RNA-based control of the expression of the type III secretion system of Yersinia pseudotuberculosis Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Jörn Hoßmann aus Bützow 1. Referentin: Professorin Dr. Petra Dersch 2. Referent: Professor Dr. Michael Steinert eingereicht am: 22.02.2017 mündliche Prüfung (Disputation) am: 29.06.2017 Druckjahr 2018 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch die Mentorin der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Maurer CK, Fruth M, Empting M, Avrutina O, Hoßmann J, Nadmid S, Gorges J, Hermann J, Kazmeier U, Dersch P, Müller R and Hartmann RW: Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies. Future Med. Chem. 8, 931–47 (2016). Posterbeiträge Hoßmann J, Pimenova M, Steinmann R, Opitz W, Heroven AK, Dersch P: Thermal and secretion-dependent regulation of the master regulator LcrF in Yersinia pseudotuberculosis. (Poster). 4th National Yersinia Meeting, Hamburg (2014). Hoßmann J, Pimenova M, Steinmann R, Opitz W, Heroven AK, Dersch P: Thermal and secretion-dependent regulation of the master regulator LcrF in Yersinia pseudotuberculosis. (Poster). Sensory and regulatory RNAs of procaryotes meeting, Braunschweig (2015). Hoßmann J, Pimenova M, Steinmann R, Opitz W, Heroven AK, Dersch P: Thermal and secretion-dependent regulation of the master regulator LcrF in Yersinia pseudotuberculosis. (Poster). 32nd Annual Meeting of NSCMID, Umea (2015). Table of contents Table of contents Table of contents ...................................................................................................I List of figures ...................................................................................................... V List of tables ..................................................................................................... VII List of abbreviations ...................................................................................... VIII 1 Introduction .................................................................................................. 1 1.1 The genus Yersinia ..................................................................................................... 1 1.2 Route of infection ....................................................................................................... 3 1.2.1 Initial infection phase ........................................................................................... 3 1.2.2 Ongoing infection phase ....................................................................................... 4 1.2.2.1 The Ysc-Yop virulon ............................................................................................................ 5 1.3 Regulation of Type III secretion .............................................................................. 10 1.3.1 The virulence master regulator LcrF .................................................................. 11 1.3.2 Yop-dependent regulation of the injectisome .................................................... 13 1.3.3 The Carbon storage system ................................................................................ 15 1.3.4 The RNA degradosome ...................................................................................... 18 1.4 Aim of the study ....................................................................................................... 22 2 Material and Methods ................................................................................ 23 2.1 Materials ................................................................................................................... 23 2.1.1 Chemicals and buffers ........................................................................................ 23 2.1.2 Enzymes, Kits and Antibodies ........................................................................... 23 2.1.3 Organisms ........................................................................................................... 24 2.1.3.1 Escherichia coli strains ...................................................................................................... 24 2.1.3.2 Yersinia pseudotuberculosis strains ................................................................................... 24 2.1.4 Plasmids ............................................................................................................. 25 2.1.5 Oligonucleotides ................................................................................................. 26 2.1.6 Media and Antibiotics ........................................................................................ 27 I Table of contents 2.2 Methods .................................................................................................................... 28 2.2.1 General microbial methods ................................................................................ 28 2.2.1.1 Cultivation of bacterial cells .............................................................................................. 28 2.2.1.2 Storage of bacterial strains ................................................................................................. 28 2.2.1.3 Determination of bacterial cell density .............................................................................. 28 2.2.1.4 Chemically competent E. coli strains ................................................................................. 28 2.2.1.5 Electrocompetent Y. pseudotuberculosis strains ................................................................ 29 2.2.1.6 Transformation by heat shock of E. coli ............................................................................ 29 2.2.1.7 Transformation of Y. pseudotuberculosis by electroporation ............................................. 30 2.2.1.8 Mutagenesis of Y. pseudotuberculosis ............................................................................... 30 2.2.2 Molecular biological methods ............................................................................ 31 2.2.2.1 Determination of nucleic acid concentration and purity .................................................... 31 2.2.2.2 Polymerase chain reaction (PCR)....................................................................................... 31 2.2.2.3 Isolation of plasmid DNA .................................................................................................. 32 2.2.2.4 Gel extraction and purification of DNA ............................................................................. 32 2.2.2.5 Agarose gel electrophoresis for separation of DNA fragments .......................................... 33 2.2.2.6 Cloning techniques ............................................................................................................. 33 2.2.2.6.1 Restriction digest .......................................................................................................... 33 2.2.2.6.2 Dephosphorylation of plasmid DNA ............................................................................ 34 2.2.2.6.3 Ligation ......................................................................................................................... 34 2.2.2.6.4 Construction of plasmids .............................................................................................. 34 2.2.2.7 Isolation of total RNA from Y. pseudotuberculosis ........................................................... 36 2.2.2.8 Phenol-chloroform extraction of RNAs ............................................................................. 36 2.2.2.9 Hot Phenol RNA extraction ............................................................................................... 37 2.2.2.10 Agarose gel electrophoresis of RNA .................................................................................. 38 2.2.2.11 Northern blot analysis ........................................................................................................ 38 2.2.2.12 RNA stability assay ............................................................................................................ 39 2.2.2.13 Quantitative real-time PCR ................................................................................................ 40 2.2.2.14 In vitro transcription of RNAs ........................................................................................... 42 2.2.2.15 3'-Biotinylation of RNAs ................................................................................................... 43 2.2.2.16 Detection of Biotin-labeled RNAs ..................................................................................... 44 2.2.2.17 RNA electromobility shift assay (RNA-EMSA) ................................................................ 44 II Table of contents 2.2.2.18 CsrA-RIP-Seq .................................................................................................................... 45 2.2.2.19 Measurement of promoter activity using β-galactosidase reporter fusions ........................ 46 2.2.3 Protein biochemical methods ............................................................................. 47 2.2.3.1 Measurement of protein concentration (Bradford assay) ..................................................