Agrobacterium tumefaciens is a common soil bacterium that causes crown gall disease on many dicotyledonous plant species. It has been exploited for use in genetic engineering because it is able to promote transfer of any DNA segment to plant chromosomes. Many molecular factors in both the plant and bacterium contribute to successful infection and DNA transfer. Two bacterial factors were studied for this dissertation. The first was bacterial synthesis of cytokinins due to the induced expression of the gene tzs. Cytokinin production by some A. tumefaciens strains at the time of infection was hypothesized to enhance infection or influence host range. To test this hypothesis, heterologous strains engineered to express tzs and a native strain from which tzs was deleted were evaluated for tumor induction on a number of plant species. Major differences were seen when radish roots were infected with the tzs deletion strain. Morphological differences of the galls formed suggested a possible role for tzs in T-DNA transfer efficiency. β-glucuronidase assays with a mannopine synthase promoter-uidA fusion gene were used to study T-DNA transfer early in the infection process. No biological role for tzs was confirmed with either tumorigenesis or T-DNA transfer assays. However, optimized conditions for GUS fluorometric assays were established and the infection process was characterized. The second factor studied was a β-glucosidase gene in A. tumefaciens strain 83/73. This strain is highly infective on conifer hosts, whereas others are not. It is also able to hydrolyze the conifer phenolic compound, coniferin, to an active virulence gene inducer, coniferyl alcohol. This appears to be a major host range determining factor. As a first step in proving this role, the gene for the β-glucosidase enzyme responsible for hydrolysis of coniferin in strain B3/73 was cloned and sequenced. The deduced protein sequence has homology to other β-glucosidases, including a conserved active site. / Graduation date: 1991

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis--Wisconsin. / Includes bibliographical references (leaves 90-98).

