京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17905号 / 農博第2028号 / 新制||農||1018(附属図書館) / 学位論文||H25||N4801(農学部図書室) / 30725 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 加納 健司, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20569号 / 医科博第79号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 江藤 浩之, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21378号 / 農博第2302号 / 新制||農||1070(附属図書館) / 学位論文||H30||N5151(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 橋本 渉, 教授 入江 一浩, 教授 保川 清 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

Yes / In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport. / This work was supported by Biotechnology and Biological Sciences Research Council grant BBS/B/14418 (Membrane Protein Structure Initiative).

In den Firmicutes wird der Induktorausschluss (Katabolitrepression) durch das am Serin46 phosphorylierte HPr (PTS) vermittelt. Der genaue Mechanismus war jedoch unklar. Um diese Frage auf der Grundlage von isolierten Proteinen zu klären, wurde ein zum Escherichia coli Maltose-/Maltodextrin-ABC-Transporter homologes System aus Lactobacillus casei BL23 (MalE1-MalF1G1K12) als Modellsystem genutzt. Im Rahmen der Promotion wurde über isothermale Titrationskalorimetrie und Fluoreszenzspektroskopie gezeigt, dass das Bindeprotein MalE1 lineare und zyklische Maltodextrine, aber keine Maltose bindet. Experimentell ermittelte dreidimensionale Strukturen von MalE1 im Komplex mit diesen Zuckern belegten eine vergleichbar geschlossene Konformation und dienten zusätzlich als Grundlage, um die fehlende Maltosebindung zu erklären. Die Stimulierung der ATPaseaktivität des in Liposomen und Nanodiscs eingebauten Komplexes wurde jedoch hauptsächlich durch eine MalE1-Beladung mit linearen Maltodextrinen bewirkt. Eine bis zu 85 %ige Inhibierung der ATPaseaktivität durch P-Ser46-HPr belegte erstmals in vitro eine Interaktion von mehr als einem phosphorylierten Protein mit dem Transporter. Analog zum EIIAGlc-Inhibitor des homologen Systems aus E. coli wurden über Quervernetzungsexperimente und massenspektrometrische Analysen Interaktionen mit dem MalK1-Dimer als interagierende Komplexeinheit in der Nähe des Walker A-Motivs nachgewiesen. Über Fluoreszenzmessungen in Anwesenheit des ATP-Analogons TNP-ATP wurde eine unbeeinflusste ATP-Bindung und damit eine fehlende Blockade der γ-Phosphatbindestelle des Walker-A Motivs durch die Phosphorylgruppe von P-Ser46-HPr bestimmt. Die folgende Substitution verschiedener positiv geladener MalK1-Reste, die als potenzielle Interaktionsstellen für die Phosphorylgruppe fungieren könnten, identifizierte K63 in der Nähe des Walker A-Motivs als ersten möglichen Partner. Der genaue Mechanismus der Inhibierung bleibt jedoch unklar. / Catabolite repression is a global mechanism which controls the utilization of carbohydrates in bacteria. In Firmicutes HPr, a component of the phosphoenolpyruvate carbohydrate phosphotransferase system, prevents the uptake of less preferred sugars but only when it is phosphorylated at serine46. However the exact mechanism was unclear. To address this question the purified ATP-binding cassette transporter from Lactobacillus casei BL23 (MalE1-MalF1G1K12) was used as a model system, which is homologous to the Escherichia coli maltose/maltodextrin ABC importer. Isothermal titration calorimetry and fluorescence spectroscopy revealed that the binding protein MalE1 binds linear and cyclic maltodextrins but not maltose. Experimentally determined three-dimensional structures from MalE1 in complex with these sugars show a comparably closed conformation and served as a basis to explain the lack of maltose binding. The stimulation of the ATPase activity of the transporter incorporated in liposomes and nanodiscs however, was mainly caused by MalE1 loaded with linear maltodextrins. For the first time an inhibition of ATPase activity by P-Ser46-HPr up to 85 % and an interaction of more than one phosphorylated protein with the transporter was demonstrated. Analogous to the EIIAGlc inhibitor of the homologous system from E. coli, cross-linking experiments and mass spectrometric analyzes revealed interactions with the MalK1 dimer near the Walker A motif. Fluorescence measurements in the presence of the ATP analogue TNP-ATP, however, revealed an unaffected ATP binding and thus a lack of blockade of the γ-phosphate binding site (Walker A motif) by the phosphoryl group from P-Ser46-HPr. The following substitution of several positively charged MalK1 residues that could act as potential sites of interaction for the phosphoryl group, identified K63 near the Walker A motif as the first potential partner. The exact mechanism of inhibition, however, remains unclear.