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Charakterisierung Des Umweltkeims Bordetella Petrii

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Charakterisierung Des Umweltkeims Bordetella Petrii ______________________________________________________________________________ BAYERISCHE JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG FAKULTÄT FÜR BIOLOGIE LEHRSTUHL FÜR MIKROBIOLOGIE ______________________________________________________________________________ Charakterisierung des Umweltkeims Bordetella petrii . Untersuchungen zur genomischen Variabilität und zum Bvg Regulon Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Melanie Lechner aus Villingen-Schwenningen Würzburg, 2008 Eingereicht am: ........................................................................................................................ Mitglieder der Promotionskommission: Vorsitzender: ............................................................................................................................ 1. Gutachter: Prof. Dr. Roy Gross 2. Gutachter: Prof. Dr. Roland Benz Tag des Promotionskolloquiums: .............................................................................................. Doktorurkunde ausgehändigt am: .............................................................................................. Erklärung Ich versichere, dass die vorliegende Arbeit von mir selbständig und nur unter Verwendung der angegebenen Hilfsmittel und Quellen angefertigt und verfasst wurde. Diese Dissertation hat weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegen. Neben dem akademischen Grad "Diplom-Biologin Univ." habe ich keine weiteren akademischen Grade erworben oder zu erwerben versucht. Würzburg, ....................................................................... Der experimentelle Teil der Arbeit wurde am Lehrstuhl für Mikrobiologie am Theodor-Boveri- Institut für Biowissenschaften der Bayerischen Julius-Maximilians-Universität Würzburg von April 2004 bis September 2007 durchgeführt. Wir müssen unbedingt Raum für Zweifel lassen, sonst gibt es keinen Fortschritt, kein Dazulernen. Man kann nichts Neues herausfinden, wenn man nicht vorher eine Frage stellt. Und um zu fragen, bedarf es des Zweifelns. “ (Richard P. Feynman) Danksagung An dieser Stelle möchte ich all jenen Menschen danken, die zum guten Gelingen dieser Arbeit beigetragen haben. Zunächst möchte ich mich bei Herrn Prof. Dr. Roy Gross bedanken, der mir dieses spannende Thema überlassen hat und mir mit guten Ratschlägen zur Seite stand. Vielen Dank auch an Herrn Prof. Dr. Roland Benz für die freundliche Übernahme des Zweitgutachtens und Herrn Prof. Dr. Werner Göbel für die Bereitstellung des Arbeitsplatzes. Dem SFB 479 danke ich für die finanzielle Unterstützung dieser Arbeit. Ein ganz herzliches Dankeschön geht an Jenni Pohlert, meine Mitstreiterin bei der Erforschung von B. petrii , die mir mit den Jahren eine wertvolle Freundin wurde. Dank auch an die Mitarbeiter der AG Gross und AG Beier für die angenehme Arbeitsatmosphäre und die fachliche Unterstützung. Besonders bedanken möchte ich mich bei Steffi Müller für ihre Freundschaft und weil das Leben schließlich kein Ponyhof ist, bei Karin Schmitt und Susanne Bauer, die die B. petrii -Experimente fortführen und mich während der Schreibphase auf dem neuesten Stand der Forschung gehalten haben und natürlich bei meinen jetzigen und ehemaligen Laborkolleginnen Kristina Keidel und Aleks Horvat, mit denen die Arbeit im Labor immer Spaß gemacht hat. Allen Mitgliedern des Lehrstuhls möchte ich für die freundliche Aufnahme und die Unterstützung bei meiner Arbeit danken. Besonders erwähnen möchte ich hier Dani Löffler, die Zellkultur-Fachfrau, Sonja Mertins, die mich in die Geheimnisse der Realtime-PCR eingewiesen hat sowie Andrea Spory, Barbara Gareiß und Jennifer Schär für die Unterstützung bei meinen „heißgeliebten“ 2D-Gel-Experimenten. Nicht zu vergessen unsere beiden Spülfrauen Frau Steinbrecher und Frau Stapf, ohne die das Arbeiten am Lehrstuhl ungleich schwerer gewesen wäre. Zum Schluss möchte ich mich bei meinen Lieben bedanken. Bei meinen Großeltern für ihr stetes Interesse an meinem Leben und bei meinem „großen“ kleinen Bruder Flo für die musikalische Weiterbildung. Ein ganz besonderes Dankeschön geht an meine Eltern für ihre unermüdliche Unterstützung, weil ich mich immer auf sie verlassen konnte und sie geduldig allen meinen B. petrii -Geschichten gelauscht haben. Meinem Freund Stefan danke ich von Herzen, weil er immer für mich da war und mit ihm alles irgendwie leichter erschien. Euch möchte ich diese Arbeit widmen. Inhaltsverzeichnis 1 ZUSAMMENFASSUNG........................................................................................................ 1 2 EINLEITUNG ........................................................................................................................ 