes. Ces ICE possèdent une relaxase non canonique, appartenant à la famille MOBT, apparentée aux initiateurs de réplication à cercle roulant de la famille Rep_trans. L'objectif de ma thèse était d'élucider le fonctionnement de la relaxase RelSt3 afin de décrypter les mécanismes moléculaires d'initiation du transfert conjugatif médié par une relaxase MOBT. Mes recherches ont conduit à l'identification du site de liaison de RelSt3 sur l'origine de transfert (oriT) d'ICESt3. Ce site, appelé bind, a pour originalité d'être distant du site nic, ce qui n'est pas le cas des autres familles de relaxases. RelSt3 présente un domaine HTH à son extrémité N-terminal. J'ai montré que ce domaine est requis pour la fixation de RelSt3 sur le site bind, et important pour son activité catalytique. Des tests de conjugaison ont démontré que ce domaine HTH est crucial pour le transfert conjugatif d'ICESt3. Des prédictions structurales de ce domaine en complexe avec l'ADN ont conduit à l'identification de l'interface d'interaction avec le site bind, confirmée par mutagénèse dirigée. J'ai également démontré que RelSt3 présente une activité de coupure-religature et qu'elle se fixe de façon covalente sur l'extrémité 5' du brin clivé, démontrant ainsi que cette enzyme participe aux étapes initiale et terminale de la conjugaison. Dans la littérature, il a été démontré que les relaxases interagissent fréquemment avec d'autres protéines accessoires, codées par l'ICE ou la bactérie hôte pour former le relaxosome. Le deuxième objectif de ma thèse était d'identifier des partenaires de RelSt3. L'analogie avec ICEBs1 chez Bacillus subtilis a permis d'identifier deux protéines candidates OrfL et OrfM codées par ICESt3, ainsi qu'une hélicase cellulaire, probablement impliquée dans la réplication en cercle roulant, nommée PcrA. Une caractérisation de ces protéines candidates a été effectuée en utilisant des approches biochimiques et biophysiques. Le réseau d'interaction entre l'ensemble de ces protéines a été dressé en utilisant des approches in vitro, ainsi que l'approche double hybride in vivo. Ces données nous permettent d'avoir un premier aperçu des constituants du relaxasome d'ICESt3. J'ai par ailleurs montré que OrfL et OrfM stimulent l'activité catalytique de RelSt3 in vitro, et qu'elles sont toutes les deux essentielles à la conjugaison d'ICESt3.Ce travail nous apporte une meilleure compréhension des mécanismes moléculaires en jeu lors de la conjugaison d'un ICE pilotée par une relaxase de la famille MOBT
Study of the molecular mechanisms of initiation of ICESt3 conjugative transfer from Gram+ bacteria, mediated by a MOBT relaxase.
No full textLes génomes bactériens évoluent principalement grâce au transfert horizontal de gènes. La conjugaison bactérienne en est un des mécanismes majeurs. Elle est notamment médiée par les éléments intégratifs et conjugatifs (ICE). En plus de leur transfert, les ICE codent d'autres fonctions conférant à leur hôte un avantage adaptatif, comme par exemple des résistances aux antibiotiques dont la diffusion est un enjeu majeur en santé publique. Il est donc nécessaire de comprendre comment les ICE se transfèrent si l'on veut limiter leur dissémination. Le transfert d'un ICE d'une cellule donatrice vers une cellule réceptrice implique son excision du chromosome, son transfert puis son intégration dans les génomes des deux cellules partenaires. Les données de la littérature révèlent que l'initiation de ce transfert est médiée par un complexe nucléoprotéique appelé relaxosome, dont la protéine clé est la relaxase, une transestérase codée par l'élément. Le rôle de la relaxase est d'effectuer une coupure simple brin sur l'ADN de l'ICE au niveau d'un site conservé, appelé nic. Ce clivage libère une extrémité 3'OH libre, servant d'amorce pour initier la réplication en cercle roulant. Le complexe ADN-relaxase est alors dirigé vers le pore de conjugaison. Au cours de ma thèse j'ai étudié un ICE modèle, ICESt3 de Streptococcus thermophilus qui appartient à la superfamille ICESt3/Tn916/ICEBs1, très répandue chez les Firmicutes. Ces ICE possèdent une relaxase non canonique, appartenant à la famille MOBT, apparentée aux initiateurs de réplication à cercle roulant de la famille Rep_trans. L'objectif de ma thèse était d'élucider le fonctionnement de la relaxase RelSt3 afin de décrypter les mécanismes moléculaires d'initiation du transfert conjugatif médié par une relaxase MOBT. Mes recherches ont conduit à l'identification du site de liaison de RelSt3 sur l'origine de transfert (oriT) d'ICESt3. Ce site, appelé bind, a pour originalité d'être distant du site nic, ce qui n'est pas le cas des autres familles de relaxases. RelSt3 présente un domaine HTH à son extrémité N-terminal. J'ai montré que ce domaine est requis pour la fixation de RelSt3 sur le site bind, et important pour son activité catalytique. Des tests de conjugaison ont démontré que ce domaine HTH est crucial pour le transfert conjugatif d'ICESt3. Des prédictions structurales de ce domaine en complexe avec l'ADN ont conduit à l'identification de l'interface d'interaction avec le site bind, confirmée par mutagénèse dirigée. J'ai également démontré que RelSt3 présente une activité de coupure-religature et qu'elle se fixe