Oligosaccharides are important biomolecules that are targets and also components of many medical treatments, including treatments for cancer, HIV, and inflammation. While the demand for medically-relevant oligosaccharides is increasing, these compounds have proven difficult to synthesize. Whole-cell oligosaccharide synthesis is a promising method that requires relatively inexpensive substrates and can complete the synthesis in just one step. However, whole-cell oligosaccharide synthesis employing common microorganisms like E. coli have been plagued by low yields. This dissertation investigates an alternative microorganism for oligosaccharide production: Agrobacterium sp. ATCC 31749. This Agrobacterium strain produces high levels of curdlan polysaccharide, demonstrating its natural ability to produce the sugar nucleotide precursor for oligosaccharide production. The two main objectives of this dissertation are 1) to develop biocatalysts for oligosaccharide synthesis by engineering ATCC 31749 and 2) to determine what factors affect poly- and oligosaccharide production in this Agrobacterium strain. ATCC 31749 was engineered to produce two oligosaccharides of medical importance: N-acetyllactosamine and galactose-α 1,3-lactose. Oligosaccharide production in the biocatalyst was further improved with additional metabolic engineering. Substrate uptake was increased through expression of a lactose permease, and availability of the sugar nucleotide substrate improved with gene knockout of the curdlan synthase gene. Both of these engineering efforts led to increased oligosaccharide synthesis in the Agrobacterium biocatalyst. Overall, the engineered Agrobacterium strains synthesized gram-scale quantities of the oligosaccharide products in just one step and requiring only a few inexpensive substrates and cofactors. Additional improvement of the oligosaccharide-producing biocatalysts required further investigation of the factors influencing poly- and oligosaccharide production in ATCC 31749. In this dissertation, several environmental and intracellular factors are identified that affect both oligosaccharide and curdlan production. Sucrose was the preferred carbon source for oligosaccharide synthesis, and the addition of citrate to the synthesis reaction led to significant improvement in oligosaccharide production. To identify the genetic factors and possible mechanisms regulating curdlan production, the genome of ATCC 31749 was sequenced. The genome sequence was utilized for transcriptome analysis of ATCC 31749. In the transcriptome analysis, genes significantly up- and down-regulated during curdlan production were identified. Subsequent gene knockout experiments showed several factors to be important for curdlan synthesis, namely the nitrogen signaling cascade, polyphosphate, and the GTP-derived second messengers (p)ppGpp and c-di-GMP. In addition to the development of biocatalysts for oligosaccharide production, this investigation provides insight into the complex mechanisms regulating exopolysaccharide synthesis.

A cultura do maracujazeiro é afetada pela virose causada pelo Cowpea aphid-borne mosaic virus (CABMV), provocando a redução da qualidade e produtividade dos frutos e, em alguns casos, pode inviabilizar o cultivo comercial desta espécie. Uma alternativa para o controle de doenças viróticas é o desenvolvimento de plantas resistentes pela transformação genética. O objetivo deste trabalho foi a obtenção de plantas transgênicas de maracujazeiro (Passiflora edulis f. flavicarpa), utilizando 2 construções gênicas contendo a região codificadora do gene da proteína capsidial do CABMV. A construção pCABMV-asCP, que contém um fragmento na orientação antisenso e a construção pCABMV-dsCP, que contém fragmentos senso e antisenso do gene da proteína capsidial, separados por um íntron, uma construção hairpin. Para os experimentos de transformação genética, via Agrobacterium tumefaciens, foram utilizados os explantes de segmentos de hipocótilo e discos de folhas jovens das variedades FB-100, IAC-275 e IAC-277. Após 2 - 3 dias de co-cultivo em meio de cultura MS (MURASHIGE; SKOOG, 1962) contendo acetosseringona (100 mM), os explantes foram transferidos para meio de cultura de seleção e regeneração constituído de sais minerais e vitaminas de MS, suplementado com canamicina (100 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L), pH 5,8 e os reguladores BAP, TDZ e combinação de BAP e TDZ. Após 4 - 6 semanas, as gemas adventícias desenvolvidas foram transferidas para meio de cultura de alongamento MSM + GA3 (1,0 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L). As plantas desenvolvidas foram aclimatizadas e analisadas por PCR, utilizando-se primers específicos para a detecção dos transgenes. Foram identificadas 30 plantas transgênicas PCR positivas para do gene nptII, sendo 11 positivas para o fragmento antisenso da proteína capsidial do CABMV e 2 positivas para o fragmento da construção gênica hairpin. Até o momento, a integração dos transgenes foi confirmada por Southern blot em 4 plantas. Paralelo aos experimentos de transformação genética, foram avaliadas plantas de maracujazeiro das variedades IAC-275 e IAC-277, obtidas em experimentos anteriores, com a construção gênica pCABMV-CP, que contém o gene da proteína capsidial do CABMV. A análise de Southern blot de 14 plantas destes experimentos, confirmou a integração do transgene em 13 plantas, as quais foram propagadas e inoculadas mecanicamente com 3 isolados do CABMV (SP, RJ, CE). A linhagem T16 foi resistente as 3 inoculações, com os 3 isolados testados. Clones desta linhagem foram analisados por RT-PCR, comprovando a transcrição do transgene / The yellow passionfruit is affected by Cowpea aphid-borne mosaic virus (CABMV), which causes a decrease in fruit quality and productivity and in some cases making it unpractical for commercial cultivation of this species. Ann alternative for the control of virus diseases is the development of resistant plants through genetic transformation techniques. The objective of this work was to obtain passion fruit (Passiflora edulis f. flavicarpa) transgenic plants with two different gene constructs derived from the CABMV coat protein coding region. pCABMV-asCP contains the gene fragment in an antisense direction and pCABMV-dsCP contains a sense and antisense coat protein gene fragments, separated by intron, a hairpin construct. The genetic transformation experiments with Agrobacterium tumefaciens, were done in hypocotyl segments and young leaf disks explants from varieties FB-100, IAC-275 and IAC-277. After two to three days in co-culture in MS culture medium (MURASHIGE; SKOOG, 1962) supplemented with acetoseringone (100 mM) the explants were subcultured to medium for selection and regeneration, composed of MS minerals and vitamins, supplemented with kanamycin (100 mg/L), cefotaxime (500 mg/L) and silver nitrate (4.0 mg/L), at pH 5.8, and the growth regulators BAP, TDZ and combination of BAP and TDZ. After four to six weeks, the adventitious buds developed were transferred to an elongating medium composed of MSM salts supplemented with GA3 (1.0 mg/L), cefotaxime (500 mg/L) and silver nitrate (4.0 mg/L). Plants were acclimatized and analyzed by PCR, with specific primers for the transgenes detection. Thirty transgenic plants were identified as PCR positive for the nptII gene, with 11 being positive for the antisense fragment of the CABMV coat protein gene and two positive for the hairpin gene construct fragment. Currently, the transgene integration was confirmed by Southern blot analysis in four plants. Simultaneously to the genetic transformation experiments, passionfruit plants, varieties IAC-275 e IAC-277, obtained from previous experiments with pCABMV-CP, which contains CABMV coat protein gene, were analized. The Southern blot analysis of 14 plants from these experiments confirmed the transgene integration in 13 plants, which were propagated and mecanically inoculated with three CABMV isolates (SP, RJ, CE). Line T16 was resistant to three inoculations with all three isolates tested. Clones from this line were analyzed by RT-PCR which confirmed the transgene transcription