Phytohormone sind wichtige Signalmoleküle bei der durch Agrobacterium tumefaciens vermittelten Tumorgenese. Zum einen sind sie direkt am onkogenen Prozess beteiligt, indem sie die Proliferation von transformierten Zellen fördern und physiologische Anpassungen im entstehenden Tumor steuern. Auf der anderen Seite vermitteln Phytohormone aber auch Abwehrreaktionen der Pflanze als Folge eines Befalls mit onkogenen Pathogenen. Um diese verschiedenen Wirkungen der Phytohormone während der Tumorgenese besser zu verstehen, wurde die Genexpression durch Microarrays zu unterschiedlichen Zeitpunken dieses Prozesses an der Modellpflanze Arabidopsis thaliana charakterisiert und die Rolle ausgewählter Phytohormone, wie Abscisinsäure, Salizylsäure, Jasmonsäure, Ethylen und H2O2 durch Mutanten in entsprechenden Signalwegen funktionell untersucht. Die Ergebnisse dieser Arbeit deuten darauf hin, dass die bekannten Pathogenabwehrwege bei Befall durch onkogene Agrobacterien mit einer zeitlichen Verzögerung aktiviert werden. Diese Verzögerung wird wahrscheinlich durch das vom Bakterium abgegebene Auxin reguliert, und somit könnte dieses Auxin die Integration der T-DNA indirekt fördern. Sind die pflanzlichen Abwehrmechanismen jedoch vor dem Transformationsprozess aktiviert, wie z.B. in cpr5–Mutanten, kann die T-DNA nicht integrieren und es entsteht kein Tumor. Beim Wildtyp akkumulieren in Folge der T-DNA Integration mit Pathogenabwehr assoziierte Signalmoleküle, wie H2O2, Ethylen und Salizylsäure, nicht aber Jasmonsäure. Die Analyse des Tumorwachstums an Mutanten mit unterschiedlichen Defekten in diesen Signalwegen zeigte jedoch, daß Ethylen und Salizylsäure keinen Einfluß auf das Tumorwachstum haben. Vielmehr regulieren Ethylen und H2O2 morphologische Anpassungen und Adaptationen an Trockenstress in Tumoren. Die von Agrobacterium tumefaciens induzierten Tumore beziehen außer Nährstoffe, vor allem Wasser von der Wirtspflanze. Das Fehlen einer intakten Epidermis oder Kutikula führt allerdings zu unkontrolliertem Wasserverlust. Da aber weder der Tumor noch die Pflanze welken, muss eine Trockenstressadaptation stattzufinden. In dieser Arbeit konnte gezeigt werden, dass die Phytohormone Abscisinsäure (ABA) und Ethylen an diesem Prozess beteiligt sind. Zum einen regulieren sie die Akkumulation von Osmoregulatoren, sowie Suberineinlagerungen in den äußeren Zellschichten des Tumors, wodurch eine dem Periderm ähnliche Schutzschicht entsteht. Diese Suberinisierung wird im Tumor wahrschein-lich von ABA induziert, wie Experimente an Arabidopsis Wurzeln belegten. Die Microarray-Analysen ergaben, dass im Tumor ein spezielles Muster an ABA- und Trockenstress-induzierten Markergene exprimiert wird, sowie einigen Aquaporinen, die den erhöhten Wasserbedarf des Tumors regulieren könnten. Das verminderte Tumorwachstum an abi- and aba-Mutanten belegt die Bedeutung von ABA-Signalen für die Homeostase des Wasser-haushalts im Tumor. / Phytohormones are important signaling molecules involved in Agrobacterium tumefaciens mediated tumor development. On the one hand, they are directly involved in the infection process by supporting proliferation of transformed plant cells and mediating physiological adaptations of the developing tumor. On the other hand, phytohormones also mediate defense responses of the host plant upon bacterial infection. In order to get further insight into these supplementary roles of phytohormones during tumor development, microarray techniques have been used to analyze changes in gene expression of Arabidosis thaliana upon infection with Agrobacterium tumefaciens at distinct time points during tumor development. The functional relevance of selected phytohormones, e.g. abscisic acid, salicylic acid, jasmonic acid, ethylene and H2O2 was analyzed by the use of Arabidopsis mutants with defects in the respective signaling pathways. This work suggests a delayed activation of the well known defence response pathways to take place upon infection by agrobacteria. This delay is most likely mediated by auxin, which is synthesized and secreted by the bacteria. Hence, auxin indirectly promotes T-DNA integration by causing delay of the plant´s defence responses. However, if pathogen defence is active before agrobacterial infection, e.g. in cpr5 mutant plants, T-DNA integration is prevented and tumor growth cannot be observed. Signaling molecules associated with defence responses, e.g. H2O2, ethylene and salicylic acid, but not jasmonic acid accumulate due to T-DNA integration in wild type plants. However, Arabidopsis mutants with defects along the ethylene or salicylic acid signalling pathways revealed wild type like tumor development neglecting their involvement in tumor associated defence responses. Rather, this work supports the hypothesis that ethylene and H2O2 are involved in regulating tumor morphology and drought stress adaptations. Crown gall tumours induced by Agrobacterium tumefaciens represent a sink that is provided with nutrients and water by the host plant. The lack of an intact epidermis or cuticle results in uncontrolled loss of water. However, neither the tumor nor the host plant display wilting. This phenomenon points to drought stress adaptations in both, tumours and the host plant. In order to understand the protecting molecular mechanisms against desiccation, the gene expression pattern of Arabidopsis thaliana tumors was compared with the profile of stress metabolites: Arabidopsis tumors accumulated high amounts of ABA, the ethylene precursor ACC, osmoprotectants and form a suberized periderm-like layer. Suberization of the outer tumor cell layers most likely is mediated by ABA since external application of ABA induced suberization of Arabidopsis roots. However, the expression level of the classical marker genes, known to respond to drought stress and/or ABA, was lower in tumors. Instead another set of drought and/or ABA-inducible genes, was higher transcribed. Elevated transcription of several ABA-dependent aquaporin genes might indicate that ABA controls the water balance of the tumor. The retarded tumor growth on abi and aba mutant plants underlined the importance of a tumor-specific ABA signaling pathway. Taken together, we propose that ABA is an important signal for protection of tumors against desiccation and thus supports tumor development.

Thesis (M.S.)--Virginia Polytechnic Institute, 1953. / Typewritten. Vita. References: leaves 88-89. Also available via the Internet.

Berlin, Freie Univ., Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format. Computerdatei im Fernzugriff.

Berlin, Freie Universiẗat, Diss., 2004. / Dateiformat: zip, Dateien im PDF-Format.

No description available.

There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-phydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoinhydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolaseencoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes.

No description available.