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Global and Molecular Views on the Control of Gene Expression

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Global and Molecular Views on the Control of Gene Expression Carbon Catabolism in Bacillus subtilis: Global and Molecular Views on the Control of Gene Expression Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Oliver Schilling aus Bamberg Göttingen 2007 D 7 Referent: Prof. Dr. Jörg Stülke Korreferent: Prof. Dr. Ralf Ficner Tag der mündlichen Prüfung: 05.07.07 Danksagung An erster Stelle möchte ich ganz herzlich Prof. Dr. Jörg Stülke für die Stellung des interessanten Themas und für die gute Betreuung der Arbeit danken. Er hat mir für die Verwirklichung meiner Ideen alle nötigen Freiheiten gelassen und brachte mir dadurch großes Vertrauen entgegen. Gleichwohl konnte ich immer auf seine Hilfsbereitschaft zählen, wenn sie gebraucht wurde. Desweiteren bedanke ich mich bei Herrn Prof. Dr. Ralf Ficner für die freundliche Übernahme des Korreferats und für die Zusammenarbeit bei einem Projekt zur strukturellen Aufklärung des GlcT-RAT Komplexes. Für die finanzielle Unterstützung der Arbeit bedanke ich mich beim Fonds der Chemischen Industrie und bei der Deutschen Forschungsgemeinschaft. Bei Dr. Armin Ehrenreich bedanke ich mich für die Einführung in die Welt der Microarrays und für seine stete Hilfe bei allen technischen Fragen rund ums Spotten, Labeln und Hybridisieren. Prof. Dr. Elmar Heinzle, PD Dr. Christoph Wittmann und Oliver Frick danke ich für die wertvolle Zusammenarbeit, aber auch dafür, dass ich eine Woche in ihrer Arbeitsgruppe an der Universität Saarbrücken mitarbeiten durfte. In diesem Zusammenhang bedanke ich mich auch bei Michael Fritz und Tae Hoon Yang für die freundliche Mithilfe bei diesem Projekt. Für die Bereitstellung von Bakterienstämmen bedanke ich mich bei Prof. Dr. Richard Herrmann (Universität Heidelberg), Dr. Ulf Gerth (Universität Greifswald) und Dominique Le Coq (Laboratoire de Génétique des Microorganismes, Thiverval-Grignon). Bei Dr. Matthias Görlach und Dr. Matthias Stoldt (Fritz-Lipmann-Institut, Jena) bedanke ich mich für die hilfreichen Gespräche zur NMR-Strukturanalyse. Für ihren experimentellen Beitrag durch Zuckertransport-Messungen möchte ich mich bei PD Dr. Fritz Titgemeyer und Tina Hertrich bedanken. Ein ganz besonderes Dankeschön geht an Christina Herzberg, die sich in besonderer Weise engagierte und damit wesentlich zu den Ergebnissen in dieser Arbeit beigetragen hat. Sie gehört sicher in die Top-Ten der TAs dieser Welt. Vielen Dank auch für die mentale Unterstützung und all die praktischen Tipps und Ratschläge fürs Leben. Mein Dank geht auch an Dr. Thorsten Mascher und seine Arbeitsgruppe, welche man immer wieder für ein Spielchen am Kicker begeistern kann. Es war interessant zu beobachten, welch große Fortschritte man dabei in kurzer Zeit erzielen kann. Desweiteren möchte ich mich bei Thorsten für die Einführung in die Tiefen der LFH-PCR bedanken. Bei Dr. Boris Görke bedanke ich mich für die interessanten Diskussionen zu allerlei molekularbiologischen Methoden. Seine Art der praktischen Durchführung ist dabei äußerst konträr zu der, die ich aus dem ursprünglichen „Botchers Lab“ kennengelernt habe. Prof. Dr. Wolfgang Hillen, an dessen Lehrstuhl an der Universität Erlangen einige Experimente für diese Arbeit durchgeführt wurden, danke ich für die Bereitstellung der dortigen Laborausstattung und Arbeitsumgebung. Den „Erlangern“ Steffi Bachem, Hans- Matti Blencke, Christian Detsch, Ines Langbein, Michael Müller, Irene Reif, Matthias Schmalisch, Shane Hanson und Ingrid Wacker danke ich für all die kleinen und großen Beiträge zu dieser Arbeit. Vielen Dank auch an den Erlanger Kern, der mit mir gemeinsam die Reise ins norddeutsche Ausland gewagt hat: Dank an Hans-Matti, der mir in den ersten Wochen in Göttingen ein Dach über dem Kopf gegeben hat, und von dem ich auch gerne etwas von seiner nordischen Gelassenheit übernommen hätte. Dank auch an Fabian Commichau, der mir als erster gezeigt hat, dass es auch außerhalb von Bayern intelligentes Leben gibt. Last but not least danke ich Matthias Schmalisch für seine ausgedehnte Diskussionsbereitschaft zu nahezu jedem Thema unter der Sonne und für seine Überredungskünste, ohne die ich einige Dinge sicher noch nicht ausprobiert hätte (Rollersoccer???). Auch an unseren USA-Aufenthalt werde ich noch lange zurückdenken. Ein grosses Dankeschön geht aber auch an die "alteingesessenen Göttinger" Birgit Veith und Mark Hoffmeister, die mir zu Beginn in ihrem Labor Asyl gewährt und mich schrittweise in die neuen Gegebenheiten eingeführt haben. Bedanken möchte ich mich natürlich auch bei meinen Praktikanten Timo Hupfeld, Dirk Jessen, Desiree Krausse und Hanna Vörsmann, die ich betreuen durfte, und die teilweise erheblichen Beitrag zu dieser Arbeit geleistet