Les systèmes à deux composants jouent un rôle prépondérant dans l'adaptation des bactéries à leur environnement. L'objectif de ce travail de thèse était d'identifier et de caractériser des systèmes à deux composants chez la bactérie phytostimulatrice Azospirillum nécessaires à l'adaptation à la rhizosphère de sa plante-hôte. L'analyse de la distribution génomique des gènes appartenant à la famille des systèmes à deux composants dans les génomes d'Azospirillum disponibles a révélé l'existence d'un grand nombre de gènes codant des hisitidine kinases hybrides, et une analyse plus approfondie a montré une organisation multidomaines complexe de cette famille de protéines. Afin de comprendre leur rôle chez Azospirillum, nous avons, dans un premier temps, sélectionné et inactivé quatre gènes codant des histidine kinases hybrides présentant une architecture multidomaines complexe. A l'aide d'une approche multidisciplinaire combinant génétique, biochimie et phylogénie, nous avons mis en évidence pour la première fois chez Azospirillum, un système atypique à trois-composants nommé PreSKR contrôlant un grand nombre de processus impliqués dans la survie et la colonisation de la rhizosphère, qui agirait en modulant le taux intracellulaire de c-di-GMP. Dans un second temps, nous nous sommes focalisés sur une histidine kinase hybride exprimée au contact de la plante hôte ; cette protéine, appelée RsiK, s'avère être impliquée dans la perception de surfaces et la régulation de la formation de biofilms. L'analyse du régulon par RNA-seq a révèlé que 78 gènes étaient contrôlés par ce système. La prévalence de la famille des histidine kinases hybrides chez Azospirillum couplée à l'approche fonctionnelle réalisée sur deux d'entre elle souligne l'importance des phosphorelais encore largement méconnus chez les bactéries rhizosphériques / Bacterial two-component systems play an important role in the ability of bacteria to adapt to various environments. The aim of this thesis was to identify and characterize two-component systems involved in the adaptation of the phytostimulatory bacteria Azospirillum to its host plant. Analysis of the genomic distribution of genes encoding two-component systems across Azospirillum available genomes revealed the existence of a high number of genes encoding hybrid histidine kinases, and further analyses highlighted a complex multi-domain organization of this family of proteins. In order to understand their role in Azospirillum, as a first step we selected and inactivated four genes encoding complex hybrid histidines kinases. Using a multidisciplinary approach which combines genetics, biochemistry and phylogeny, we brought to light for the first time in Azospirillum, an atypical three-component system named PreSKR which controls a wide variety of processes involved in survival and rhizosphere colonization likely by modulating c-di-GMP levels. As a second step, we focused on a gene encoding a hybrid histidine kinase named RsiK which is induced in contact with its host plant. RsiK is involved in surface sensing and biofilm formation regulation. Transcriptomic analysis of rsiK regulon by RNA-seq showed that 78 genes were under the control of this system. The prevalence of genes encoding hybrid histidine kinase family in Azospirillum, coupled with the functional characterization of two of them, highlight the importance of phosphorelays, still largely unrecognized in rhizospheric bacteria

Les cytokinines sont des hormones régulant de nombreux processus physiologiques. Elles sont en particulier impliquées dans certaines interactions plantes-microorganismes pathogènes. Leur perception est assurée par les récepteurs Histidine Kinases (HK) dont la fonction est prédominante dans la transduction de signaux moléculaires et environnementaux. Ce travail a permis une caractérisation complète des cinq récepteurs aux cytokinines de type CHASE-Histidine Kinase (MdCHK) et d’un osmosenseur (MdHK1) chez le pommier, espèce d’intérêt économique soumise à l’attaque de nombreux pathogènes. La combinaison d’approches moléculaires et bio-informatiques a révélé les particularités fonctionnelles et les rôles spécifiques des MdCHK. Ces connaissances ont permis de sélectionner une souche de levures exprimant le récepteur MdCHK2 en tant que biosenseur de cytokinines. Ce nouvel outil biotechnologique optimisé pour la détection rapide de cytokinines a conduit à la mise en évidence d’une production de ces composés par Erwinia amylovora, bactérie pathogène du pommier. Le criblage de bactéries pathogènes humaines grâce au biosenseur a également révélé leur production par Staphylococcus aureus et Streptococcus agalactiae. Afin d’optimiser ce biosenseur, une approche mécanistique de MdCHK2 a été entreprise et dévoile l’importance de certains domaines dans ses spécificités de perception. / Cytokinins are hormones regulating numerous physiological processes. They are particularly involved in some plant-pathogen interactions. Their perception is ensured by Histidine Kinase (HK) receptors which play a prevailing role in transducing molecular and environmental signals. This work enabled the full characterization of the five cytokinin CHASE-Histidine Kinase receptors (MdCHK) and the osmosensor (MdHK1) in apple tree, a species of economic interest subjected to many pathogen attacks. Combining molecular and bioinformatics approaches led us to reveal the functional features and specific roles of the MdCHK. This knowledge allowed us to select a yeast expressing the MdCHK2 receptor as a cytokinin biosensor. This new biotechnological tool optimized for fast cytokinin detection led us to expose the production of such molecules in Erwinia amylovora, a pathogenic bacterium of apple. Screening human pathogenic bacteria with the biosensor also unveiled their production in Staphylococcus aureus and Streptococcus agalactiae. In order to optimize this biosensor, we initiated a mechanistic approach on MdCHK2 and showed the importance of some domains in its perception specificities.

