Xylella fastidiosa é uma bactéria gram-negativa, não flagelada, agente causal de doenças de importância econômica como a doença de Pierce nas videiras e a clorose variegada dos citros (CVC) nas laranjeiras. O objetivo do presente trabalho foi realizar análises in silico das sequências promotoras dos genes deste fitopatógeno em uma tentativa de arrecadar novas evidências para o melhor entendimento da dinâmica de regulação transcricional de seus genes, incluindo aqueles envolvidos em mecanismos de patogenicidade e virulência. Para tanto, duas estratégias foram utilizadas para predição de elementos cis-regulatórios em regiões promotoras do genoma da cepa referência 9a5c, comprovadamente associada à CVC. A primeira, conhecida como phylogenetic footprinting, foi empregada para identificação de elementos regulatórios conservados em promotores de unidades transcricionais ortólogas, levando em consideração o conjunto de genes de X. fastidiosa e 7 espécies comparativas. O critério para identificação de unidades transcricionais ortólogas, isto é, unidades trancricionais oriundas de espécies distintas e cujos promotores compartilham elementos cis-regulatórios, foi paralelamente estudado utilizando-se informações regulatórias das bactérias modelos: Pseudomonas aeruginosa, Bacillus subtilis e Escherichia coli. Os resultados obtidos com análise de phylogenetic footprinting nos permitiu acessar a rede regulatória transcricional da espécie de forma compreensiva (global). Foram estabelecidas 2990 interações regulatórias, compreendendo 80 motivos distribuídos nos promotores de 56.8% das unidades transcricionais do genoma de X. fastidiosa. Na segunda estratégia recuperamos informações regulatórias experimentalmente validadas em E. coli e complementamos o conhecimento de dez regulons de X. fastidiosa, através de uma metodologia de scanning (varredura), dos quais algumas interações regulatórias já haviam sido previamente descritas por outros trabalhos. Destacamos os regulons de Fur e CRP, reguladores transcricionais globais, que se mostraram responsáveis pela modulação de genes relacionados a mecanismos de invasão e colonização do hospedeiro vegetal entre outros. Por fim, análises comparativas em regiões regulatórias correspondentes entre cepas foram realizadas e diferenças possivelmente associadas a particularidades fenotípicas foram identificadas entre 9a5c e J1a12, um isolado de citros não virulento, e 9a5c e Temecula1, um isolado de videira causador da doença de Pierce. / Xylella fastidiosa is a gram-negative, non-flagellated bacterium responsible for causing economically important diseases such as Pierce\'s disease in grapevines and Citrus Variegated Clorosis (CVC) in sweet orange trees. In the present work we performed in silico analysis on promoter sequences of protein-coding genes from this phytopathogen, including those involved in virulence and pathogenic mechanisms, in an attempt to better understand the underlying transcriptional regulatory dynamics. Two strategies for cis-regulatory elements prediction were applied on promoter sequences from 9a5c strain genome, a proven causal agent of CVC. The first one, known as phylogenetic footprinting, involved the prediction of regulatory motifs conserved on promoter sequences of orthologous transcription units from X. fastidiosa and a set of 7 comparatives species. The criteria to identify orthologous transcription units, i. e., those from different species and whose promoter sequences share at least one common regulatory motif, was studied based on regulatory information available for model organisms: Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli. The results obtained with the phylogenetic footprinting analysis permitted us to access the underlying transcriptional regulatory network from the species in a comprehensive manner (genome-wide), with a total of 2990 regulatory interactions corresponding to 80 predicted motifs distributed on promoter sequences of 56.8% of all transcription units. In the second strategy regulatory information from E. coli was recovered and used to expand the knowledge of ten regulons in X. fastidiosa, through a scanning process, of which some regulatory interactions were previously described by independent studies. We emphasize some genes related to host invasion and colonization present in the Fur and CRP regulons, two global transcription regulators. Lastly, comparative analysis on corresponding regulatory regions among strains were performed and differences possibly associated to phenotypic variation were identified between 9a5c and J1a12, a non-virulent strain isolated from orange trees, and between 9a5c and Temecula1, a strain associated to Pierce\'s disease on grapevines.