de façon covalente sur l'extrémité 5' du brin clivé, démontrant ainsi que cette enzyme participe aux étapes initiale et terminale de la conjugaison. Dans la littérature, il a été démontré que les relaxases interagissent fréquemment avec d'autres protéines accessoires, codées par l'ICE ou la bactérie hôte pour former le relaxosome. Le deuxième objectif de ma thèse était d'identifier des partenaires de RelSt3. L'analogie avec ICEBs1 chez Bacillus subtilis a permis d'identifier deux protéines candidates OrfL et OrfM codées par ICESt3, ainsi qu'une hélicase cellulaire, probablement impliquée dans la réplication en cercle roulant, nommée PcrA. Une caractérisation de ces protéines candidates a été effectuée en utilisant des approches biochimiques et biophysiques. Le réseau d'interaction entre l'ensemble de ces protéines a été dressé en utilisant des approches in vitro, ainsi que l'approche double hybride in vivo. Ces données nous permettent d'avoir un premier aperçu des constituants du relaxasome d'ICESt3. J'ai par ailleurs montré que OrfL et OrfM stimulent l'activité catalytique de RelSt3 in vitro, et qu'elles sont toutes les deux essentielles à la conjugaison d'ICESt3.Ce travail nous apporte une meilleure compréhension des mécanismes moléculaires en jeu lors de la conjugaison d'un ICE pilotée par une relaxase de la famille MOBT.Bacterial genomes evolve mainly through horizontal gene transfer. Bacterial conjugation is one of the major mechanisms for these transfers. Conjugation is mediated by integrative and conjugative elements (ICE). In addition to their transfer function, ICEs encode other functions that may provide an adaptive advantage to their host, such as resistance to antibiotics whose dissemination is a major public health issue. It is therefore necessary to understand how ICEs are transferred in order to limit their dissemination.The transfer of an ICE from a donor cell to a recipient cell requires its excision from the chromosome, its transfer from one cell to the other and then its integration into the genomes of the two partner cells. According to the literature, the initiation of ICE transfer is mediated by a nucleoprotein complex called relaxosome, whose key protein is the relaxase, a transesterase encoded by the element. The role of the relaxase is to perform a single-stranded cleavage on the DNA of the ICE at a conserved site, called nic. This cleavage releases a free 3'OH end, used as a primer to initiate rolling circle replication. The DNA-relaxase complex is then driven to the conjugation pore.During my PhD thesis, I studied ICESt3 from Streptococcus thermophilus which belongs to the ICESt3/Tn916/ICEBs1 superfamily, widespread among Firmicutes. These ICEs encode a non-canonical relaxase belonging to the MOBT family, which is related to the rolling circle replication initiators of the Rep_trans family. The general objective of my thesis was to elucidate the function of the RelSt3 relaxase in order to decipher the molecular mechanisms of initiation of conjugative transfer mediated by a MOBT relaxase.My work led to the identification of the RelSt3 binding site on ICESt3 origin of transfer (oriT). This site, called bind, is peculiar in that it is distant from the nic site, which is not the case for other relaxase families. RelSt3 possesses an HTH domain at its N-terminus. I have shown that this domain is required for the binding of RelSt3 to its bind site, and that it is important for its catalytic activity. Conjugation assays demonstrated that this HTH domain is crucial for the conjugative transfer of ICESt3. Structural predictions of the HTH domain in complex with DNA led to the identification of the interaction interface with the bind site, confirmed by mutagenesis. I also demonstrated that RelSt3 exhibits a nicking-closing activity and that it covalently binds to the 5' end of the cleaved strand, demonstrating that this enzyme participates in both initial and final steps of conjugation.In the literature, it has been shown that relaxases interact frequently with other accessory proteins, encoded by the ICE or by the host bacteria, participating in relaxosome formation. The second objective of my thesis was to identify RelSt3 partners. Comparisons with available data on ICEBs1 from Bacillus subtilis allowed to identify two candidate proteins, OrfL and OrfM, that may belong to the relaxosome of ICESt3, as well as a cellular helicase, PcrA , probably involved in the rolling circle replication. A characterization of these proteins was performed using biochemical and biophysical approaches. The interaction network between all of these proteins was established using in vitro approaches, as well as with the in vivo two-hybrid approach. These data provide a first insight into the components of the ICESt3 relaxasome. I also showed that OrfL and OrfM stimulate the catalytic activity of RelSt3 in vitro, and that they are both essential for ICESt3 conjugation.This work lead to a better understanding of the molecular mechanisms required during the conjugation of an ICE driven by a MOBT family relaxase
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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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