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The Influence of Riboregulation on Fitness and Virulence in Neisseria Meningitidis

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The Influence of Riboregulation on Fitness and Virulence in Neisseria Meningitidis The influence of riboregulation on fitness and virulence in Neisseria meningitidis Der Einfluss der Riboregulation auf Fitness und Virulenz von Neisseria meningitidis Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades (Dr. rer. nat.) der Graduate School of Life Sciences (GSLS), Bayrische Julius-Maximilians-Universität Würzburg Sektion: Infektion und Immunität Vorgelegt von Saskia Corinna Bauriedl aus Regensburg Würzburg, 2019 Eingereicht am: 23.09.2019 Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. med. Georg Gasteiger Gutachter: Prof. Dr. rer. nat. Dr. med. Christoph Schoen Gutachter: Prof. Dr. rer. nat. Jörg Vogel Gutachter: Prof. Dr. rer. nat. Joachim Morschhäuser Tag des Promotionskolloquiums: …………………………. Doktorurkunde ausgehändigt am:….……………………… Eidesstattliche Erklärung I hereby confirm that my thesis entitled ‘The influence of riboregulation on fitness and viru- lence in Neisseria meningitidis’ is the result of my own work. I did not receive any help or support from commercial consultants. All sources and/or materials applied are listet and spec- ified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Würzburg, ………………………………………………… Saskia Bauriedl Declaration Hiermit erkläre ich an Eides statt, die Dissertation „Einfluss der Riboregulation auf Fitness und Virulenz von Neisseria meningitidis“ eigenständig, d.h. insbesondere selbstständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet zu haben. Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prü- fungsverfahren vorgelegen hat. Würzburg, den ………………………………………………… Saskia Bauriedl Contents 1. Summary ........................................................................................................................... 7 2. Zusammenfassung ............................................................................................................ 9 3. Introduction .................................................................................................................... 11 3.1. The pathogenic commensal Neisseria meningitidis ................................................... 11 3.2. Gene regulation in N. meningitidis ............................................................................ 12 3.3. RNA-mediated regulation of gene expression ........................................................... 13 3.4. Base-pairing small RNAs........................................................................................... 15 3.5. Trans-acting sRNAs that titrate proteins ................................................................... 19 3.6. RNA binding proteins (RBPs) required for the regulatory functions of sRNAs ....... 19 3.7. Scope of the study ...................................................................................................... 24 4. Materials .......................................................................................................................... 25 4.1. Laboratory equipment ................................................................................................ 25 4.2. Chemicals and consumables ...................................................................................... 27 4.3. Kits and enzymes ....................................................................................................... 29 4.4. Media and stock solutions .......................................................................................... 30 4.5. Gels, buffers and solutions ......................................................................................... 34 4.6. Strains ........................................................................................................................ 44 4.7. Plasmids ..................................................................................................................... 46 4.8. Oligonucleotids .......................................................................................................... 47 5. Methods ........................................................................................................................... 52 5.1. Cultivation of bacteria ................................................................................................ 52 5.2. Determination of the optical density at 600nm .......................................................... 52 5.3. Preperation of chemically compentent E. coli cells ................................................... 52 5.4. Transformation of E. coli ........................................................................................... 53 5.5. Transformation of N. meningitidis ............................................................................. 53 5.6. Preperation of meningococcal genomic DNA ........................................................... 53 5.7. Preperation of plasmid DNA...................................................................................... 54 5.8. Polymerase chain reaction (PCR) .............................................................................. 54 5.9. Agarose gel electrophoresis of DNA ......................................................................... 56 5.10. Sequencing of PCR products and plasmids ............................................................. 57 5.11. Cloning procedure: digestion of DNA, gel extraction and ligation ......................... 57 5.12. Mutant construction ................................................................................................. 58 5.13. Total RNA isolation with the hot phenol method .................................................... 65 5.14. DNase digestion ....................................................................................................... 66 5.15. RNA gel electrophoresis .......................................................................................... 66 5.16. Northern blot analysis for sRNA detection .............................................................. 66 5.17. Agarose northern blot for mRNA detection ............................................................. 67 5.18. Generation of radiolabelled DNA oligonucleotides for RNA detection .................. 68 5.19. Generation of radiolabelled RNA transcripts (riboprobes) for RNA detection ....... 69 5.20. Rifampicin stability assay ........................................................................................ 69 5.21. Differential RNA-sequencing .................................................................................. 69 5.22. ProQ UV-CLIP-sequencing ..................................................................................... 70 5.23. Analysis of crosslink-specific mutations (UV-CLIP) .............................................. 72 5.24. RNA-sequencing (RNA-seq) ................................................................................... 72 5.25. Differential expression analysis (RNA-seq) ............................................................ 73 5.26. Functional enrichment analysis ................................................................................ 73 5.27. In vitro transcription and 5’end labelling of RNA ................................................... 73 5.28. Gel Mobility Shift Assays ........................................................................................ 74 5.29. In-line probing assay ................................................................................................ 74 5.30. Coomassie staining and Western Blot analysis ....................................................... 75 5.31. Quantitative proteomics ........................................................................................... 75 5.32. Phenotypic characterization ..................................................................................... 77 5.33. Computational analyses ........................................................................................... 82 6. Results .............................................................................................................................. 84 6.1. The primary transcriptome of N. meningitidis strain 8013 ........................................ 84 6.2. The repertoire of meningococcal sRNAs ................................................................... 87 6.3. The repertoire of Hfq-associated RNAs in N. meningitidis strain 8013 .................... 94 6.4. The Hfq-associated sRNAs NMnc0017 and NMnc0018 regulate the putative colonization factor PrpB ................................................................................................. 100 6.5. ProQ is a second global RNA-binding protein in N. meningitidis ........................... 107 7. Discussion ...................................................................................................................... 137 7.1. An unexpected large repertoire of antisense TSS .................................................... 137 7.2. Contribution of sRNAs to genetic diversity and virulence of N. meningitidis ........ 138 7.3. Hfq as a major RNA binding protein in N. meningitidis ......................................... 140 7.4. Functional analysis of the Hfq-associated sRNAs NMnc0017 and NMnc0018 ...... 141 7.5. ProQ as a major regulator of bacterial 3’ends.........................................................
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