3-Methyl indole (3-MI) is a malodorant in food and animal waste and Clostridium scatologenes ATCC 25775 is the model organism for the study of 3-MI production. 3-MI is an anaerobic degradation product of L-tryptophan and can cause pulmonary disorders and death in cattle and goats. To elucidate the 3-MI biosynthesis pathway and the underlying genes, it is necessary to develop a system to allow genetic modification in Clostridium scatologenes ATCC 25775. Bacteriophages and transposons are useful tools to achieve this goal. Isolation of Clostridium scatologenes ATCC 25775 bacteriophage was attempted by prophage induction and enrichments using environmental sources. To induce prophages, cultures of Clostridium scatologenes ATCC 25775 were exposed to an effective concentration of mitomycin C at 2μg/ml and 5μg/ml. Induction with temperature was performed at 42ºC and 55ºC. Bacteriophage liberation, determined by a decrease in optical density was not observed in response to mitomycin C or by different growth temperatures. Nineteen environmental samples were tested for the presence of a bacteriophage that could infect Clostridium scatologenes ATCC 25775. The first cycle of enrichments suggested a decrease in cell density, consistent with the presence of a bacteriophage but this was not observed in further iterations. Plaque assays were performed to confirm the presence of phage, but no plaques were observed. Although, different experimental conditions were tested, a transducing bacteriophage capable of infecting Clostridium scatologenes ATCC 25775 was not isolated. Transposons have been successfully used to generate mutants in Clostridium difficle. Therefore, we attempted to introduce transposons Tn5 and Tn916 into Clostridium scatologenes ATCC 25775 using electroporation. Transposon mutagenesis using Tn916 did not yield antibiotic resistant colonies. In contrast, commercially available transposon Tn5 gave antibiotic resistant colonies. However, further screening of the colonies using transposon specific primers in PCR reactions, did not yield any PCR product. We were unsuccessful in developing a genetic modification system in Clostridium scatologenes ATCC 25775 using bacteriophage or transposons.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173 / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当

Les piwi-interacting RNAs (piRNA) interagissent avec les protéines qui font partie de la branche PIWI de la famille des Argonautes. Ils participent à la répression des transposons dans la ligne germinale. Chez la souris, les protéines PIWI sont indispensables pour la fertilité des mâles et sont responsables de la méthylation de l'ADN au niveau des promoteurs des transposons. Les piRNA sont produis à partir de deux mécanismes: la biogenèse primaire et le cycle d'amplification ping-pong. Nous avons fait des études fonctionnelles sur deux protéines qui participent à la voie des piRNA: l'hélicase d'ARN MOV10L1 et l'immunophiline FKBP6. Nous avons décrit le rôle de MOV10L1 en tant que facteur de biogenèse primaire. La disruption génétique du domaine hélicase de MOV10L mène à une activation des transposons et à une perte de la méthylation de l'ADN. Ainsi, les piRNA ne sont pas produits chez le mutant, ce qui suggère que MOV10L1 est essentielle pour la biogenèse des piRNA. Tdrd1 interagit avec les protéines PIWI et joue un rôle dans la voie des piRNA en recrutant quelques facteurs à partir de son domaine MYND N-terminal. Nous avons montré que le domaine MYND de Tdrd1 interagit avec FKBP6, une protéine qui appartient à une famille d'isomérases de prolines qui sont présentes dans des complexes de chaperonnes. Les études biochimiques réalisées indiquent que FKBP6 est inactive et qu'elle utilise son domaine isomérase comme un module structural qui interagit avec les protéines PIWI, alors qu'elle interagit avec Hsp90 avec son domaine TPR. L'analyse d'une souris mutante pour fkbp6 a révélé une dérépression des transposons et une perte de la méthylation de l'ADN, mais peu d'effets sur la biogenèse primaire. Ces résultats sont en accord avec un rôle de FKBP6 en aval de Tdrd1, et nous pouvons penser que ce rôle pourrait être le recrutement de Hsp90 pour participer au cycle d'amplification ping-pong.

