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N. Meningitidis Stringent response regulation and its impact on ex vivo survival in the commensal pathogen Neisseria meningitidis Regulation der stringenten Kontrolle und ihre Auswirkungen auf das ex vivo Überleben des kommensalen Erregers Neisseria meningitidis Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Laura Violetta Hagmann (geb. Kischkies) Frankfurt am Main Würzburg, 2016 Eingereicht am: …………………………… Mitglieder der Promotionskommission: Vorsitzender: …………………………………………………….. Gutachter: PD Dr. rer. nat. Dr. med. Christoph U. Schoen Gutachter: PD Dr. rer. nat. Knut Ohlsen Tag des Promotionskolloquiums: …………………………... Doktorurkunde ausgehändigt am: …………………………... EIDESSTATTLICHE ERKLÄRUNG Hiermit versichere ich, dass ich die vorliegende Dissertation selbstständig angefertigt und nur die angegebenen Quellen und Hilfsmittel verwendet habe. Ich versichere zudem, dass diese Arbeit in dieser oder ähnlicher Form in keinem anderen Prüfungsverfahren vorgelegen hat. Bis auf den Titel der Diplom-Biologin habe ich bislang keinen anderen akademischen Grad erworben oder zu erwerben versucht. Würzburg, den 13.10.2016 ……………………………………. Laura Violetta Hagmann Table of Content 1 Summary .......................................................................................................... 1 2 Zusammenfassung .......................................................................................... 2 3 Introduction...................................................................................................... 4 The commensal pathogen Neisseria meningitidis ............................................................ 4 Pathogenomics of N. meningitidis ..................................................................................... 5 Genomic flexibility and repeated DNA sequences in N. meningitidis ............................... 7 Gene regulation in N. meningitidis .................................................................................... 9 Adaption to environmental changes: the stringent response .......................................... 11 Stringent response and virulence .................................................................................... 12 Aims of this study ............................................................................................................ 14 4 Materials ......................................................................................................... 15 Laboratory equipment ..................................................................................................... 15 Chemicals and consumables .......................................................................................... 16 Kits and enzymes ............................................................................................................ 18 Buffers and solutions ....................................................................................................... 19 Culture media .................................................................................................................. 27 Proteose peptone medium composition ....................................................................... 27 GCBL medium composition .......................................................................................... 28 GCB++ agar ................................................................................................................... 29 LB medium composition ............................................................................................... 29 Super Optimal Broth (SOB) medium ............................................................................ 30 Minimal medium for meningococci (MMM) ................................................................... 30 Modified Neisseria Defined Medium (mNDM) .............................................................. 31 Cell culture solutions and media ................................................................................... 31 Antibiotic supplements .................................................................................................... 33 Oligonucleotides .............................................................................................................. 33 Plasmids .......................................................................................................................... 35 Microorganisms ............................................................................................................... 37 Strains used in this study .............................................................................................. 37 Cell lines .......................................................................................................................... 37 5 Methods .......................................................................................................... 38 Cultivation of bacteria ...................................................................................................... 38 Estimation of bacterial cell number by determination of the optical density at 600 nm ............................................................................................................................. 38 Preparation of chemically competent E. coli cells ........................................................... 38 Transformation of E. coli ................................................................................................. 39 Transformation of N. meningitidis ................................................................................... 39 Preparation of meningococcal genomic DNA ................................................................. 40 Isolation of plasmid DNA ................................................................................................ 40 Bacterial lysates .............................................................................................................. 42 Polymerase chain reaction (PCR) .................................................................................. 42 Visualization and purification of PCR products ............................................................... 44 Sequencing of PCR products and plasmids ................................................................... 44 Cloning procedure: digestion of DNA, gel extraction and ligation .................................. 44 Construction of mutants .................................................................................................. 46 Construction of isogenic deletion mutants ................................................................... 46 Construction of mutants using megaprimer PCR......................................................... 46 Construction of relA 6xHis-tagged mutants ................................................................. 47 Southern Blot .................................................................................................................. 48 RNA preparation from liquid culture ............................................................................... 48 cDNA synthesis............................................................................................................... 50 Transcriptional start site mapping ................................................................................... 51 Quantitative real-time PCR (qRT-PCR) .......................................................................... 51 Northern Blot ................................................................................................................... 52 Generation of DIG-labeled DNA probes ......................................................................... 52 Detection of DIG-labeled DNA by chemiluminescence .................................................. 53 cDNA microarray............................................................................................................. 53 Western Blot ................................................................................................................... 57 Phenotypic characterization ............................................................................................ 57 In vitro growth experiments .......................................................................................... 57 Cell adhesion and invasion assay ................................................................................ 57 Static biofilm assay for N. meningitids ......................................................................... 58 Collection and processing of human specimens .......................................................... 59 Ex vivo survival assays ................................................................................................ 59 (p)ppGpp Extraction and Quantification ......................................................................... 59 Computational analyses ................................................................................................. 60 6 Results ............................................................................................................ 62 The stringent response in N. meningitidis ...................................................................... 62 Both strains express an almost identical RelA protein ................................................. 62 RelA is functional and the sole ppGpp synthase in N. meningitidis ............................. 63 SpoT is essential
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