Uma das espécies que atualmente vem despertando interesse econômico por seu elevado valor de mercado é o maracuzajeiro doce (Passiflora alata Curtis). Entretanto, a cultura é afetada por diferentes doenças que prejudicam a produtividade e a qualidade dos frutos, sendo a doença causada pelo Cowpea aphid-borne mosaic virus (CABMV) a que mais afeta a cultura do maracujazeiro no Brasil. O presente trabalho teve como objetivo a obtenção de plantas transgênicas de P. alata visando resistência ao CABMV. O processo de transformação genética utilizado foi via Agrobacterium tumefaciens, estirpe EHA105, contendo o cassete de expressão com um fragmento do gene da proteína capsidial do CABMV, numa construção tipo hairpin e o gene de seleção nptII que confere resistência ao antibiótico canamicina. Para os experimentos de transformação genética foram utilizados como explantes segmentos de hipocótilo e segmentos internodais. Após 2 a 3 dias de co-cultivo em meio de cultura MS (MURASHIGE; SKOOG, 1962) contendo acetosseringona (100 mM), os explantes foram transferidos para meio de cultura de seleção e regeneração constituído de sais minerais e vitaminas de MS, suplementado com benzilaminopurina (BAP - 1mg/L) + thidiazuron (TDZ - 0,5 mg/L) + canamicina (100 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L), pH 5,8. Após 4 a 6 semanas de incubação, determinou-se o número de explantes responsivos e as gemas adventícias desenvolvidas foram transferidas para meio de cultura de alongamento MSM + GA3 (1,0 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L). As plantas desenvolvidas foram aclimatizadas e analisadas por PCR, utilizando primers específicos para a detecção do fragmento do gene da proteína capsidial do CABMV e do gene de seleção (nptII). Foram identificadas 47 plantas transgênicas PCR positivas para do gene nptII. Até o momento, a integração do gene nptII foi confirmada por Southern blot em 9 plantas / One species that is currently attracting interest due to its high economic value is the sweet passionflower (Passiflora alata Curtis). However, the culture is affected by different diseases that harm the productivity and fruit quality. The disease caused by Cowpea aphid-borne mosaic virus (CABMV) is the one that more affect the culture of passionflower in Brazil. This work aimed to obtain transgenic plants of P. alata resistant to the CABMV. The genetic transformation process was via Agrobacterium tumefaciens, strain EHA105, containing the expression cassette with a fragment of the coat protein gene of CABMV, in a hairpin construct and the selection gene nptII, which confers resistance to the antibiotic kanamycin. In the experiments of genetic transformation hypocotyl segments and internodal segments were used as explants. After 2-3 days of co-cultivation in MS medium (MURASHIGE; SKOOG, 1962) containing acetosyringone (100 mM), the explants were transferred to the selection and regeneration culture medium consisting of mineral salts and vitamins of MS medium supplemented with benzylaminopurine (BAP - 1 mg/L) + thidiazuron (TDZ - 0.5 mg/L) + kanamycin (100 mg/L) + cefotaxime (500 mg/L) + silver nitrate (4.0 mg /L), pH 5.8. After 4-6 weeks of incubation, it was determined the number of responsive explants. Shoots developed were transferred to elongating culture medium MSM + GA3 (1.0 mg/L) + cefotaxime (500 mg/L) + nitrate silver (4.0 mg/L). The developed plants were acclimatized and analyzed by PCR using specific primers to detect the fragment of CABMV and the selection gene (nptII). It was identified 47 transgenic plants PCR positive for the gene nptII. Until this moment, the integration of the nptII gene was confirmed by Southern blot in 9 plants

Made available in DSpace on 2014-08-20T13:33:00Z (GMT). No. of bitstreams: 1 dissertacao_raquel_rosa_costa.pdf: 808388 bytes, checksum: 18bdb94a8d12b8f7a746b7ee2613012a (MD5) Previous issue date: 2011-06-27 / he main objective of this work was to introduce the gene bvlI with confirmed potential antifungal of lectin from Bauhinia variegata, Apple Tree (Malus domestica Borkh) cv. Gala, to establish resistance to fungus Coletotrichum gloeosporioides, causing Leaf Spot. Multiplication experiments were performed in vitro regeneration of leaf explant culture and two protocols via Agrobacterium tumefaciens transformation aimed at inserting the gene bvll. The multiplication of shoots the medium that has best results was MS supplemented with 0.1 mg L-1 ANA and 8.3 mg L-1 2iP and, for the regeneration of the best responses were obtained with using MS, supplemented with 30 g L-1 sorbitol, 0.5 mg L-1 ANA and 5 mg L-1 TDZ, reaching a rate of 94.5% of regenerations. As regards the processing protocols, which showed best results was the P1 Protocol, which through analysis of PCR were obtained 15% shoots containing the gene bv/l. However, the expression of BVLI protein was not detected by Dot Blot, indicating the need for further procedure, such as refinement of protein extraction protocol, as well as utilization other techniques of analyses, such as Real Time. / O objetivo principal do presente trabalho foi introduzir o gene bvlI com potencial antifúngíco confirmado da lectina de Bauhinia variegata, em macieira (Malus domestica Borkh), cv. Gala, para estabelecer resistência ao fungo Coletotrichum gloeosporioides, causador da Mancha Foliar. Foram realizados experimentos de multiplicação in vitro, regeneração de explantes foliares e, dois protocolos de transformação via Agrobacterium tumefaciens visando a inserção do gene bvll. Na multiplicação de brotações o meio que apresentou melhores resultados foi o MS suplementado com 0,1 mg L-1 ANA e 8,3 mg L-1 de 2iP e, para a regeneração as melhores respostas foram obtidas com meio MS, suplementado com 30 g L-1 de sorbitol, 0,5 mg L-1 ANA e 5 mg L1 de TDZ, atingindo um percentual de 94,5% de regenerações. Quanto aos protocolos de transformação, o que apresentou melhores resultados foi o protocolo P1, no qual através da análise de PCR foram obtidas 15% de brotações contendo o gene bv/l. No entanto, a expressão da proteína BVLI não foi detectada através de Dot Blot, indicando a necessidade de novos procedimentos, como refinamento do protocolo de extração de proteínas, bem como utulização de outras técnicas de análises, como Real Time.