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T19:12:55Z : No. of bitstreams: 1 dias_es_me_sjrp.pdf: 1177689 bytes, checksum: f1f230e591b3ef47a8155f3bc9916e14 (MD5) / Três modelos têm sido propostos para explicar o modo de expansão dos elementos de transposição nos genomas – master gene, transposon e intermediário. O modelo master gene aplica-se à situação em que existe apenas uma única sequência ativa, de uma subfamília particular, que dá origem às demais sequências, as quais não apresentam capacidade de mobilização, enquanto o modelo transposon assume que todas as sequências de uma subfamília são ativas. Por outro lado, o modelo intermediário, considera que algumas cópias apresentam capacidade de mobilização e outras permanecem inativas. Os modelos propostos podem ser relacionados aos mecanismos de transposição, copy-and-paste e cut-and-paste, característicos das classes de elementos transponíveis I e II, respectivamente. Contudo, os estudos de dispersão intragenômica se concentram em elementos da classe I sem LTRs. Com o objetivo de ampliar o entendimento da dinâmica de dispersão genômica dos elementos de transposição e buscando verificar se os modelos propostos para a dispersão dos retroposons ajustam-se aos transposons e aos retrotransposons, foram analisados o transposon Bari e o retrotransposon 412 no genoma das espécies do grupo melanogaster de Drosophila e em populações naturais de D. melanogaster e D. simulans. Assim, buscas por seqüência similares a ambos os elementos selecionados foram realizadas nos genomas seqüenciados de seis espécies do grupo melanogaster; bem como, foram amplificadas, clonadas e sequenciadas cópias presentes nas populações naturais de ambas as espécies. Foram reconstruídas as relações evolutivas entre as sequências dos genomas, como também entre aquelas das populações naturais por meio do programa Network, utilizando o algoritmo Median Joining. Nossos resultados indicam a ausência de um modelo único de dispersão para ambos os elementos... / Three models have been proposed to explain the expansion of transposable elements within genomes - master gene, transposon and intermediate. The master gene model applies to situations in which there is only one active sequence of a particular subfamily, which gives rise to other sequences which have no capacity for mobilization, while the transposon model assumes that all sequences of a subfamily are active. On the other hand, the intermediate model considers that some copies have the capacity for mobilization and others remain inactive. The proposed models can be related to mechanisms of transposition, copy-and-paste and cut-and-paste, characteristic of class I and II of transposable elements, respectively. However, studies of intragenomic dispersion concentrate on elements of the class I without LTRs. Aiming at enhancing the understanding of the transposable elements genomic dynamics of dispersion and trying to verify whether the proposed models for dispersion of retroposons fit to transposons and retrotransposons, we analyzed the retrotransposon 412 and Bari transposon in the genome of the species melanogaster group of Drosophila and in natural populations of D. melanogaster and D. simulans. Thus, searches for sequences similar to both elements were done in the sequenced genomes of six species of the melanogaster group; as well as copies present in natural populations of both species were amplified, cloned and sequenced. We reconstructed the evolutionary relationships between the sequences of the genomes, as well as those amplified from samples of wild populations through the Network program, using the Median Joining algorithm. Our results indicate the absence of a single model of dispersion for both transposable elements, Bari and 412, in the different species analyzed, as well as in the populations of both species... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-07Bitstream added on 2014-06-13T18:56:06Z : No. of bitstreams: 1 cordeiro_jx_me_jabo.pdf: 844386 bytes, checksum: ae05c2706130041d387b83b68d235663 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Bacillus thuringiensis é uma bactéria gram-positiva e possui habilidade de produzir proteínas denominadas Cry ou delta-endotoxinas, durante a fase de esporulação. Estas proteínas são utilizadas no controle biológico de várias ordens de insetos. No entanto, pouco se conhece a respeito das vias metabólicas envolvidas na produção dessas proteínas, por exemplo, na natureza, é possível encontrar isolados que produzem grande quantidade de proteínas Cry, como também é possível encontrar outros isolados que produzem poucas quantidades. Considerando que essa diferença é devida a uma regulação gênica diferenciada entre isolados, a identificação dessa regulação pode levar a um incremento na produção de proteínas. Neste trabalho, foram analisados 10 clones mutantes comparados com o isolado selvagem (Bt1) quanto à mortalidade de lagartas neonatas de Spodoptera frugiperda considerando também a quantidade de proteínas Cry produzidas por estes mutantes. Os resultados demonstram que dois clones identificados como B9 e E2, demonstraram um bom controle dessas lagartas, mesmo aquelas que permaneceram vivas, ocorreu um atraso em seu desenvolvimento. Quanto à produção de proteínas, os clones apresentaram perfil protéico entre ~45 kDa e ~120 kDa, perfil característico encontrado entre estirpes ativas contra Lepidópteros. No entanto, os clones que apresentaram bons níveis de mortalidade (B9 e E2) mostraram somente a banda de ~65 kDa similar ao isolado selvagem, que pode ser a responsável pela sua ação inseticida. Os resultados obtidos demonstram a possibilidade de efetuar melhoramento genético em B. thuringiensis por meio de mutagênese mediada por transposon e abre caminho para o estudo das vias metabólicas envolvidas na produção de proteínas Cry. / Bacillus is a gram-positive bacterium and produces internal crystal inclusions named crystal (Cry) proteins or d - endotoxins during the sporulation phase. These proteins as used to control several agriculture insect pests. However little is known concerning the metabolic pathways involved in the production of these proteins. For example, in nature, it is possible to find isolates that produce large quantities of the Cry proteins as well as it is possible to find ones that produce limited quantities. Since such difference might be related to gene regulation the identification of regulation factors found in these isolated should lead to an increment on the crystal protein production found in isolated naturally exhibiting lower production. In this work, ten mutant clones were analyzed compared to