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Charakterisierung Eines Universellen 'Cyclase-Transducer-Elements' (CTE)

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Charakterisierung Eines Universellen 'Cyclase-Transducer-Elements' (CTE) Charakterisierung eines universellen 'Cyclase-Transducer-Elements' (CTE) in Klasse IIIa Adenylatcyclasen Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Dipl. Biol. (t.o.) Miriam Sabrina Ziegler aus Stuttgart Tübingen 2016 1 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 13.02.2017 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Peter Ruth 2. Berichterstatter: Prof. Dr. Joachim E. Schultz Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig verfasst und alle verwendeten Quellen, Hilfsmittel sowie Ergebnisse von Dritten angegeben habe. Tübingen, den 13.02.2017 Miriam S. Ziegler 3 Danksagung Mein ganz besonderer Dank gilt meinem Doktorvater, Herrn Prof. Dr. Joachim E. Schultz, für die Möglichkeit, dieses spannende Themengebiet für meine Promotion zu nutzen. Er gewährte mir den Freiraum, eigene Ideen in der Forschung zu verfolgen und stand mir mit wissenschaftlichen Diskussionen, die neue Denkanstöße gaben, stets hilfreich zur Seite. Ich bedanke mich für die Möglichkeit zur Teilnahme an Kongressen. Bei Herrn Prof. Dr. Peter Ruth bedanke ich mich ganz herzlich für die Erstellung des Zweitgutachtens. Herrn Prof. Dr. Klaus Hantke und Herrn PD Dr. Bertolt Gust danke ich für die Abnahme der Promotionsprüfung. Herrn Hantke danke ich weiterhin für viele hilfreiche, fachliche Diskussionen und die gute Zusammenarbeit im Labor. Herrn Prof. Dr. Hubert Hilbi danke ich für die Bereitstellung der LqsS-Klone. Für die erfolgreiche und produktive Kooperation bedanke ich mich bei Frau Dr. Kajal Kanchan, Herrn Jens Baßler und Herrn Prof. Dr. Andrei N. Lupas. Bei Frau Anita Schultz bedanke ich mich für die Bereitstellung verschiedener Konstrukte und für ihre Hilfsbereitschaft bei Klonierungsproblemen. Frau Dr. Yinglan Guo danke ich für die freundschaftlichen und fachlichen Diskussionen. Bei Frau Ursula Kurz bedanke ich mich für einen reibungslosen Laborbetrieb, die Übernahme entlastender Klonierungs- und Aufreinigungsschritte sowie für ihre fachlichen und freundschaftlichen Ratschläge sehr herzlich. Allen Mitarbeitern der Arbeitskreise Ruth, Lukowski und Drews danke ich für die nette Zusammenarbeit und gute Atmosphäre auf der 7. Ebene. Bei allen aktuellen und ehemaligen Mitarbeitern des AK Schultz bedanke ich mich für die freundliche Aufnahme in die Arbeitsgruppe und das angenehme Arbeitsklima. Meinen Kolleginnen Dr. Karin Winkler, Dr. Janani Natarajan, Dr. Simone Breitkopf und Dr. Stephanie Beltz danke ich für viele fachliche Anregungen und für ihre Freundschaft im und außerhalb des Labors. Frau Dr. Laura García Mondéjar und Frau Dr. Simone Breitkopf danke ich fürs Einarbeiten in die Labormethoden, Frau Dr. Stephanie Beltz für die Überlassung der LqsS-Konstrukte, Marta Cerrolaza für ihr hohes Engagement. Meiner Familie und Dr. Jakob Birke danke ich von Herzen für die Aufmunterungen und das Wissen, dass wir immer füreinander da sind. Danke! 5 Inhaltsverzeichnis Inhaltsverzeichnis I. ABKÜRZUNGSVERZEICHNIS .................................................................................................................................. 8 II. ZUGANGSNUMMERN, DOMÄNENÜBERSICHT UND AMINOSÄURESEQUENZEN MIT POSITIONSNUMMERN .................................................................................................................................................... 10 1 EINLEITUNG ............................................................................................................................................................. 12 1.1 SIGNALTRANSDUKTION ....................................................................................................................................................... 12 1.2 MODULARITÄT VON ENZYMEN: HAMP, S-HELIX UND CTE ....................................................................................... 13 1.2.1 HAMP Domäne .......................................................................................................................................................... 13 1.2.2 S-Helix .......................................................................................................................................................................... 14 1.2.3 Cyclase-Transducer-Element (CTE) ................................................................................................................. 15 1.3 ADENYLATCYCLASEN .......................................................................................................................................................... 16 1.3.1 Mykobakterielle Adenylatcyclasen Rv1625c und Rv3645 ....................................................................... 19 1.3.2 Cyanobakterielle CyaG aus Spirullina spp. (Arthrospira) ....................................................................... 20 1.4 CHEMOTAXISREZEPTOREN VON ESCHERICHIA COLI ....................................................................................................... 21 1.5 QUORUM SENSING REZEPTOREN LQSS UND CQSS ......................................................................................................... 23 1.6 ZIEL DER ARBEIT ................................................................................................................................................................. 25 2 MATERIAL UND METHODEN ............................................................................................................................. 27 2.1 CHEMIKALIEN UND KITS ..................................................................................................................................................... 27 2.2 GERÄTE.................................................................................................................................................................................. 28 2.3 SOFTWARE ............................................................................................................................................................................ 29 2.4 BAKTERIENSTÄMME ............................................................................................................................................................ 30 2.5 PLASMIDE .............................................................................................................................................................................. 30 2.6 OLIGONUKLEOTIDE .............................................................................................................................................................. 32 2.7 MEDIEN UND ALLGEMEINE WACHSTUMSBEDINGUNGEN .............................................................................................. 36 2.7.1 Medien .......................................................................................................................................................................... 36 2.7.2 Wachstumsbedingungen ...................................................................................................................................... 37 2.7.3 Kompetente Zellen .................................................................................................................................................. 37 2.7.4 Dauerkulturen .......................................................................................................................................................... 38 2.8 MOLEKULARBIOLOGISCHE METHODEN ........................................................................................................................... 38 2.8.1 Isolierung von Plasmid-DNA ............................................................................................................................... 38 2.8.2 Bestimmung der DNA-Konzentration ............................................................................................................. 38 2.8.3 Polymerasekettenreaktion (PCR) ...................................................................................................................... 38 2.8.4 Agarosegelelektrophorese ................................................................................................................................... 43 2.8.5 Restriktionsverdau .................................................................................................................................................. 43 2.8.6 Ligation von DNA-Fragmenten .......................................................................................................................... 44 2.8.7 Transformation von Escherichia coli ............................................................................................................... 44 2.8.8 Plasmid-DNA Sequenzierung .............................................................................................................................. 44 2.9 PROTEINBIOCHEMISCHE METHODEN ............................................................................................................................... 45 2.9.1 Expression .................................................................................................................................................................. 45 2.9.2 Zellernte .....................................................................................................................................................................
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