Les bactéries pathogènes opportunistes (BPO) sont responsables d'une grande part de la pathologie infectieuse bactérienne. Les BPO d'origine environnementale doivent subir des changements profonds de mode de vie pour s'adapter et coloniser l'homme. Comprendre les conditions de cette adaptation permettra de préciser la notion d'opportunisme infectieux et le rôle des BPO environnementales dans l'émergence des pathogènes.Les genres Ochrobactrum et Agrobacterium regroupent des bactéries présentant une grande variété de modes de vie et établissant différentes relations avec la cellule eucaryote. Ces bactéries connues pour vivre dans l'environnement sont par ailleurs des pathogènes opportunistes de l'homme principalement responsables d'infections chez les individus immunodéprimés. Dans le cadre de ce travail nous avons entrepris une étude populationnelle par une approche de génétique multilocus sur des collections de souches cliniques et environnementales de différentes origines géographiques. Les structures de population obtenues ont été confrontées à divers caractères phénotypiques reliés à la virulence et/ou l'adaptation chez l'homme, la température de croissance, la formation de biofilm et la virulence vis-à-vis des modèles Caenorhabditis elegans et macrophages humains.Ochrobactrum anthropi et Ochrobactrum intermedium sont les deux principales espèces d'intérêt médical du genre Ochrobactrum. La population d'O. anthropi est de type épidémique qui s'organise en deux complexes clonaux (CCs). Si le CC1 regroupe à la fois des souches de diverses origines, le CC4 ne contient que des souches cliniques. Cette sous-population apparait associée à l'homme même si les caractères phénotypiques étudiés ne révèlent pas de différences entre ces deux sous populations. De la même façon, ces deux CCs ne se distinguent pas par leur comportement en modèle macrophage ou par leur diversité génomique. O. intermedium, tout comme O. anthropi, présente une forte diversité génétique toutefois, aucun regroupement des souches en fonction de leur origine n'est mis en évidence pour cette espèce. La diversité des souches cliniques apparait aussi importante que celle de l'ensemble de la population. Plusieurs arguments suggèrent une niche étroite pour cette espèce, notamment une faible diversité génomique. Par ailleurs, le faible nombre de souches environnementales associé à une meilleure croissance planctonique à 37°C qu'à 25°C et 30°C suggèrent que l'homme pourrait constituer cette niche. L'étude de la virulence d'O. intermedium en modèle macrophage ou C. elegans met en évidence différents comportements, pour autant ceux-ci ne semblent pas liés à la structure de population. Certaines souches sont capables de se multiplier dans le modèle macrophage.L'étude du genre Agrobacterium par une approche multilocus sur une collection représentative des différents modes de vie de ces bactéries met en évidence, tout comme pour O. anthropi, une sous population clinique qui regroupe près de 80% des souches de cette origine. D'autres arguments tels que la croissance à 42°C confirment que le génovar A7 peut correspondre à une sous-population associée à l'homme. Les données obtenues seront confrontées aux connaissances sur d'autres bactéries pathogènes opportunistes d'origine environnementale comme Pseudomonas aeruginosa, Stenotrophomonas maltophilia et les bactéries du complexe Burkholderia cepacia qui présentent également des sous populations associées à l'homme et/ou à certaines pathologies humaines. L'existence de ces sous populations suggère une spécialisation qui sera discutée dans le contexte de la spéciation des bactéries pathogènes afin de revisiter le concept d'opportunisme infectieux. / The opportunistic bacterial pathogens (OBP) cause the main part of bacterial infectious diseases. Environmental-borne OBP should encounter dramatic changes in lifestyle in order to colonize human beings. The conditions of this adaptation should precise concepts about OBP and emerging pathogens.The genera Ochrobactrum and Agrobacterium groups bacteria with versatile lifestyles that establish diverse relationships with the eukaryotic cells. These environmental-borne OBP caused diverse infectious diseases in immune-compromised patients. In this study, we undertook an approach of multilocus genetic on large population of environmental and clinical strains of Ochrobactrum and Agrobacterium. The population structures were compared to phenotypic traits related to adaptation and virulence in man, such as growth temperature, biofilm formation and virulence tested in Caenorhabditis elegans and human macrophages models.Ochrobactrum anthropi and Ochrobactrum intermedium are the two main Ochrobactrum species to be involved in human diseases. O. anthropi displays an epidemic population structure organized in two large clonal complexes (CCs). CC4 groups only human associated strains whereas CC1 contain environmental and clinical strains. Population genetics suggested that CC4 is a human-associated clone although phenotypic, genomic and virulence traits do not differ between CC1 and CC4 strains.As O. anthropi, O. intermedium displays a high genetic diversity without correlation between the genetic structure and the origin of strains. The level of genetic diversity among clinical strains appears as high as observed in the whole population. Several data such as a low level of genomic diversity suggested that O. intermedium is associated to a narrow ecological niche. The low number of environmental strains described for this species as well as an optimal growth at 37°C suggested that human beings could be the main niche for O. intermedium. Virulence in macrophage and C. elegans models showed diverse behaviour whereas some strains are able to survive and multiply in macrophages model.Multilocus genetics in a population of Agrobacterium spp. that displays diverse lifestyles, revealed a human associated population as observed for O. anthropi. The clinical genovar A7 groups 80% of the clinical strains included in the study, this strains growing at 42°C. Data obtained in this study will be confronted to the knowledge about other environmental-borne OBP such as Pseudomonas aeruginosa, Stenotrophomonas maltophilia and bacteria belonging to the species complex Burkholderia cepacia. All these bacteria displayed sub-populations associated to man or to a particular human disease. These sub-populations suggest a specialization process that will be described in the context of the speciation of bacterial pathogen in order to revisite the concept of « opportunisme infectieux ».

L’agent pathogène Agrobacterium tumefaciens induit, chez les plantes, le développement de tumeurs dans lesquelles il prolifère, en intégrant un fragment de son plasmide Ti de virulence dans le génome de son hôte. Les tissus transformés synthétisent des composés originaux, appelés opines, qui sont utilisés comme nutriments spécifiques par la bactérie. Une vingtaine d’opines sont connues à ce jour, et chacune d’elle peut être métabolisée par des souches d’Agrobacterium tumefaciens possédant les gènes de transport et de catabolisme qui lui sont associés, ce qui apparait comme un avantage compétitif dans la colonisation de la tumeur. La présence de ces gènes dépend du type de plasmide Ti que la souche pathogène possède.Agrobacterium tumefaciens B6 possède un pTi de type octopine, qui porte les gènes de métabolisme des mannityl-opines, qui sont la mannopine, l’acide mannopinique, l’agropine et l’acide agropinique. La mannopine et l’acide mannopinique sont synthétisés par la même enzyme, et ont pour précurseurs respectivement la désoxy-fructosyl-glutamine (DFG) et le désoxy-fructosyl-glutamate (DFGA), tous deux opines de la famille de la chrysopine. La DFG est aussi un composé d’Amadori répandu et assimilable par de nombreux organismes. La mannopine sert de précurseur pour la synthèse de l’agropine. Enfin, la mannopine, l’acide mannopinique et l’agropine peuvent toutes trois se lactamiser spontanément en acide agropinique.Malgré la similarité chimique de ces quatre opines, chacune est transportée par une protéine périplasmique de fixation (PBP) associée à un transporteur ATP-binding Cassette (ABC) différent. La PBP sélectionne et fixe une opine pour l’apporter au transporteur ABC, qui permet le passage de l’opine dans le cytoplasme grâce à l’hydrolyse de deux molécules d’ATP. La spécificité du transporteur entier est déterminée par la PBP.Des études génétiques chez des souches possédant un pTi de type octopine ont montré que le système PBP-transporteur ABC AgaABCD est spécifique de l’acide agropinique, AgtABCD spécifique de l’agropine, MoaABCD spécifique de l’acide mannopinique et que MotABCD transporte la mannopine et également l’acide mannopinique. Chez la souche C58, qui ne possède pas un pTi de type octopine, le système de transport SocAB, codé par des gènes situés sur le plasmide cryptique At, transporte la DFG comme nutriment, et semble aussi capable d’importer la mannopine.Mon travail de thèse a permis, dans un premier temps, de caractériser les fortes affinités et la spécificité des PBP AgaA et AgtB pour l’acide agropinique, de la PBP MoaA pour l’acide mannopinique et de la PBP SocA pour la DFG, mais aussi la non spécificité de MotA pour la mannopine, l’acide mannopinique et la DFG, ce qui remet en question les affinités précédemment décrites pour AgtB et SocA. Dans un deuxième temps, ce travail a apporté les bases moléculaires et structurales des complexes PBP-mannityl-opines, complexes jamais caractérisés auparavant. Enfin, dans un troisième temps, la structure de la PBP AttC chez la souche C58, annotée comme mannopine-like, a été déterminée, et les expériences d’interaction ont montré qu’elle n’interagit avec aucune mannityl-opine, ce qui conduit à une révision de son annotation.Mes travaux apportent un éclairage nouveau sur l’import des mannityl-opines chez Agrobacterium tumefaciens. Le fait qu’aucun des transporteurs étudié ne permette l’import de l’agropine laisse penser qu’il existe une autre PBP ou un autre système de transport encore inconnu assurant cette fonction, ouvrant la voie vers de nouvelles études sur les pTi de type octopine et agropine. / Agrobacterium tumefaciens pathogenic agent confers the development of tumors in plants, in which it proliferates, integrating a fragment of its virulence Ti plasmid into its host genome. Transformed tissues synthesize original compounds, called opines, used as specific nutrients by the bacterium. More than twenty opines are known so far, and each one of them can be metabolized by A. tumefaciens strains possessing its associated transport and catabolism genes, which appears as a competitive advantage in the tumor colonization. The presence of these genes relies on the Ti plasmid type a pathogenic strain possesses. A. tumefaciens B6 possesses an octopine-type pTi, which harbors the metabolism genes of the mannityl-opines, which are mannopine, mannopinic acid, agropine and agropinic acid. Mannopine and mannopinic acid are synthesized by the same enzyme, and their precursors are deoxy-fructosyl-glutamine (DFG) and deoxy-fructosyl-glutamate (DFGA) respectively, both opines of the chrysopine family. DFG is also a wide-spread Amadori compound which can be uptaken by numerous organisms. Mannopine is a precursor for agropine synthesis. Finally, mannopine, mannopinic acid and agropine can spontaneously lactamize into agropinic acid.Despite the chemical similarity of these four opines, each one is transported by a different periplasmic binding protein (PBP) associated with an ATP-binding cassette (ABC) transporter. The PBP selects and binds one opine to bring it to the ABC transporter, which allows the passage of the opine to the cytoplasm due to two ATP molecules hydrolysis. The whole transporter specificity is determined by the PBP.Genetic studies in strains possessing an octopine-type pTi showed that AgaABCD PBP-ABC transporter system is specific to agropinic acid, AgtABCD to agropine, MoaABCD to mannopinic acid and that MotABCD transports mannopine and also mannopinic acid. In C58 strain, which do not possess an octopine-type pTi, SocAB transport system, coded by genes located on the cryptic pAt plasmid, allows the transport of DFG as a nutrient, and seems able to import mannopine too.My thesis work allowed, first, to characterize the strong affinities and the specificity of PBPs AgaA and AgtB to agropinic acid, PBP MoaA to mannopinic acid and PBP SocA to DFG, and also MotA unspecificity toward mannopine, mannopinique acid and DFG, which leads to a revision of the previously described affinities of AgtB and SocA. Secondly, this work brought molecular and structural basis of PBP-mannityl-opine complexes, never described before. Finally, the structure of PBP AttC, annotated as a mannopine binding-like protein in C58, was determined, and interactions experiments showed that it binds no mannityl-opines, leading to a revision of its annotation.My work sheds light on the mannityl-opines importation in Agrobacterium tumefaciens. The fact that none of the studied transport system allows agropine import lets think that there is another unknown PBP or another unknown whole transport system assuming this role, opening new ways to new studies about octopine- and agropine-type pTis.

Le phytopathogène Agrobacterium tumefaciens est l'agent causal de la maladie appelée galle du collet, et est capable d'infecter plus de 90 familles de plantes dicotylédones. Cette ∝-protéobactérie appartient à la famille Rhizobiaceae. A. tumefaciens est un complexe de différentes espèces regroupées en 10 génomovars (G1 à G8 et G13). A. tumefaciens C58 appartient au groupe du G8. Son génome est constitué de 4 réplicons : 1 chromosome circulaire, 1 chromosome linéaire et des 2 plasmides dispensables : pAt (pour A.tumefaciens) et pTi (pour Tumor inducing, qui est requis pour la virulence). Pour explorer de nouveaux aspects du mode de vie d’A. tumefaciens, et en particulier l'interaction entre la bactérie et sa plante hôte, deux approches différentes ont été utilisées pour identifier, caractériser et analyser les gènes qui pourraient jouer un rôle dans l'adaptation des bactéries à la tumeur. Une expérience de l'évolution par des passages en série de trois souches différentes de l'agent pathogène sur la plante hôte Solanum lycopersicum a été effectuée afin de clarifier la dynamique évolutive du génome au cours de l'infection. Parallèlement, une étude de différents transcriptomes (in planta et in vitro) a été réalisée et étudiée pour élucider des gènes bactériens candidats impliqués dans l'interaction de la bactérie avec la plante et divers composés produits dans la tumeur. Ce travail tente de donner une vue plus générale du processus d'adaptation de la bactérie à la niche écologique qui est la tumeur. / Agrobacterium tumefaciens is the causal agent of the plant disease called crowngall, and it’s able to infect more than 90 families of dicotyledonous plants. It is an α-Proteobacterium and belongs to the Rhizobiaceae family. A. tumefaciens is a complex of different species grouped in 10 genomovars (G1 to G8, and G13). A. tumefaciens C58 belongs to the G8 group. Its genome consists in 4 replicons: 1 chromosome circular, 1 chromosome linear and 2 dispensable plasmids: pAt (for A. tumefaciens) and pTi (for Tumor inducing), which is required for virulence. To explore new aspects of the A. tumefaciens lifestyle, and in particular the interaction between the bacteria and its plant host, two different approaches have been used to identify, characterize and analyze genes that could play a role in the adaptation of the bacteria to tumor lifestyle. An evolution experiment by serial passages of three different strains of thepathogen on the host plant Solanum lycopersicum has been carried out to clarify the evolutionary dynamics of the genome during the course of infection. In parallel, a study of different transcriptomes (in planta and in vitro) was performed and studied to elucidate bacterial candidate genes involved in the interaction of the bacteria with the plant and various compounds produced in the tumor. This work attempts to give a more general view of the process of adaptation of the bacteria to the ecological niche that is the tumor.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Agrobacterium tumefaciens-mediated transformation technology has been used in a variety of applications throughout the fields of cellular and molecular plant biology as well as plant physiology. Research is conducted in order to extend this application range and overcome some of the intrinsic limitations of the Agrobacterium transformation system. Predominantly, these limitations can be attributed to the host range specificity of A. tumefaciens, as well as adverse effects induced on explant tissue by active plant defence mechanisms, triggered by the plant-pathogen-interaction. Typically, this active defence mechanism culminates in the hypersensitive response (HR), characterised by localised cell death and necrosis. Not all Agrobacterium species, however, share the same host range and some have evolved the ability to infect plant species not normally considered hosts of A. tumefaciens. This host range specificity can be exploited to extend the application of existing Agrobacterium transformation systems. In an attempt to establish an efficient transformation system for Vitis vinifera which, has proven very difficult to transform with A. tumefaciens, indigenous A. vitis strains have been evaluated as possible host-specific transformation agents. Strains of Agrobacterium vitis should be suitable for this type of endeavour, since they have evolved several unique characteristics directly linked to the infection of their hosts. These include the ability to utilise, tartrate, a host abundant carbon source, as well as the production of an acid polygalacturonase that could play a role during the infection process. The proposition that the evolution of A. vitis is a fairly recent event is also confirmed by the relatively little divergence observed between A. tumefaciens and A. vitis. In this study, a selection of A. vitis strains were evaluated in screenings designed to accentuate desirable traits in strains such as good infectivity of grapevine material (presumably an indicator of an efficient mechanism of gene transfer to be exploited in an engineered transformation system) as well as a favourable reaction (causing no necrosis) on grapevine somatic embryos. Two strains produced large tumours on grapevine cuttings and caused little necrosis on the somatic embryos. Significant variation in infectivity as well as callus necrosis was observed between the strains as well as in a genotype-specific manner on the host material. This genotypic-specific effect of either host or pathogen could be an indication of the degree of specialisation developed by plant pathogens to infect specific hosts. On the basis of these results, it was possible to select an A. vitis strain for further biochemical and genetic characterisation. Simple biochemical analysis classified the strain as an octopine strain. DNA-DNA hybridisation techniques combined with a plasmid walking technique resulted in the partial characterisation of the T-DNA of the selected A. vitis strain. A partial restriction enzyme map of the T-DNA was constructed and the T-DNA and flanking areas were cloned. Significant differences, most notably, the absence of a TB-area as well as the absence of the agrocinopine (aes) gene from the 5' area of the T-DNA, were observed. Partial sequencing data indicated the presence of at least four conserved T-DNA genes located on the TA-DNA, as well as the presence of three bacterial insertion (IS-)elements flanking the region. Two of these IS elements, both related to the IS 110 family of IS elements have not yet been reported in A. vitis. In fact, these two elements seem to be the 5' and 3' ends of a disrupted element and could therefore have played an evolutionary role in the development of this strain. This study provides fundamental background for the development of a more efficient transformation system specific for grapevine, exploiting same of-the unique characteristics of one of its pathogens, A. vitis. / AFRIKAANSE OPSOMMING: Agrobacterium tumefaciens-gebaseerde transformasiesisteme word in "n wye reeks van toepassings in die velde van sellulêre- en molekulêre plantbiologie asook plantfisiologie aangewend. Navorsing word voortdurend onderneem om die inherente beperkinge van die Agrobacterium-transformasiesisteem te oorkom en sodoende die toepassingsveld van die sisteem verder te verbreed. Die beperkinge tipies aan dié sisteem kan hoofsaaklik toegeskryf word aan die gasheerspesifisteit van A. tumeteciens, asook die negatiewe reaksies op eksplantmateriaal wat deur die plant se aktiewe verdedigingsmeganisme, soos ontlok deur die plant-patogeen interaksie, veroorsaak word. Hierdie aktiewe verdedigingsmeganisme lei gewoonlik tot In hipersensitiewe respons (HR) in die plant, wat deur gelokaliseerde selafsterwing en nekrose gekenmerk word. Alle Agrobacterium-spesies het egter nie almal dieselfde gasheerreeks nie en sommige rasse het as gevolg van evolusionêre ontwikkelings die vermoë verkry om plantspesies wat normaalweg buite die gasheerreeks van A. tumefaciens val, te infekteer. Hierdie tipe gasheerspesifisiteit kan uitgebuit word om die toepassingsmoontlikhede van bestaande Agrobacterium-transformasiesisteme te verbreed. In In poging om In effektiewe transformasiesisteem vir Vitis vinifera, In moeilik transformeerbare gewas, te ontwikkel, is inheemse rasse van Agrobacterium vitis ondersoek as moontlike gasheerspesifieke transformasie-agente. Rasse van A. vitis behoort uiters geskik te wees vir so "n toepassing, aangesien hulle verskeie unieke eienskappe, wat direk aan die infeksie van die gasheer gekoppel is, vertoon. Van hierdie eienskappe is onder meer die vermoë om tartraat, In koolstofbron volop in druifplante, te benut. A. vitis produseer verder ook In suur poligalaktorunase wat vermoedelik In rol in die infeksieproses speel. Die voorstel dat die evolusionêre ontwikkeling van A. vitis In redelike onlangse gebeurtenis is, word onderskryf deur die betreklike homogenisiteit met A. tumefaciens. In hierdie studie is "n groep A. vitis-rasse met behulp van siftingsprosedures wat daarop gemik is om gesogte eienskappe in rasse uit te wys, beoordeel. Die vermoë om druifplantmateriaal te infekteer (wat vermoedelik "n aanwyser van "n effektiewe meganisme van geenoordraging is wat in "n gemanipuleerde transformasiesisteem benut kan word), sowel as 'n gunstige reaksie (d.w.s geen nekrose) op druifplant somatiese embrio's is van die gesogte eienskappe waarvoor gesoek word. Twee rasse het groot tumors op druifplant-stingelsegmente veroorsaak terwyl hulle bykans geen weefselskade op somatiese embrio's geïnduseer het nie. Betekenisvolle verskille in infektiwiteit en in kallusnekrose is tussen die rasse sowel as in 'n genotipe-spesifieke-verhouding waargeneem. Hierdie genotipe-spesifieke effek, kenmerkend van óf die gasheer óf die patogeen, kan aanduidend wees van die vlak van spesialisasie wat heers by die infeksie van spesifieke gashere. Na aanleiding van bogenoemde resultate was dit moontlik om 'n A. vitis-ras te selekteer wat verder aan biochemiese en genetiese analises onderwerp kon word. Eenvoudige biochemiese analises het dit moontlik gemaak om die ras as oktopien te klassifiseer. DNA-DNA hibridisasietegnieke gekombineerd met 'n unieke plasmiedwandeltegniek het gelei tot die gedeeltelike karakterisering van die geselekteerde A. vitisras. In Gedeeltelike restriksie-ensiem (RE) kaart van die T-DNA kon gevolglik opgestel word. Die T-DNA en die aangrensende gedeeltes is boonop gekloneer. Betekenisvolle verskille, spesifiek die afwesigheid van In TB area, sowel as die afwesigheid van die agrosinopien-sintasegeen (acs) aan die 51-kant van die T-DNA, is waargeneem. Gedeeltelike basispaaropeenvolgingsdata het egter die teenwoordigheid van minstens vier gekonserveerde T-DNA-gene, asook die teenwoordigheid van drie bakteriese invoegingselemente (IS) aan weerskante van die area, geïdentifiseer. Twee van hierdie elemente, wat beide homologie vertoon met die IS110 familie van IS elemente, is nog nie vantevore in A. vitis aangetref nie. Dit wil boonop blyk of dié twee elemente die 51 - en 31 - areas van In onderbroke element vorm, wat dus In moontlike aanduiding is van hul potensiële rol in die evolusionêre ontwikkeling van die ras. Hierdie studie verskaf basiese inligting wat daartoe kan lei dat 'n doeltreffender transformasiesisteem spesifiek vir druifplante ontwikkel word deur van die unieke kenmerke van een van sy patogene, A. vitis, uit te buit.

