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Small Regulatory Rnas Controlling Complex Phenotypes in Vibrio Cholerae

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Small Regulatory Rnas Controlling Complex Phenotypes in Vibrio Cholerae Small regulatory RNAs controlling complex phenotypes in Vibrio cholerae Dissertation Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Roman Herzog aus Augsburg München, Juli 2020 Diese Dissertation wurde angefertigt unter der Leitung von Prof. Dr. Kai Papenfort im Bereich von Department Biologie I an der Ludwig-Maximilians-Universität München Gutachter: 1. Prof. Dr. Kai Papenfort 2. Prof. Dr. Heinrich Jung Datum der Abgabe: 21.07.2020 Tag der mündlichen Prüfung: 15.12.2020 II Eidesstattliche Erklärung Ich versichere hiermit an Eides statt, dass die vorgelegte Dissertation von mir selbstständig und ohne unerlaubte Hilfe angefertigt wurde. Des Weiteren erkläre ich, dass ich nicht anderweitig ohne Erfolg versucht habe, eine Dissertation einzureichen oder mich der Doktorprüfung zu unterziehen. Die folgende Dissertation liegt weder ganz, noch in wesentlichen Teilen einer anderen Prüfungskommission vor. Roman Herzog, München, den 20. Juli 2020 Statutory Declaration I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources. As well I declare, that I have not submitted a dissertation without success and not passed the oral exam. The present dissertation (neither the entire dissertation nor parts) has not been presented to another examination board. Roman Herzog, Munich, 20 July 2020 III Contents Eidesstattliche Erklärung ........................................................................................................III Statutory Declaration .............................................................................................................III Contents ............................................................................................................................... IV Nomenclature ....................................................................................................................... VI Abbreviations ...................................................................................................................... VII List of Publications ................................................................................................................ XI Declaration of contributions ................................................................................................. XII Summary ............................................................................................................................. XV Zusammenfassung ............................................................................................................. XVII 1 Introduction ..................................................................................................................... 1 1.1 Post-transcriptional gene regulation in γ-proteobacteria ................................................. 1 1.1.1 The RNA chaperone Hfq ............................................................................................... 2 1.1.2 Gene-regulatory mechanisms of trans-encoded base-pairing sRNAs .......................... 4 1.2 The life-cycle of Vibrio cholerae ........................................................................................ 8 1.3 Quorum sensing in V. cholerae .......................................................................................... 8 1.3.1 Autoinducer molecules ................................................................................................. 8 1.3.2 Recognition and integration of quorum sensing signals ............................................ 10 1.3.3 AphA and the virulence cascade................................................................................. 12 1.4 Biofilm formation............................................................................................................. 13 1.5 Cell shape ......................................................................................................................... 14 1.6 Cell envelope stress responses ........................................................................................ 16 1.6.1 σE response ................................................................................................................. 16 1.6.2 Vxr response ............................................................................................................... 18 1.7 Aim of this work ............................................................................................................... 19 2 Three autoinducer molecules act in concert to control virulence gene expression in Vibrio cholerae ................................................................................................................................ 21 3 RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae ... 22 4 A conserved RNA seed-pairing domain directs small RNA-mediated stress resistance in enterobacteria ...................................................................................................................... 45 IV 5 Concluding discussion .................................................................................................... 82 5.1 The role of the DPO pathway in V. cholerae ................................................................... 83 5.1.1 Post-transcriptional control of the aphA messenger ................................................. 83 5.1.2 DPO-dependent gene regulation in V. cholerae ......................................................... 86 5.1.3 On the role of DPO and pyrazines as interspecies communication signals ............... 90 5.2 The role of sRNAs in the cell envelope stress response of V. cholerae ........................... 92 5.2.1 The VadR sRNA is a regulator of cell shape and biofilm formation and increases antibiotic tolerance ................................................................................................................. 93 5.2.2 The partially redundant MicV and VrrA sRNAs form the repressing arm of the σE response ................................................................................................................................. 96 5.3 Conclusions and outlook ................................................................................................. 98 References for Chapters 1 and 5 .......................................................................................... 100 Supplemental Information – Chapter 3 ................................................................................ 116 Danksagung ........................................................................................................................ XIX Curriculum Vitae .................................................................................................................. XX V Nomenclature Base-pairing interactions of an sRNA with an mRNA are indicated in the format sRNA/mRNA, e.g., VqmR/aphA. Numbers above sRNA sequences and promoter regions denote the distance relative to the transcriptional start site. Numbers above mRNA sequences indicate the relative distance to the translational start site. Gene deletions are indicated by the “∆” symbol, e.g., ΔvqmR. Genes or genomic fragments that follow a “p” are expressed from a plasmid, e.g., pVqmR. “P” indicates a promoter, e.g., PvqmR. VI Abbreviations (v/v) volume per volume (w/v) weight per volume [32P] phosphorus-32 °C degree Celsius A adenosine a.k.a. also known as A.U. arbitrary units A-22 S-(3,4-dichlorobenzyl) isothiourea AA aminoacetone AI autoinducer AI-2 autoinducer-2 AI-3 autoinducer-3 Ala-AA N-alanyl-aminoacetone Amp ampicillin AMP antimicrobial peptide ANOVA analysis of variance bp base-pair C cytosine CAI-1 cholera autoinducer-1 c-di-GMP cyclic diguanylate monophosphate cDNA complementary DNA CDS coding sequence Cef cefalexin CFU colony forming units Cm chloramphenicol Co-IP co-immunoprecipication conc. concentration CPM counts per million CT cholera toxin Da Dalton DNA deoxyribonucleic acid VII DNase deoxyribonuclease DPD 4,5-dihydroxy-2,3-pentanedionine DPO 3,5-dimethylpyrazin-2-ol dRNA-seq differential RNA-sequencing DTT dithiothreitol e.g. exempli gratia ECF extracytoplasmic function σ factor EDTA ethylene diamine tetraacetic acid ESR envelope stress response EtOH ethanol f.c. final concentration FDR false discovery rate fMet N-formylmethionine g gravitational force equivalent G guanosine GFP green fluorescent protein GO gene ontology h hour HCD high cell density IGR intergenic region Kan kanamycin kb kilobase-pairs LB lysogeny broth LCD low cell density LNA locked nucleic acid log2 binary logarithm LPS lipopolysaccharide M marker M.U. miller units MGO methylglyoxal min minute mRNA messenger RNA n number VIII N any nucleotide nt nucleotide n.a. not available OD600 optical density at 600 nanometer OMP outer membrane (β-barrel) protein ONPG O-nitrophenyl-ß-D-galactopyranoside PBP penicillin binding protein PBS phosphate buffered saline PCR polymerase chain reaction PenG penicillin G PG peptidoglycan qRT-PCR quantitative real-time PCR QS quorum sensing R purine nucleotide RBS ribosome binding site Rcs regulator of capsule synthesis RIP-seq RNA immunoprecipitation followed by high-throughput sequencing RNA ribonucleic acid RNAP RNA polymerase RNAPα alpha subunit of RNA polymerase RNase ribonuclease rpm revolutions per minute rRNA ribosomal RNA SAM (S)-adenosylmethionine SD standard deviation or Shine-Dalgarno SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
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