Bien que présents dans l'organisme en quantités infinitésimales, les métaux de transition jouent un rôle physiologique essentiel. On les trouve généralement sous forme de chélates avec des coordinats biologiques de masse molaire élevée dont l'étude est particulièrement difficile. Il est cependant possible de modéliser les sites actifs de ces macromolécules à l'aide de petites molécules plus faciles à étudier. Nous avons synthétisé des pseudo-peptides à base d'histamine pour simuler les sites de coordination de protéines telles que la sérum albumine. Ces peptidoamines sont également intéressantes du fait de leur activité antioxydante. L'étude des propriétés complexantes de ces molécules vis-à-vis du Cu(II) et du Ni(II) a permis de mettre en évidence une variété de complexes parfois très différents par leur nature et leur stabilité. À partir de ces résultats, nous avons proposé la synthèse de nouveaux tensioactifs possédant des propriétés complexantes par greffage d'un chaîne hydrophobe sur des peptides contenant l'histidine. Les têtes polaires correspondent aux pseudo-peptides peptides ß-alanyl-histidine et glycyl-glycyl-histidine. Les concentrations micellaires critiques ont été déterminées par tensiométrie et fluorimétrie et les diagrammes de phase binaires ont été tracés. Les propriétés complexantes ont été démontrées et les diagrammes de répartition de espèces ont été déterminés à partir de molécules modèles à courte chaîne hydrophobe. Enfin des tensioactifs trimodulaires ont été préparés en greffant sur les molécules précédentes une partie de type polyoxyéthylène, pour augmenter l'hydrophilie.

Transition metals such as copper, zinc and nickel are required in many enzymatic processes that require redox changes. When transition metal concentration exceeds a certain threshold, their redox and metal binding properties make these elements extremely toxic. Bacteria regulate the cellular concentration of these important, yet toxic, elements using elaborate homeostatic systems. One such mechanism is the chemiosmotic extrusion of copper by the Cus system in the Gram-negative bacterium Escherichia coli. This work studies the regulation of the Cus system in response to copper and silver ions. Copper is an essential cofactor required in many enzymatic processes. But its redox properties can lead to toxicity. Silver is chemically similar to copper, but is not bioactive and its presence in cells can lead to extreme cytotoxicity. Transcription from cusCFBA genes is controlled by the CusR/CusS TCS in response to elevated levels of copper or silver in the periplasmic space of E. coli. Extracellular signals are transduced into the cell through phosphotransfer reactions between the prototypical histidine kinase CusS and the response regulator CusR. Copper sensing by the periplasmic domain of CusS is proposed to initiate signal transduction in the Cus system. Despite the frequency with which bacteria employ histidine kinases to sense their environment, signal recognition and incorporation by the protein is not well understood. The goal of this research is to investigate the role of CusS in regulating metal homeostasis in E. coli and characterize the periplasmic domain of the protein to determine its metal binding properties. The experiments described in this work reveal that the CusS is essential for copper and silver resistance and regulates expression from the cusCFBA promoter region. Signal recognition occurs by direct metal binding by the periplasmic domain of CusS. Metal binding causes a change in the secondary structure of the domain and its tendency to dimerize is enhanced under these conditions. The possibility of signal attenuation by interaction with the metallochaperone CusF is also discussed. These data help construct a model for signal transduction in the Cus system and help characterize, for the first time, a metal-responsive sensor histidine kinase in E. coli.

Two component systems (TCSs) are regulatory pathways in bacteria and lower eukaryotes that integrate multiple stimuli and bring about appropriate responses to promote adaptation of the bacteria to their niches. They are commonly insulated from cross-talk and form discrete regulatory systems where the sensor histidine kinase (SK) and the response regulator (RR) share high fidelity for one another. The GacS network controls the switch between acute and chronic virulence of P. aeruginosa. The network is unusual in having a 'core' SK, GacS, which is modulated directly by one other SK, RetS. Here the complex relationship between GacS and RetS is dissected to reveal three distinct mechanisms by which they interact. Two of these mechanisms involve the dephosphorylation of GacS-P by RetS and it is these mechanisms that are important in vivo for the regulation of biofilm formation, rsmY and rsmZ expression, swarming, and virulence in both Galleria mellonella and an acute model of infection in mice. This study reveals an unprecedented level of complexity in the ability of RetS to interact with GacS and suggests that RetS has a number of mechanisms by which it can downregulate the GacS network output. Furthermore, the interactions of additional SKs that have previously been linked to the GacS network were investigated. Here I demonstrate that many of these kinases can interact with one another but that RetS remained the only kinase tested that could directly interact with GacS. The interactions observed between kinases could be either stimulatory, having a synergistic impact on phosphorylation levels, or inhibitory. I also show that kinase-kinase interactions allow for the regulation of phosphorylation of downstream proteins. Finally, we searched for additional SKs that may be able to interact with the GacS network. Here I identify three new kinases, which show differing interactions with the kinases of the GacS network. The discovery of additional SKs in the GacS network indicates that the network is likely to respond to a far greater number of different signals than previously realised as it decides between acute and chronic virulence.

and key words Mouse polyomavirus-derived virus-like particles composed from major capsid protein VP1 (MPyV VP1-VLPs) are interesting structures for use as a delivery system of various cargos into cells. VP1 protein self-assembles into icosahedral particles of 45 nm in diameter that are hollow highly regular nanoparticles. In this work, model small molecule cargo, Cyclodextrin-Based Bimodal Fluorescence/MRI Contrast Agent, was encapsidated into MPyV VP1-VLPs. The cargo was stably associated with VLPs and was delivered into mammalian cells using these VLPs. To prevent VLPs entrapment in endolysosomal compartments and increase the potential of VLPs applications, MPyV VP1 protein was modified by insertion of histidine-tag (6 histidine long sequence surrounded by glycine and serine) sequences into VP1 surface loop DE, because histidine modification of synthetic systems had enhancing effect on endosome escape and cargo delivery. With the use of in Bac-to-Bac® baculovirus expression system His-VP1 protein was expressed in insect cells and a variety of VP1-assemblies was obtained: long tubules and small 20nm VLPs formed from VP1 with 4 histidine-tags in DE loop, and novel VP1 nanostructure, which we named nano-jumpers, formed from VP1 with 2 histidine-tags. Nonetheless the endosome escape properties of...

O nosso grupo tem investigado os efeitos da Clorfeniramina (CPA), antagonista H1, e da L-histidina (LH), uma droga precursora da síntese de histamina, sobre a ansiedade e a memória emocional. Diante disso, os objetivos desse estudo foram investigar os efeitos LH administrada i.p. e da CPA microinjetada no hipocampo dorsal sobre a ansiedade e a memória emocional de camundongos submetidos ao labirinto em cruz elevado (LCE). O experimento foi realizado em dois dias consecutivos. No primeiro dia (T1) 71 camundongos machos da cepa Suíço-Albino pesando 25-35g foram pré-tratados i.p. com salina (SAL) ou LH (500mg/Kg). Após duas horas, os sujeitos receberam microinjeção de SAL ou CPA (0,016nmol; 0,052nmol; 0,16nmol/0,1l) no hipocampo dorsal. Após cinco minutos, os animais foram expostos ao LCE por cinco minutos. Vinte