haben. Ich möchte auch meinem ersten und einzigen Diplomanden Sebastian Hübner dafür danken, dass er durch seine selbständige Arbeitsweise wenig Betreuungszeit in Anspruch genommen hat, wovon eigene Projekte profitieren konnten. Mein Dank gilt selbstverständlich in gleicher Weise auch allen nicht namentlich genannten Kollegen für das gute und lockere Laborklima. Zu guter Letzt möchte ich mich noch bei meiner Familie für ihre beständige Unterstützung bedanken. Vielen Dank auch an Kathrin, die mich aufopferungsvoll auch durch schwere Zeiten begleitet und mir die nötige mentale Kraft verliehen hat. Table of contents I Table of contents List of abbreviations ______________________________________________________ III List of publications ________________________________________________________VI Summary _________________________________________________________________ 1 Zusammenfassung__________________________________________________________ 3 1 Introduction _____________________________________________________________ 5 1.1 Sugar metabolism in Bacillus subtilis _______________________________________ 5 1.1.1 An overview _______________________________________________________ 5 1.1.2 Regulation of sugar metabolism in B. subtilis: cause and effect _______________ 9 1.2 RNA and its role in gene regulation _______________________________________ 13 1.2.1 The structural features of single stranded RNA___________________________ 14 1.2.2 Non-coding RNAs _________________________________________________ 16 1.2.3 RNA switches_____________________________________________________ 18 1.2.3.1 Regulation by RNA thermometers _________________________________ 21 1.2.3.2 RNA mediated regulation ________________________________________ 22 1.2.3.3 Metabolite mediated regulation____________________________________ 23 1.2.3.4 Protein mediated regulation ______________________________________ 26 1.2.3.5 RNA aptamers: control by artificial regulatory RNA elements ___________ 27 1.3 PTS-controlled antitermination in B. subtilis ________________________________ 29 1.3.1 The PTS and its role in sugar transport _________________________________ 29 1.3.2 Regulation of the ptsGHI operon by transcriptional antitermination___________ 30 1.3.3 The family of homologous antiterminator systems of B. subtilis _____________ 33 1.4 Aim of this work ______________________________________________________ 36 2 Transcriptional and metabolic responses of Bacillus subtilis to the availability of organic acids: Transcription regulation is important but not sufficient to account for metabolic adaptation _____________________________________________________ 37 3 Multiple-mutation reaction: A method for the simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae _______________________ 57 4 A protein-dependent riboswitch controlling ptsGHI operon expression in Bacillus subtilis: RNA structure rather than sequence provides interaction specificity _______________________________________________________________________ 64 5 Keeping signals straight in transcription regulation: specificity determinants for the interaction of a family of conserved bacterial RNA-protein couples ______________ 89 Table of contents II 6 Discussion _____________________________________________________________ 121 6.1 Effects of organic acids on the central metabolism of B. subtilis ________________ 121 6.2 Specificity in gene regulatory systems ____________________________________ 131 6.3 Outlook ____________________________________________________________ 142 7 References _____________________________________________________________ 143 8 Supplementary material _________________________________________________ 176 Curriculum vitae _________________________________________________________ 205 List of abbreviations III List of abbreviations A adenine asRNA antisense RNA ATP adenosine triphosphate BSA bovine serum albumin bp base pair C carbon C cytosine cDNA copy DNA CCR carbon catabolite repression CCR combined chain reaction cm centimeter Cm chloramphenicol CTT cytidine triphosphate Cy3/Cy5 fluorescent dyes of the cyanine dye family DHAP dihydroxyacetone phosphate DIG digoxigenin DNA desoxyribonucleic acid DNase desoxyribonuclease I dsRNA double stranded RNA EI enzyme I EII enzyme II EDTA ethylenediaminetetraacetic acid Em erythromycin FAD flavin adenine dinucleotide FBP fructose-1,6-bisphosphate FMN flavin mononucleotide Fig. figure G guanine Glc glucose GlcN6P glucosamine-6-phosphate GMP guanosine monophosphate GTP guanosine triphosphate H hydrogen h hour IMP inosine monophosphate IPTG isopropyl-β-D-1-thiogalactopyranoside kb kilo base kDa kilodalton Km kanamycin LB Luria Bertani LFH long flanking homology Lin lincomycin µl microliters µm micrometers M molar mCi milli Curie MCS multiple cloning site
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