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Yersinia Enterocolitica

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Yersinia Enterocolitica TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Mikrobielle Ökologie Regulation und Freisetzung des insektiziden Komplexes in Yersinia enterocolitica Mandy Starke Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.- Prof. Dr. W. Liebl Prüfer der Dissertation: 1. apl. Prof. Dr. T. Fuchs 2. Univ.-Prof. Dr. M. Hofrichter (nur schriftliche Beurteilung) Technische Universität Dresden Univ.-Prof. Dr. S. Scherer (nur mündliche Prüfung) 3. Univ.-Prof. Dr. J. Heesemann (i.R.) Ludwigs-Maximilians-Universität München Die Dissertation wurde am 10.11.2014 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 17.03.2015 angenommen. “Alles Wissen und alle Vermehrung unseres Wissens endet nicht mit einem Schlusspunkt, sondern mit einem Fragezeichen.“ -Hermann Hesse- Inhaltsverzeichnis I Inhaltsverzeichnis ABBILDUNGSVERZEICHNIS ........................................................................................................... V TABELLENVERZEICHNIS .............................................................................................................. VII ABKÜRZUNGSVERZEICHNIS ..................................................................................................... VIII ZUSAMMENFASSUNG .................................................................................................................... XII SUMMARY ....................................................................................................................................... XIV VORWORT ........................................................................................................................................... 1 1 EINLEITUNG ........................................................................................................................... 3 1.1 Die Gattung Yersinia ........................................................................................................................... 3 1.1.1 Yersinia enterocolitica .......................................................................................................................... 3 1.1.2 Pathogenese und Virulenzfaktoren ...................................................................................................... 4 1.1.3 Temperaturabhängige Regulation von Virulenzfaktoren in Yersinien.................................................. 6 1.1.4 Physiologie von Yersinien bei niedrigen Temperaturen ....................................................................... 8 1.2 Die insektizide Pathogenitätsinsel Tc-PAIYe in Y. enterocolitica ......................................................... 10 1.2.1 LysR-ähnliche Regulatoren ................................................................................................................. 14 1.2.2 Holin/Endolysin .................................................................................................................................. 16 1.2.3 Evolution der Tc-PAI ........................................................................................................................... 19 1.3 Zielsetzungen dieser Arbeit............................................................................................................... 23 2 MATERIAL UND METHODEN ......................................................................................... 25 2.1 Mikrobiologische Methoden ............................................................................................................. 25 2.1.1 Bakterienstämme ............................................................................................................................... 25 2.1.2 Kulturbedingungen, Antibiotika und Medien ..................................................................................... 25 2.1.3 Einfrieren von Bakterien ..................................................................................................................... 26 2.2 Molekulargenetische Methoden ....................................................................................................... 27 2.2.1 Plasmide und Vektoren ...................................................................................................................... 27 2.2.2 Präparation von Nukleinsäuren .......................................................................................................... 27 2.2.2.1 Plasmidisolation .......................................................................................................................... 27 2.2.2.2 Isolierung chromosomaler DNA aus Yersinia spp. ...................................................................... 27 2.2.2.3 RNA-Isolation aus Yersinia spp.................................................................................................... 28 2.2.2.4 Konzentrations- und Reinheitsbestimmung von Nukleinsäuren ................................................ 29 2.2.3 Agarosegelelektrophorese.................................................................................................................. 30 2.2.4 Polymerase-Kettenreaktion ................................................................................................................ 31 2.2.4.1 Standard-PCR .............................................................................................................................. 31 II Inhaltsverzeichnis 2.2.4.2 Quantitative Echtzeit-PCR ........................................................................................................... 32 2.2.4.3 Aufreinigung von PCR-Produkten ............................................................................................... 35 2.2.5 Bestimmung des Transkriptionsstartes .............................................................................................. 36 2.2.6 Enzymatische Modifikation von DNA ................................................................................................. 38 2.2.6.1 Spaltung von DNA mit Restriktionsendonukleasen .................................................................... 38 2.2.6.2 Dephosphorylierung von DNA .................................................................................................... 39 2.2.6.3 Ligation ....................................................................................................................................... 39 2.2.7 DNA-Transfer ...................................................................................................................................... 39 2.2.7.1 Transformation ........................................................................................................................... 39 2.2.7.2 Konjugation ................................................................................................................................. 41 2.2.8 Konstruktion von Yersinia-Stämmen .................................................................................................. 42 2.2.8.1 Herstellung von Insertionsmutanten .......................................................................................... 42 2.2.8.2 Herstellung von Reporterstämmen in Y. enterocolitica .............................................................. 42 2.2.8.3 Herstellung von Deletionsmutanten ........................................................................................... 44 2.2.9 Sequenzierung von DNA ..................................................................................................................... 45 2.2.10 Bioinformatische Methoden ............................................................................................................... 46 2.2.11 Quantifizierung der Biolumineszenz und Fluoreszenz in vitro ........................................................... 46 2.2.11.1 Visualisierung der Genexpression mit dem Xenogen In Vivo Imaging System ....................... 46 2.2.11.2 Quantifizierung der Genexpression mit dem Wallac Victor3 .................................................. 47 2.2.11.3 Bestimmung des Gehaltes an ATP .......................................................................................... 48 2.2.11.4 Fluoreszenzmikroskopie ......................................................................................................... 48 2.3 Proteinchemische Methoden ............................................................................................................ 49 2.3.1 Präparation von Proteinen aus Y. enterocolitica ................................................................................ 49 2.3.1.1 Herstellung von Gesamtzellextrakten aus Flüssigkultur ............................................................. 49 2.3.1.2 Isolierung von Überstandsproteinen .......................................................................................... 49 2.3.2 Überexpression von Proteinen ........................................................................................................... 49 2.3.2.1 Klonierung der pBAD/HisA (TetR)-Konstrukte ............................................................................. 50 2.3.2.2 Expression heterologer Proteine mit Hilfe von pBAD/HisA (TetR) und pET28b .......................... 50 2.3.2.3 Zellaufschluss mittels French Pressure Cell ................................................................................
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