5 2.1 DIE GATTUNG BORDETELLA ............................................................................................... 5 2.2 DAS B. BRONCHISEPTICA -CLUSTER .................................................................................... 6 2.2.1 Klinische Relevanz des B. bronchiseptica-Clusters ................................................. 7 2.2.2 Virulenzfaktoren des B. bronchiseptica-Clusters................................................... 10 2.2.2.1 Adhäsine............................................................................................................. 10 2.2.2.2 Toxine................................................................................................................. 12 2.2.2.3 Weitere Faktoren ................................................................................................ 14 2.3 DIE „NEUEN “ BORDETELLA -ARTEN .................................................................................. 15 2.3.1 B. petrii................................................................................................................... 17 2.3.2 Virulenzfaktoren der „neuen“ Bordetella-Arten.................................................... 19 2.4 DAS BVG AS-ZWEIKOMPONENTENSYSTEM ...................................................................... 21 2.4.1 Phasenvariation und phänotypische Modulation................................................... 21 2.4.2 Signaltransduktion und Struktur des BvgAS-Systems............................................. 22 2.4.3 Transkriptionelle Genregulation über das BvgAS-System..................................... 23 2.4.4 Der bvgAS-Lokus innerhalb der Gattung Bordetella............................................. 27 2.5 MECHANISMEN DER BAKTERIENEVOLUTION .................................................................... 28 2.5.1 Genomische Inseln - Integrative und konjugative Elemente .................................. 31 2.5.1.1 Das clc -Element von Pseudomonas sp. Stamm B13.......................................... 33 2.5.2 Evolutionäre Tendenzen in der Gattung Bordetella............................................... 34 2.6 ZIELSETZUNG DER ARBEIT ............................................................................................... 37 3 MATERIAL.......................................................................................................................... 39 3.1 GERÄTE ........................................................................................................................... 39 3.2 BAKTERIENSTÄMME ........................................................................................................ 39 3.3 ZELLLINIEN ..................................................................................................................... 40 3.4 VEKTOREN UND REKOMBINANTE PLASMIDE .................................................................... 40 3.5 OLIGONUKLEOTIDE .......................................................................................................... 41 3.5.1 Plasmidspezifische Oligonukleotide....................................................................... 41 3.5.2 Klonierungs- und Screening- Oligonukleotide....................................................... 41 3.5.3 Oligonukleotide zur Sequenzierung von 16S rDNA ............................................... 43 3.5.4 Oligonukleotide zur Herstellung von Southern Blot Sonden zur Untersuchung der genomischen Inseln ................................................................................................ 43 3.5.5 Oligonukleotide für Realtime-PCR ........................................................................ 44 3.5.6 Sonstige Oligonukleotide........................................................................................ 44 3.6 VERBRAUCHSMATERIALIEN ............................................................................................. 46 3.6.1 Chemikalien............................................................................................................ 46 3.6.2 Enzyme.................................................................................................................... 46 3.6.3 Molekularbiologische Kits ..................................................................................... 46 3.7 WACHSTUMSMEDIEN UND ZUSÄTZE ................................................................................ 46 3.7.1 Wachstumsmedien .................................................................................................. 46 3.7.2 Antibiotika .............................................................................................................. 48 3.8 MOLEKULARGEWICHTSMARKER .....................................................................................
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