quatro horas depois, o mesmo protocolo experimental foi adotado na reexposição (T2). Os animais foram distribuídos aleatoriamente em 8 grupos de acordo com o tratamento farmacológico: SAL/SAL (n=9), SAL/CPA1 (n=9), SAL/CPA2 (n=10), SAL/CPA3 (n=8), LH/SAL (n=10), LH/CPA1 (n=8), LH/CPA2 (n=8) e LH/CPA3 (n=9). As três doses de CPA microinjetadas no hipocampo dorsal não alteraram a porcentagem de tempo gasto nos braços abertos (%TBA) na exposição ao LCE: T1 SAL/CPA1 (46,13±4,45); SAL/CPA2 (47,59±4,89); SAL/CPA3 (44,30±6,65) quando comparados com o grupo controle SAL/SAL (35,84±2,77) e não alteraram o número de entradas nos braços fechados (EBF) SAL/CPA1 (8,56±1,06); SAL/CPA2 (9,70±1,10); SAL/CPA3 (9,38±1,25) - quando comparados com o grupo controle SAL/SAL (10,56±1,11). A administração i.p. de LH aumentou a %TBA em T1 para o grupo LH/SAL (59,79±4,71), quando comparado ao grupo controle SAL/SAL (35,84±2,77), mas não alterou o EBF: LH/SAL (9,20±1,78) e SAL/SAL (10,56±1,11). Os animais do grupo LH/CPA3 diminuíram %TBA (32,25±4,81) em T1 quando comparados com o grupo LH/SAL (59,79±4,71). Os animais do grupo SAL/CPA1 não apresentaram diminuição da %TBA em T2 (T1: 46,13±4,45; T2: 39,38±6,53). O mesmo foi observado para os sujeitos dos grupos LH/CPA2 (T1: 50,10±3,99; T2: 40,97±8,22) e LH/CPA3 (T1: 32,25±4,81; T2: 32,16±6,93). Nós concluímos que: a CPA microinjetada no hipocampo dorsal de camundongos não apresenta efeito sobre a ansiedade; a administração intraperitoneal de LH apresenta efeito ansiolítico em camundongos expostos ao LCE e que esse efeito é revertido pela maior dose de CPA (0,16nmol/0,1l); são necessárias maiores doses de CPA para que haja prejuízo na memória emocional de camundongos reexpostos ao LCE quando os níveis de histamina no hipocampo dorsal estão elevados. / Our group has been investigating the effects of Chlorpheniramine (CPA), a histaminergic H1 antagonist, and of L-Histidine (LH), a histamine precursor, on anxiety-related behaviors and emotional memory. Thus the aim of this study was to investigate the effects of LH i.p. injections and of dorsal intra-hippocampal microinjections of Chlorpheniramine (CPA) on anxiety-related behaviors and emotional memory in male mice using elevated plus-maze (EPM). The experiment was performed in two days. On the first day (T1) 71 male Swiss Albino mice of body weight 25- 35g were pre-treated with saline (SAL) i.p. or LH (500mg/Kg) i.p. After two hours they were treated with dorsal intra-hippocampal microinjections of SAL or CPA (0.016nmol; 0.052nmol; 0.16nmol/0,1l). Five minutes after intra-hippocampal microinjections the animals were exposed to EPM for 5 minutes. Twenty four hours later the same protocol was repeated (T2). The animals were randomly assigned to 8 groups based on drug treatment: SAL/SAL (n=9), SAL/CPA1 (n=9), SAL/CPA2 (n=10), SAL/CPA3 (n=8), LH/SAL (n=10), LH/CPA1 (n=8), LH/CPA2 (n=8) and LH/CPA3 (n=9). All three doses of intra-hippocampal microinjections of CPA did not change the percentage of time spent in the open-arms (%OAT) on T1 SAL/CPA1 (46.13±4.45); SAL/CPA2 (47.59±4.89); SAL/CPA3 (44.30±6.65) when compared to control group SAL/SAL (35.84±2.77) and did not change the enclosed arm entries (EAE) SAL/CPA1 (8.56±1.06); SAL/CPA2 (9.70±1.10); SAL/CPA3 (9.38±1.25) when compared to control group SAL/SAL (10.56±1.11). Intraperitoneal injections of LH increased %OAT on T1 on LH/SAL group (59.79±4.71), when compared to control group SAL/SAL (35.84±2.77), but not EAE LH/SAL (9.20±1.78) and SAL/SAL (10.56±1.11). Animals treated with LH and CPA3 (LH/CPA3) decreased %OAT (32.25±4.81) on T1 when compared to LH/SAL (59.79±4.71) group. SAL/CPA1 animals did not decreased %OAT on T2 (T1: 46.13±4.45; T2: 39.38±6.53). The same happened to LH/CPA2 (T1: 50.10±3.99; T2: 40.97±8.22) and LH/CPA3 (T1: 32.25±4.81; T2: 32.16±6.93) groups. Thus, we conclude that: dorsal intra-hippocampal microinjection of Chlorpheniramine has no effect on anxiety-related behaviors in male mice; intraperitoneal injection of L-Histidine has an anxiolytic-like effect in male mice exposed to elevated plus-maze, that is reversed by the higher dose of Chlorpheniramine (0.16nmol/0,1l); higher doses of CPA are necessary to impair emotional memory when the levels of hippocampal histamine are elevated.

O nosso grupo tem investigado os efeitos da Clorfeniramina (CPA), antagonista H1, e da L-histidina (LH), uma droga precursora da síntese de histamina, sobre a ansiedade e a memória emocional. Diante disso, os objetivos desse estudo foram investigar os efeitos LH administrada i.p. e da CPA microinjetada no hipocampo dorsal sobre a ansiedade e a memória emocional de camundongos submetidos ao labirinto em cruz elevado (LCE). O experimento foi realizado em dois dias consecutivos. No primeiro dia (T1) 71 camundongos machos da cepa Suíço-Albino pesando 25-35g foram pré-tratados i.p. com salina (SAL) ou LH (500mg/Kg). Após duas horas, os sujeitos receberam microinjeção de SAL ou CPA (0,016nmol; 0,052nmol; 0,16nmol/0,1l) no hipocampo dorsal. Após cinco minutos, os animais foram expostos ao LCE por cinco minutos. Vinte quatro horas depois, o mesmo protocolo experimental foi adotado na reexposição (T2). Os animais foram distribuídos aleatoriamente em 8 grupos de acordo com o tratamento farmacológico: SAL/SAL (n=9), SAL/CPA1 (n=9), SAL/CPA2 (n=10), SAL/CPA3 (n=8), LH/SAL (n=10), LH/CPA1 (n=8), LH/CPA2 (n=8) e LH/CPA3 (n=9). As três doses de CPA microinjetadas no hipocampo dorsal não alteraram a porcentagem de tempo gasto nos braços abertos (%TBA) na exposição ao LCE: T1 SAL/CPA1 (46,13±4,45); SAL/CPA2 (47,59±4,89); SAL/CPA3 (44,30±6,65) quando comparados com o grupo controle SAL/SAL (35,84±2,77) e não alteraram o número de entradas nos braços fechados (EBF) SAL/CPA1 (8,56±1,06); SAL/CPA2 (9,70±1,10); SAL/CPA3 (9,38±1,25) - quando comparados com o grupo controle SAL/SAL (10,56±1,11). A administração i.p. de LH aumentou a %TBA em T1 para o grupo LH/SAL (59,79±4,71), quando comparado ao grupo controle SAL/SAL (35,84±2,77), mas não alterou o EBF: LH/SAL (9,20±1,78) e SAL/SAL (10,56±1,11). Os animais do grupo LH/CPA3 diminuíram %TBA (32,25±4,81) em T1 quando comparados com o grupo LH/SAL (59,79±4,71). Os animais do grupo SAL/CPA1 não apresentaram diminuição da %TBA em T2 (T1: 46,13±4,45; T2: 39,38±6,53). O mesmo foi observado para os sujeitos dos grupos LH/CPA2 (T1: 50,10±3,99; T2: 40,97±8,22) e LH/CPA3 (T1: 32,25±4,81; T2: 32,16±6,93). Nós concluímos que: a CPA microinjetada no hipocampo dorsal de camundongos não apresenta efeito sobre a ansiedade; a administração intraperitoneal de LH apresenta efeito ansiolítico em camundongos expostos ao LCE e que esse efeito é revertido pela maior dose de CPA (0,16nmol/0,1l); são necessárias maiores doses de CPA para que haja prejuízo na memória emocional de camundongos reexpostos ao LCE quando os níveis de histamina no hipocampo dorsal estão elevados. / Our group has been investigating the effects of Chlorpheniramine (CPA), a histaminergic H1 antagonist, and of L-Histidine (LH), a histamine precursor, on anxiety-related behaviors and emotional memory. Thus the aim of this study was to investigate the effects of LH i.p. injections and of dorsal intra-hippocampal microinjections of Chlorpheniramine (CPA) on anxiety-related behaviors and emotional memory in male mice using elevated plus-maze (EPM). The experiment was performed in two days. On the first day (T1) 71 male Swiss Albino mice of body weight 25- 35g were pre-treated with saline (SAL) i.p. or LH (500mg/Kg) i.p. After two hours they were treated with dorsal intra-hippocampal microinjections of SAL or CPA (0.016nmol; 0.052nmol; 0.16nmol/0,1l). Five minutes after intra-hippocampal microinjections the animals were exposed to EPM for 5 minutes. Twenty four hours later the same protocol was repeated (T2). The animals were randomly assigned to 8 groups based on drug treatment: SAL/SAL (n=9), SAL/CPA1 (n=9), SAL/CPA2 (n=10), SAL/CPA3 (n=8), LH/SAL (n=10), LH/CPA1 (n=8), LH/CPA2 (n=8) and LH/CPA3 (n=9). All three doses of intra-hippocampal microinjections of CPA did not change the percentage of time spent in the open-arms (%OAT) on T1 SAL/CPA1 (46.13±4.45); SAL/CPA2 (47.59±4.89); SAL/CPA3 (44.30±6.65) when compared to control group SAL/SAL (35.84±2.77) and did not change the enclosed arm entries (EAE) SAL/CPA1 (8.56±1.06); SAL/CPA2 (9.70±1.10); SAL/CPA3 (9.38±1.25) when compared to control group SAL/SAL (10.56±1.11). Intraperitoneal injections of LH increased %OAT on T1 on LH/SAL group (59.79±4.71), when compared to control group SAL/SAL (35.84±2.77), but not EAE LH/SAL (9.20±1.78) and SAL/SAL (10.56±1.11). Animals treated with LH and CPA3 (LH/CPA3) decreased %OAT (32.25±4.81) on T1 when compared to LH/SAL (59.79±4.71) group. SAL/CPA1 animals did not decreased %OAT on T2 (T1: 46.13±4.45; T2: 39.38±6.53). The same happened to LH/CPA2 (T1: 50.10±3.99; T2: 40.97±8.22) and LH/CPA3 (T1: 32.25±4.81; T2: 32.16±6.93) groups. Thus, we conclude that: dorsal intra-hippocampal microinjection of Chlorpheniramine has no effect on anxiety-related behaviors in male mice; intraperitoneal injection of L-Histidine has an anxiolytic-like effect in male mice exposed to elevated plus-maze, that is reversed by the higher dose of Chlorpheniramine (0.16nmol/0,1l); higher doses of CPA are necessary to impair emotional memory when the levels of hippocampal histamine are elevated.

Phosphate is an essential compound for life. Escherichia coli employs a signal transduction pathway that controls the expression of genes that are required for the high-affinity acquisition of phosphate and the utilization of alternate sources of phosphorous. These genes are only expressed when environmental phosphate is limiting. The seven genes for this signaling pathway encode the two-component regulatory proteins PhoB and PhoR, as well as the high-affinity phosphate transporter PstSCAB and an auxiliary protein called PhoU. As the sensor kinase PhoR has no periplasmic sensory domain, the mechanism by which these cells sense environmental phosphate is not known. This paper explores the hypothesis that it is the alternating conformations of the PstSCAB transporter which are formed as part of the normal phosphate transport cycle that signal phosphate sufficiency or phosphate limitation. We tested two variants of PstB that are predicted to lock the protein in either of two conformations for their signaling output. We observed that the pstBQ160K mutant, predicted to reside in an inward facing, open conformation signaled phosphate sufficiency whereas the pstBE179Q mutant, predicted to reside in an outward facing, closed conformation signaled phosphate starvation. Neither mutant showed phosphate transport.

L'endoribonucléase de restriction RegB est une enzyme produite par le bactériophage T4. Elle est impliquée dans la transition phase précoce-phase moyenne durant le cycle lytique du virus. RegB coupe avec une spécificité quasi absolue la séquence GGAG impliquée notamment dans l'initiation de la traduction chez la bactérie Escherichia coli. Nous avons caractérisé dans cette thèse de façon précise la toxicité de RegB dans la bactérie et nous avons proposé des outils pour contourner cette toxicité tels la manipulation du nombre de copies du vecteur d'expression ou l'atténuation de l'efficacité du site d'initiation de la traduction. Nous avons, par ailleurs, proposé une application de RegB pour la construction d'un vecteur de clonage à sélection positive et à expression duale dans les systèmes procaryotes et eucaryotes. L'étude par RMN du 31P de la cinétique de clivage d'un ARN par RegB a permis de définir RegB comme une "transphosphorylase libérant un phosphodiester 2', 3'-cyclique". Des études de mutagenèses dirigées et aléatoires combinées à l'évolution du gène regB dans un virus apparenté au phage T4 (le virus RB49) ont mis en évidence le rôle des résidus Glutamate 19, Histidine 48, Arginine 52 et Histidine 68 dans l'activité de RegB. Le mutant RegB H48A a été choisi pour construire un modèle structural du site actif de RegB. L'attribution séquentielle de cette protéine par RMN hétéronucléaire 1H/15N/13C a été entreprise avec succès.