Crown gall development is accompanied by hypoxia, drought and oxidative stress. These abiotic stress factors are known to have an impact on fatty acid (FA) desaturation. Thus, an alteration in the lipid profile of plant tumors was expected. A comprehensive lipid analysis of Arabidopsis thaliana crown galls induced by Agrobacterium tumefaciens showed an increase in the degree of FA desaturation. The poly unsaturated fatty acid (PUFA) linolenic acid (18:3) of endoplasmic reticulum (ER) derived phospholipids was especially affected. The increased levels of desaturated FAs were reflected by a strong induction of two genes encoding desaturases, FAD3 and SAD6. In contrast to FAD3, which encodes the ER membrane bound fatty acid desaturase enzyme that synthesizes 18:3 PUFAs in the ER, the function of SAD6 is unknown. The ability of SAD6 to complement the extreme dwarf growth phenotype of the ssi2-2 mutant allele suggests that SAD6 is a functional stearoyl-acyl-carrier-protein delta-9 desaturase (SAD) which catalyzes the first step in FA desaturation and forms stearic acid (18:1). Overexpression of the SAD6 gene in Arabidopsis (SAD6-OE) to a similar degree as in tumors resulted in a light-dependent chlorosis phenotype and caused a similar shift in the lipid profile towards unsaturated phospholipids. Posttranscriptional down-regulation of SAD6 overexpression by RNA reverted the chlorosis phenotype and the changes in the lipid profile, showing that SAD6 overexpression forms the unsaturated FA profile and the phenotype in SAD6-OE. The subcellular localization of the SAD6 protein in chloroplasts, which is obligatory for SAD function was demonstrated. SSI2, which encodes the major contributor to the 18:1 FA levels in Arabidopsis is down-regulated in crown galls pointing to a replacement of SSI2 function by SAD6 in the tumor. SAD6 transcripts were almost undetectable in Arabidopsis under normal growth condition, whereas under hypoxia the gene was strongly activated. In the tumor hypoxia most likely caused the very high transcription of SAD6. Hypoxia is known to limit FA desaturation and it is associated with an elevated reactive oxygen species (ROS) production which is detrimental for unsaturated FAs. Thus, up-regulation of SAD6 in the crown gall, most likely serves as an adaptive mechanism to activate desaturation under low oxygen concentrations and to maintain the levels of unsaturated FA under oxidative stress. The ER localized FAD3 most likely is responsible for the rise in 18:3 of the phospholipid class to cope with drought stress in crown galls. This hypothesis was supported by the loss of function mutant, fad3-2, which developed significantly smaller tumors as the wild type under low relative humidity.Taken together, this study suggests that the induction of SAD6 and FAD3 shapes the tumor lipid profile by increasing the levels of unsaturated FAs. Unsaturated fatty acids prepare the crown gall to cope with ongoing hypoxia, drought and oxidative stress during growth and development. / Die Physiologie der durch Agrobacterium tumefaciens hervorgerufenen Wurzelhalsgallen ist geprägt von Sauerstoffmangel, Trocken- und oxidativen Stress. Diese Stressfaktoren beeinflussen die Umwandlung gesättigter zu ungesättigten Fettsäuren (Desaturierung). Somit sind Änderungen im Lipidmuster des durch Agrobacterium tumefaciens ausgelösten Pflanzentumors wahrscheinlich. Eine umfassende Analyse des Wurzelhalsgallenlipidmusters ergab, dass der Anteil an ungesättigten Fettsäuren erhöht war. Am auffälligsten war vor allem die Erhöhung der mehrfach ungesättigten Fettsäure Linolensäure (18:3) in den mit dem endoplasmatischen Retikulum (ER) assoziierten Phospholipiden. Dieser Anstieg ging einher mit der stark erhöhten transkriptionellen Aktivität des FAD3-Gens, das eine membrangebundene Fettsäure-Desaturase kodiert, die Linolensäure (18:3) im ER synthetisiert. Darüber hinaus war ein weiteres funktionell unbekanntes Desaturase-Gen, SAD6, stark aktiviert. Das SAD6 Protein war in Chloroplasten lokalisiert und in der Lage den extremen Zwergwuchs-Phänotyp der ssi2-2 Mutante zum Wildtyp zu komplementieren. Damit wurde nahegelegt, dass SAD6, wie SSI2, eine funktionelle „delta-9 Stearoyl-Acyl-Carrier-Protein-Desaturase“ (SAD) ist. Die Überexpression des SAD6-Gens in Arabidopsis (SAD6-OE), vergleichbar der in Wurzelhalsgallen, führte zu einem Anstieg ungesättigter Phospholipide und einem lichtabhängigen chlorotischen Phänotyp. Eine posttranskriptionelle Reduzierung der SAD6 Überexpression durch RNAi revertierte den Chlorosephänotyp und die Veränderungen im Lipidprofil zum Phänotyp des Wildtyps. Da SSI2, welches das SAD-Enzym für die Ölsäure (18:1)-Produktion in Arabidopsis kodiert, in Wurzelhalsgallen stark herunterreguliert ist, übernimmt hier sehr wahrscheinlich SAD6 die Funktion von SSI2. Insbesondere deshalb, weil der im Tumor vorherrschende Sauerstoffmangel zu einer starken Aktivierung des SAD6-Gens führt. Die Produktion ungesättigter Fettsäuren wird unter hypoxischen Bedingungen limitiert, weshalb eine erhöhte Expression von SAD6 die reduzierte Synthese ungesättigter Fettsäuren kompensieren könnte. Hypoxie und vor allem die posthypoxische Phase führen zur Produktion reaktiver Sauerstoffspezies (ROS), die ungesättigte Fettsäuren peroxidieren, so dass das Hypoxie-sensitive SAD6-Gen darüber hinaus das Niveau ungesättigter Fettsäuren unter oxidativem Stress zu erhalten scheint. Die ER lokalisierte Desaturase FAD3 ist ursächlich für die spezifische Erhöhung von Linolensäure (18:3) in den ER assoziierten Phospholipiden und führt somit zu einer Anpassung an den Trockenstress im Tumor. Dies wird dadurch unterstützt, dass an fad3-2 Mutanten unter erhöhtem Trockenstress deutlich kleinere Tumore wachsen. Diese Studie hat gezeigt, dass die Induktion von SAD6 und FAD3 in der Wurzelhalsgalle mit einer erhöhten Produktion ungesättigter Fettsäuren einhergeht und somit die Entwicklung und das Wachstum von Wurzelhalsgallen unter Sauerstoffmangel, oxidativem Stress und Wasserverlust ermöglicht wird.