the wild type (Bt1) with respect to Spodoptera frugiperda neonatal larvae mortality considering also the amount of crystal protein produced by these mutants. The results have demonstrated that two mutant clones named B9 and E2 have produced strong control of these larvae; while at the same time eliciting a delay on the development of the ones that eventually did not dye at first. As for the Cry protein content, the cloned presented a protein profile ranging from ~45 kDa to ~120 kDa which is typical of Cry proteins acting on lepidopterans. The clones that have shown good mortality levels (B9 and E2) have exhibited only a protein band with ~65 kDa, similar to the one produced by the wild type strain, which might be related to its control activity. Finally, the results obtained seem to lead to the possibility of improving genetically B. thuringiensis using mutagenesis induced by transposon and at the same time might pave the way to the shed light on the study of metabolic pathways involved in the Cry proteins production.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Pseudomonas aeruginosa is an opportunistic pathogen that express a plethora of virulence components controlled through two-component regulatory systems that allow for sensing and responding to environmental stimuli. This study was aimed at identifying transcription regulators of algZ that encodes the histidine sensor kinase (AlgZ) of the AlgZR two-component regulatory system. To understand how the algZ gene is transcriptionally controlled, transposon mutagenesis was used to create a mutant library with varying algZ expression based on their b-Galactosidase activity. The gene PA3327 was identified as a potential regulator of algZ expression using arbitrary PCR. This gene encodes a probable non-ribosomal peptide synthetase responsible for the biosynthesis of secondary metabolites such as antibiotics. Further experiments are required to understand how PA3327 transcriptionally regulates algZ expression and its physiological role in the organism. Because the AlgZ/R system regulates virulence, it is possible to attenuate virulence by targeting the expression of algZ gene.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22882号 / 医博第4676号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 濵﨑 洋子, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les transposases sont des enzymes qui reconnaissent des séquences spécifiques sur l'ADN aux bornes des transposons (éléments à transposer) et catalysent des réactions de coupure et de transfert de brins. La mobilité des transposons entraîne la plasticité des génomes, et dans certains cas, l'exaptation de gènes de transposons contribue à l'émergence de nouvelles fonctions cellulaires. La paramécie est un modèle eucaryote unicellulaire extraordinaire pour étudier le rôle des transposases domestiquées de PiggyBac (nommées PiggyMac et PiggyMac-like) dans le réarrangement programmé de son génome. Ces dernières contribuent à l'assemblage de son génome somatique au cours du cycle sexuel. Les principaux axes de ce projet se sont centrés sur l’étude structurale de la transposase PiggyBac (issue de Trichoplusia ni) et une étude d’interaction avec des séquences particulières de son transposon. Ainsi qu’une étude structurale de la transposase domestiquée PiggyMac chez la paramécie.La première structure obtenue (domaine riche en cystéine de la transposase PiggyBac) montre une structuration en doigt de zinc de type PHD-RING. Il a été démontré in vivo et in vitro l’importance du domaine riche en cystéines (CRD) de PiggyBac pour une activité d’excision et d’intégration du transposon PiggyBac. Nous avons pu mettre en évidence que le CRD de PiggyBac cible des séquences spécifiques d’ADN qui sont localisées dans les séquences TIR (Terminal Inverted Repeat) gauche et droite du transposon. Grâce aux résultats issus de la RMN, des modèles de complexes protéine-ADN ont pu être établis.Concernant PiggyMac (transposase PiggyBac domestiquée), son domaine riche en cystéines et histidines a pu être produit doublement marqué (15N et 13C) dans E.coli. Des études structurales en RMN et l'utilisation d'un programme de modélisation moléculaire CYANA ont permis d’accéder à la structure tridimensionnelle de ce domaine.Nous montrons que celui-ci se replie en doigt de zinc entrelacé qui lie deux ions zinc avec un total de huit histidines et cystéines (résultat en cours de publication). La configuration de ce doigt de zinc est différente de celui de PiggyBac et même nouveau dans la littérature concernant ce peptide. Cette étude a permis de mettre en évidence un nouveau type de doigt de zinc. / Transposases are enzymes that recognize specific DNA sequences across transposons (elements to transpose) and catalyze cleavage and transfer reactions strands. The mobility of transposons causes the genome plasticity, and in some cases, the transposon gene exaptation contributes to the emergence of new cellular functions. Paramecium is an extraordinary unicellular eukaryotic model to study the role of transposases domesticated PiggyBac (named PiggyMac and PiggyMac-like) in the programmed rearrangement of its genome. These contribute to the assembly of the somatic genome during sexual cycle. The main axes of this project have focused on the structural study of the transposase PiggyBac (derived from Trichoplusia ni) and an interaction study with particular sequences of the transposon. And a structural study of the transposase domesticated PiggyMac in Paramecium.The first structure obtained (cysteine-rich domain of the transposase PiggyBac) shows a structure in PHD-RING-type zinc finger. It has been demonstrated in vivo and in vitro the importance of the cysteine-rich domain (CRD) of PiggyBac for excision activity and integration of the transposon PiggyBac. We were able to show that the CRD PiggyBac target specific DNA sequences that are located in the TIR sequences (Inverted Terminal Repeat) left and right of the transposon. Thanks to the NMR results, protein-DNA complexes models were established.Regarding PiggyMac (PiggyBac domesticated transposase), its cysteine ​​and histidine rich domain has been doubly labeled product (15N and 13C) in E. coli. NMR structural studies and the use of a CYANA molecular modeling program allowed to access the three-dimensional structure of this domain.We show that it folds into interlaced zinc finger that binds two zinc ions with a total of eight histidine and cysteine ​​(results being published). The configuration of this zinc finger is different from that of PiggyBac and even new in the literature concerning this peptide. The study highlighted a new type of zinc finger.