Xylella fastidiosa é uma bactéria gram-negativa, não flagelada, agente causal de doenças de importância econômica como a doença de Pierce nas videiras e a clorose variegada dos citros (CVC) nas laranjeiras. O objetivo do presente trabalho foi realizar análises in silico das sequências promotoras dos genes deste fitopatógeno em uma tentativa de arrecadar novas evidências para o melhor entendimento da dinâmica de regulação transcricional de seus genes, incluindo aqueles envolvidos em mecanismos de patogenicidade e virulência. Para tanto, duas estratégias foram utilizadas para predição de elementos cis-regulatórios em regiões promotoras do genoma da cepa referência 9a5c, comprovadamente associada à CVC. A primeira, conhecida como phylogenetic footprinting, foi empregada para identificação de elementos regulatórios conservados em promotores de unidades transcricionais ortólogas, levando em consideração o conjunto de genes de X. fastidiosa e 7 espécies comparativas. O critério para identificação de unidades transcricionais ortólogas, isto é, unidades trancricionais oriundas de espécies distintas e cujos promotores compartilham elementos cis-regulatórios, foi paralelamente estudado utilizando-se informações regulatórias das bactérias modelos: Pseudomonas aeruginosa, Bacillus subtilis e Escherichia coli. Os resultados obtidos com análise de phylogenetic footprinting nos permitiu acessar a rede regulatória transcricional da espécie de forma compreensiva (global). Foram estabelecidas 2990 interações regulatórias, compreendendo 80 motivos distribuídos nos promotores de 56.8% das unidades transcricionais do genoma de X. fastidiosa. Na segunda estratégia recuperamos informações regulatórias experimentalmente validadas em E. coli e complementamos o conhecimento de dez regulons de X. fastidiosa, através de uma metodologia de scanning (varredura), dos quais algumas interações regulatórias já haviam sido previamente descritas por outros trabalhos. Destacamos os regulons de Fur e CRP, reguladores transcricionais globais, que se mostraram responsáveis pela modulação de genes relacionados a mecanismos de invasão e colonização do hospedeiro vegetal entre outros. Por fim, análises comparativas em regiões regulatórias correspondentes entre cepas foram realizadas e diferenças possivelmente associadas a particularidades fenotípicas foram identificadas entre 9a5c e J1a12, um isolado de citros não virulento, e 9a5c e Temecula1, um isolado de videira causador da doença de Pierce. / Xylella fastidiosa is a gram-negative, non-flagellated bacterium responsible for causing economically important diseases such as Pierce\'s disease in grapevines and Citrus Variegated Clorosis (CVC) in sweet orange trees. In the present work we performed in silico analysis on promoter sequences of protein-coding genes from this phytopathogen, including those involved in virulence and pathogenic mechanisms, in an attempt to better understand the underlying transcriptional regulatory dynamics. Two strategies for cis-regulatory elements prediction were applied on promoter sequences from 9a5c strain genome, a proven causal agent of CVC. The first one, known as phylogenetic footprinting, involved the prediction of regulatory motifs conserved on promoter sequences of orthologous transcription units from X. fastidiosa and a set of 7 comparatives species. The criteria to identify orthologous transcription units, i. e., those from different species and whose promoter sequences share at least one common regulatory motif, was studied based on regulatory information available for model organisms: Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli. The results obtained with the phylogenetic footprinting analysis permitted us to access the underlying transcriptional regulatory network from the species in a comprehensive manner (genome-wide), with a total of 2990 regulatory interactions corresponding to 80 predicted motifs distributed on promoter sequences of 56.8% of all transcription units. In the second strategy regulatory information from E. coli was recovered and used to expand the knowledge of ten regulons in X. fastidiosa, through a scanning process, of which some regulatory interactions were previously described by independent studies. We emphasize some genes related to host invasion and colonization present in the Fur and CRP regulons, two global transcription regulators. Lastly, comparative analysis on corresponding regulatory regions among strains were performed and differences possibly associated to phenotypic variation were identified between 9a5c and J1a12, a non-virulent strain isolated from orange trees, and between 9a5c and Temecula1, a strain associated to Pierce\'s disease on grapevines.

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Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine). / EHEC O157:H7 are an emerging zoonotic food- and water-borne hazard highly pathogenic to humans and associated with diseases ranging from acute gastroenteritis to hemolytic uremic syndrome. From their commensal carriage by farm animals to human targets, EHEC pass through a crucial step of persistence in the open environment. Microbial endosymbiosis is one strategy used by pathogenic bacteria to survive in aquatic environments. Amoebae species are free-living protozoa found in diverse environmental habitats and known to host several water-borne pathogens. Thus amoebae could contribute to transmission of EHEC to humans. The first part of my PhD project was focused on interaction of the free-living amoebae Acanthamoeba castellanii with EHEC. The results showed that the presence of amoeba extends the persistence of EHEC that survived phagocytosis by amoebae. This demonstrates the real potential of amoebae to harbourd EHEC that may contribute to their transmission. However, absence of shiga toxins enhanced the intra-amoeba survival. Moreover, EHEC had a toxic and lethal effect on amoebae partially due to shiga toxins. The involvement of shiga toxins in the intracellular survival and mortality of amoebae suggests the value of using amoebae as a model of host/pathogen interactions to study the pathogenicity of EHEC. During their transmission cycle, EHEC encounter limitation inorganic phosphate (Pi) in the environment. Using jointly the PhoB-R two-component system (TCS) and the Pst (Pi specific transport) system, EHEC detect and respond to this Pi limitation by activating the Pho regulon. The interplay between the EHEC virulence, the Pho-Pst and/or the environmental Pi remains unknown. The second part of my project explored the role of Pho regulon (responding to Pi-limitation stress) in the virulence of EHEC. Transcriptomic analysis showed that EHEC has evolved a sophisticated response to Pi deficiency involving not only biochemical strategies that are likely critical to its survival, but also coordinating its virulence response. In these conditions, the regulator PhoB regulates directly the expression of LEE and Stx2 genes. This is confirmed by an increase in EspB secretion and Stx2 production and secretion in low Pi conditions. Moreover, the activation of Pho regulon increases biofilm formation and reduces motility in EHEC. This correlated with the induction of genes regulating curli production and repression of indole production pathway and the flagellum and PGA biosynthesis.

Im Fokus dieser Arbeit stand die Analyse von Faktoren, die den Zusammenbau des Flagellenapparates von Legionella pneumophila (Lp) regulieren. Mit einem kombinierten Replikations-/ Überlebensversuchs mit Lp Corby oder Lp Paris und ihren zugehörigen Regulationsmutanten wurde eine verminderte Fitness für dfliA und erstmals für drpoN, dfleQ defiziente Stämme nachgewiesen. Zur Validierung von Microarray-Daten aus Lp Paris wurden wachstumsphasenabhängige Transkriptions- und Translationsanalysen mit Lp Corby Wildtyp und drpoN, dfleQ, dfliA und dflaA defizienten Stämmen durchgeführt. Es wurde gezeigt, dass die basale Expression von fliA in den späteren Phasen unabhängig von RpoN und FleQ stattfindet. In dieser Arbeit konnte erstmals der Transkriptionsstartpunkt des Hauptregulators FliA bestimmt werden. Es zeigte sich eine putative RpoD (S70) Bindungsstelle. Ein Modell zur Regulation der fliA Expression wurde weiterentwickelt. Demnach kommt es in der exponentiellen Phase durch das Zusammenwirken von RpoD und DksA, aber unabhängig von FleQ, zur basalen fliA Promotoraktivität. Durch den Übergang in die transmissive Phase und direkte oder indirekte Interaktion mit FleQ sowie dem Alarmon ppGpp scheint es zu einem Austausch des Sigmafaktors S70 gegen SS und zu einer Aktivierung der fliA Expression zu kommen. Elektronenmikroskopische Studien zeigten, dass drpoN und dfleQ defiziente Mutanten wahrscheinlich aufgrund des fehlenden Basalkörpers nicht flagelliert sind. Mutanten für dfliA, dflaA und dfliD hatten ebenfalls keine Flagelle, zeigten aber eine ungewöhnliche, gerade Hook Struktur, die den Zusammenbau des Basalkörpers demonstriert. Weiterhin wurden durch in silico Studien 15 Legionella Spezies in Bezug auf das Flagellensystem und ein putatives Chemotaxissystem untersucht. So konnte L. oakridgensis als erste Art ohne beide Systeme sequenziert werden. Andererseits konnten mit LLAP12, L. bozemanii, L. gormanii und L. lytica Stämme beschrieben werden, die beide Systeme tragen. / This work focused on the analysis of factors contributing to the regulation of the flagellum self-assembly of Legionella pneumophila (Lp). With a combined replication/survival assay with Lp Corby or Lp Paris and their corresponding regulatory mutants a reduced fitness could be verified for dfliA and for the first time for drpoN, dfleQ deficient strains. For validation of microarray-data for Lp Paris with strain Lp Corby a growth phase dependent analysis of transcription and translation rates was done with wild-type and the drpoN, dfleQ, dfliA and dflaA deficient strains. A regulation of basal fliA expression independently from RpoN and FleQ was shown in the later growth phases. Furthermore the transcriptional start site of fliA could be shown for the first time. A RpoD (S70) binding site could be identified. According to a further developed model for the regulation of the fliA expression RpoD and DksA lead to a basal fliA promotor activity, independently from FleQ. Most likely, during transition to stationary phase, direct or indirect interaction with FleQ and the alarmone ppGpp results in the exchange of the sigma factor S70 and the binding of RpoS. This leads to the activation of fliA expression. Electron microscopic studies revealed that drpoN and dfleQ deficient mutants are not flagellated caused by the missing basal body. Mutants of dfliA, dflaA and dfliD were also aflagellated, but there was a uncommon straight hook structure visible which demonstrates a filament-independent assembly of the basal body. Furthermore, in silico analysis was done with 15 Legionella species with regard to the flagellum regulation system and a putative chemotaxis system. Analysis revealed that the strain L. oakridgensis is the first strain lacking both systems. On the other hand the strains LLAP12, L. bozemanii, L. gormanii and L. lytica could be characterized as strains carrying both systems.

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