The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...

The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...

Die für die Vermehrung der Paramyxoviren notwendige Freisetzung des Virusgenoms in die Wirtszelle findet nach einer Verschmelzung der Virushülle mit der Zellmembran statt. Die Membranfusion wird durch eine Konformationsänderung des membranständigen Fusionsproteins (F-Protein) der Paramyxoviren vermittelt. Der Auslöser der Strukturumwandlung des F-Proteins ist bislang unbekannt. Man nimmt an, daß eine Wechselwirkung mit dem zweiten membranständigen Protein der Hämagglutinin-Neuraminidase (HN-Protein) die Strukturumwandlung des F-Proteins induziert. Das F-Protein kann jedoch auch in Abwesenheit des HN-Proteins eine Membranfusion vermitteln. Für das Verständnis des Mechanismus der F-proteinvermittelten Fusion ist die Kenntnis der dreidimensionalen Struktur des F-Proteins notwendig. In der vorliegenden Arbeit wurden die F-Proteine der Paramyxoviren, Sendaivirus und Simianvirus 5, in fusionskompetenter Form isoliert und in kleine Lipidvesikel rekonstituiert, um deren Struktur mittels Kryoelektronenmikroskopie und Einzelpartikelanalyse aufzuklären. Die 3D-Struktur des Sendaivirus-F-Proteins konnte mit einer Auflösung von 16 Angström aufgeklärt werden. Es ist die erste 3D-Struktur des F-Proteins eines Paramyxovirus in der fusionskompetenten Form. Um geeignete Bedingungen herauszufinden, die das Auslösen der Konformationsänderung der F-Proteine bzw. das "Einfangen" von Strukturintermediaten während der Fusion ermöglichen, wurde das Fusionsverhalten von Sendaivirus und Simianvirus 5 bei unterschiedlichen Temperatur- und pH-Werten sowie in Anwesenheit von Lysolipiden mittels Fluoreszenzdequenchingassays untersucht. Ein signifikanter Anstieg der Fusionsaktivität der untersuchten Viren konnte durch eine Erhöhung der Temperatur erreicht werden. Mittels ESR-Spektroskopie unter Einsatz von spinmarkierten Lysolipiden konnte gezeigt werden, daß Lysolipide die proteinvermittelte Fusion von Hüllviren in einem späten lipidabhängigen Schritt hemmen. Diese Untersuchungen bilden damit eine Grundlage zur Aufklärung der 3D-Struktur des F-Proteins im fusionsaktiven Zustand. Desweiteren wurde die Rolle der transmembranalen und zytoplasmatischen Domäne des F-Proteins bei der Membranfusion und der Wechselwirkung mit dem HN-Protein mittels Fluoreszenzmikroskopie untersucht. Die Befunde der 3D-Strukturaufklärung und der fluoreszenzmikroskopischen Studien wurden unter anderem in Hinblick auf die Bedeutung der Wechselwirkung zwischen den F- und HN-Proteinen für die Fusion diskutiert. / Paramyxoviruses infect their host cells by fusion of the viral envelope with the cell membrane. The membrane fusion is mediated by a confomational change of a viral envelope glycoprotein called the fusion (F) protein. The trigger of the F protein conformational change is still unknown. It is suggested, that an interaction of the F protein with the second envelope glycoprotein hemagglutinin-neuraminase (HN) induces its conformational change. However the F protein can mediate membrane fusion in absense of HN. The knowledge of the three dimensional structure of the F protein is reqiured to understand the F mediated membrane fusion. In the present work the fusion competent form of the fusion proteins of the paramyxoviruses Sendai virus and Simian virus 5 were isolated and incorporated each of them into small lipid vesicles. The 3D-structure of the entire ectodomain of the Sendai virus F protein has been determined in fusion potential conformation by cryo electron microscopy of single molecules and 3D-reconstruction at a resolution of 16 Angström. To detect usefull conditions for triggering the conformational change of F, the fusion of Sendai virus and Simian virus 5 have been studied at different temperature and pH, respectively, using a fluorescence dequenching assay. A significant increase of virus fusion activity has been found due to temperature enhancement. Using ESR-spectroscopy and spin-labeled lysolipids it has been shown that lysolipids inhibit the protein mediated fusion of enveloped viruses at a late lipid-dependent intermediate. Thus lysolipids are capable to freeze a conformational intermediate of the F protein during fusion. Furthermore the role of the transmembrane and the cytoplasmic domain of the Sendai virus F protein for membrane fusion was investigated using fluorescence microscopy. The results of the fluorescence microscopy study and the detection of the 3D-structure have been discussed in view of the relevance of F-HN-interaction for membrane fusion.