In Tumoren an Arabidopsis thaliana, induziert über Agrobacterium tumefaciens (Stamm C58), ist von den 49 bekannten Lipidtransferproteinen (LTPs) nur die Expression von LTP2 stark erhöht (Deeken et al., 2006). Mutanten ohne LTP2-Transkripte (ltp2KO) entwickeln deutlich kleinere Tumore als der Wildtyp. Durch die permanenten Zellstreckungs- und Dehnungsprozesse besitzen Tumore keine intakte Epidermis (Efetova et al., 2007). Dies wiederum führt zum Verlust einer vollständigen Cuticula-Schicht, welche von der Epidermis produziert wird und dieser als Barriere zur Umwelt aufgelagert ist. Um den transpirationsbedingten Wasserverlust zu minimieren, werden in Tumoren langkettige Aliphaten in die äußeren Zellschichten eingelagert (Efetova et al., 2006). Ein ähnliches Szenario findet um Verwundungsareale statt (Kolattukudy et al., 2001). Die Gen-Expression von LTP2 wird nicht durch tumorinduzierende Agrobakterien ausgelöst. Faktoren wie Verwundung, sowie die Applikation des Trockenstress-Phytohormons Abscisinsäure (ABA) begünstigen die LTP2-Gen-Expression positiv. Außerdem ist der LTP2-Promotor in Gewebe aktiv, in welchem sekundäre Zellwandmodifikationen auftreten, sowie insbesondere in Abscissionsschichten von welkenden Organen. Ungerichtete Lipidanalysen der ltp2KO-Mutante im Vergleich zum Wildtyp zeigten nur signifikante Veränderungen in der Menge definierter Sphingolipide – obwohl bislang eine Beteiligung von LTP2 am Transfer von Phospholipiden postuliert wurde. Allerdings kann das LTP2-Protein, wie Protein-Lipid-Overlay-Analysen demonstrierten, weder komplexen Sphingolipide noch Sphingobasen binden. Neben Sphingobasen sind auch langkettige Fettsäuren Bestandteile von Sphingolipiden und diese sind wiederum Bindepartner von LTP2. Um eine eventuelle Beteiligung von LTP2 an der Bildung von Suberin von Tumoren zu zeigen, wurde dieses analysiert. Die GC-MS-Analysen des Tumor-Suberins haben jedoch veranschaulicht, dass durch das Fehlen von LTP2-Transkripten das Lipidmuster nicht beeinträchtigt wird. Eine Überexpression von LTP2 im gesamten Kormophyten war trotz drei unabhängiger experimenteller Ansätze nicht möglich. Daher wurde das Protein ektopisch in epidermalen Zellen exprimiert (CER5Prom::LTP2). Die Transgenen CER5Prom::LTP2 wiesen einige morphologische Besonderheiten auf, wie verminderte Oberflächenhydrophobizität, aberrante Blüten- und Blattmorphologien etc., die typisch für Wachsmutanten sind. GC-MS-Analysen der cuticulären Wachse dieser transgenen Pflanzen zeigten, einen erhöhten Gehalt an C24- und C26-Fettsäuren, wohingegen die korrespondierenden Aliphaten wie Aldehyde und Alkane dezimiert waren. Unterstützend zeigten Lokalisationsanalysen, dass das LTP2-Protein an/in der Plasmamembran assoziiert ist. Somit kann die These aufgestellt werden, dass LTP2 langkettigen, unverzweigten Aliphaten (Fettsäuren) an der Grenzfläche Plasmamembran/Zellwand transferiert, die zur Versieglung und Festigung von Zellwänden benötigt werden. / Out of 49 known lipid transfer protein (LTP) only the expression of LTP2 is highly increased in tumors induced on Arabidopsis thaliana via Agrobacterium tumefaciens (strain C58; Deeken et al., 2006). Mutants with no LTP2 transcripts (ltp2KO) develop significantly smaller tumors than the wild-type. Due to the permanent cell stretch and elongation processes tumors do not possess an intact epidermal layer (Efetova et al., 2007). This leads to the loss of a complete cuticle layer, which is produced by the epidermis and builds up a barrier to the environment. To minimize the transpirational water loss, long-chain aliphatic compounds are incorperated into the outer cell layers of tumors (Deeken et al., 2006). The gene expression of LTP2 is not triggered by tumor-inducing agrobacteria. Instead, factors such as wounding and the application of the phytohormone abscisic acid (ABA) induce the LTP2 gene expression. In addition, the LTP2 promoter is highly active in tissue, in which secondary cell wall modifications occur, and in the abscission zone of wilting organs. Untargeted lipid analyzes of ltp2KO mutant in comparison to the wild type showed significant changes in the amount of defined sphingolipids only - although the involvement of LTP2 has been postulated for the transfer of phospholipids. However, the LTP2 protein, as protein-lipid overlay analysis demonstrated, binds neither complex sphingolipids nor sphingobases. Instead LCFAs, which are part of sphingolipids are binding partners of LTP2. In order to show a possible involvement of LTP2 in the formation of tumor-suberin GC-MS analyzes were performed. These demonstrated that the composition of the lipid-pool is not altered in ltp2KO plants. Overexpression of LTP2 was not possible in spite of three independent experimental approaches. The protein was instead expressed ectopically in epidermal cells (CER5Prom::LTP2). The transgenes CER5Prom::LTP2 showed some morphological abnormities, such as reduced surface hydrophobicity, aberrant flowers and leaf morphologies, which are typical for wax mutants. GC-MS analyzes of the cuticular wax of those transgenic lines revealed an increased amount of C24- and C26- fatty acids. Furthermore LTP2 was localized at the plasma membrane. Thus, this thesis proposes a role of LTP2 in the transfer of long chain, unbranched aliphatics (fatty acids), which are needed to seal up and strengthen cell walls at the interface plasma membrane and cell wall.