La brucellose est infection causée par les bactéries du genre Brucella. Cette maladie est répandueà travers le monde entier chez les mammifères et est transmissible aux humains. Causant notammentstérilité, avortement et destruction des articulations, la brucellose représente un risque sanitaire majeur.La chronicité et la récurrence de cette infection provoquent une morbidité importante chez l'hommemalgré des traitements antibiotiques longs et coûteux. Actuellement, les vaccins disponibles ne sont pasconsidérés comme sûrs et efficaces. Le confinement de la maladie repose en partie sur l'identification etl’élimination des troupeaux infectés. La brucellose représente toujours d'énormes pertes économiquespour les pays où la maladie est endémique. Le développement rationnel d'un vaccin atténué efficace etsûr contre les infections à Brucella nécessite l'identification des gènes de virulence indispensables à laréplication in vivo de la bactérie.Dans un modèle d'infection intranasale bien caractérisé chez la souris imitant l'infection naturellepar voie aérienne, nous avons décrit la dynamique de l'infection. En utilisant un marqueur fluorescent,nous sommes en mesure de surveiller la multiplication bactérienne in situ et de déterminer les différentesphases de l'infection. Lors d'une infection intranasale, les macrophages alvéolaires (MA) sont le principaltype cellulaire infecté mais seule une petite proportion de la MA infectée (5 à 15%) est permissive àl'infection. Les bactéries entrant dans la réplication au cours des premières 24 heures sont massivementéliminées, mais cette importante pression sélective peut être partiellement levée par des déficitsimmunitaires génétiques par exemple pour la signalisation de l’IL-17 (réponse immunitaire de type TH17)ou même par une altération de la réponse immunitaire en induisant un phénotype asthmatique (réponseimmunitaire de type TH2).Une identification approfondie de tous les gènes essentiels nécessaires à la croissance sur desmilieux riches ou des gènes conditionnellement nécessaires à la survie lors d'une infection in vitro(macrophages RAW murins) ou in vivo (souris) a été effectuée à différents moments clés précoces du cycleinfectieux en utilisant la technique du séquençage des transposons (Tn-Seq). Sur les 3140 gènes de B.melitensis, 643 sont requis pour la croissance extracellulaire sur des milieux riches. 179 gènessupplémentaires sont indispensables à la survie dans les poumons de la souris jusqu'à 5 jours aprèsl'infection. Seule la moitié de ces gènes peuvent être identifiés à l'aide du modèle in vitro standard,illustrant la limitation d'une telle approche in vitro pour identifier les exigences d'adaptation àl'environnement hôte. L'application de l'analyse en cluster montre que la plupart de ces gènes identifiéspeuvent être recadrés en voies complètes ou impliqués dans des fonctions liées. La synthèse deslipopolysaccharides, la synthèse de certains acides aminés, la B oxydation des acides gras et la cytochromeC oxydase seraient particulièrement importantes face à l'environnement hôte. Nous avons maintenantune idée plus claire des exigences minimales pour que la bactérie infecte avec succès son hôte. Enappliquant cette approche en cas d’immunodéficience pour la signalisation de l’IL-17 ou en conditionasthmatique, nous savons maintenant que l'essentialité de certains gènes peut être levée à savoir lasynthèse du core et de la chaîne O du lipopolysaccharide et la B oxydation des acides gras respectivement.La délétion génétique de certains gènes sélectionnés (10) candidats valide les résultats de nos analysesTn-Seq. Ces analyses comparatives ont le potentiel d'identifier des souches de mutants atténués quipourraient déclencher une immunité protectrice sans la capacité de se propager ou de devenir chroniqueou d'être entièrement virulente même chez des animaux avec une immunité compromise. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished