Amna Mehmood

• Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight on soybean plants and enters the leaf tissue through stomata or open wounds, where it encounters a sucrose-rich milieu. Sucrose is utilized by invading bacteria via the secreted enzyme, levansucrase (Lsc), liberating glucose and forming polyfructan levan. During this study, it was found that lsc is under the control of the hexose metabolic repressor, HexR. The DNA binding sequence of this regulator was found upstream of lsc genes. During in vitro growth analysis using sucrose as sole carbon source and in planta growth, it was observed that the growth of the hexR mutant was impaired as compared to wild type. We could conclude that HexR acts as an in planta fitness factor and that the genes involved in extra-cellular sugar acquisition might be co-regulated with those involved in intra-cellular metabolic pathways in P. syringae. Furthermore, an in-depth study using different lsc knock-out mutants was conducted to characterize them phenotypically and biochemically. It was observed that M6 (lscB/lscC mutant) had a significantly lower in planta survival as compared to the wild type or mutants M3 (lscB mutant) and M5 (lscC mutant). The biochemical characterization of mutant-derived levan polymers by HPLC-microTOF revealed that the levan polymerized by LscB/LscC on 5% sucrose had a higher fructose-to-glucose ratio as compared to levans polymerized by LscB or LscC, respectively, suggesting a concerted action of both enzymes. Additionally, levan polymerized by LscB/LscC produced on 5% sucrose accumulated a higher amount of fed antibiotics as compared to any of the levan polymers derived from LscB or LscC alone. We concluded that levansucrases are important for the survival and symptom development of PG4180 in in planta. The thesis additionally dealt with method development for identification of protein translocation across the membrane for levansucrase.