Transcriptional regulation by sigma factor phosphorylation controls polymyxin resistance and swarming behavior in Vibrio parahaemolyticus DISSERTATION zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von SHANKAR CHANDRASHEKAR IYER aus Chennai, India Marburg/Lahn im Juni 2019 Originaldokument gespeichert auf dem Publikationsserver der Philipps-Universität Marburg http://archiv.ub.uni-marburg.de Dieses Werk bzw. Inhalt steht unter einer Creative Commons Namensnennung Keine kommerzielle Nutzung Weitergabe unter gleichen Bedingungen 3.0 Deutschland Lizenz. Die vollständige Lizenz finden Sie unter: http://creativecommons.org/licenses/by-nc-sa/3.0/de/ Die Untersuchungen zur vorliegenden Arbeit wurden von Oktober 2015 bis Juli 2019 am Max-Planck-Institut für Terrestrische Mikrobiologie unter der Leitung von Dr. Simon Ringgaard durchgeführ. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation angenommen am: Erstgutachter: Dr. Simon Ringgaard Zweitgutachter: Prof. Dr. Anke Becker Weitere Mitglieder der Prüfungskommission: Prof. Dr. Gert Bange Prof. Dr. Hans-Ulrich Mösch Tag der mündlichen Prüfung am: 06.09.2019 Die während der Promotien erzielten Erbebnisse wurden zum Teil in folgenden Originalpublikationen veröffentlicht: Shankar Chandrashekar Iyer, Delia Casas-Pastor, David Kraus, Petra Mann, Kathrin Schirner, Timo Glatter, Georg Fritz, and Simon Ringgaard Transcriptional regulation by σ factor phosphorylation in bacteria. Nature Microbiology. In revision. To Amma, Appa and my dear Kat Table of Contents _______________________________________________________________ Table of Contents ABBREVIATIONS ........................................................................................................... 11 ABSTRACT ..................................................................................................................... 13 ZUSAMMENFASSUNG .................................................................................................. 14 1. CHAPTER 1: INTRODUCTION .............................................................................. 15 1.1. Post-translational modifications ...................................................................... 16 1.1.1. Phosphorylation as a post-translational modification ............................... 17 1.2. Protein kinases................................................................................................ 18 1.2.1. Discovery of protein kinases in eukaryotes ................................................ 18 1.2.2. Discovery of protein kinases in bacteria ................................................... 19 1.2.3. Classification of kinases: Chemical nature of the side chains ................. 19 1.2.4. The high energy phosphate donor............................................................ 20 1.2.5. Protein kinases as drug targets ................................................................ 20 1.2.6. Structure of eukaryotic protein kinases .................................................... 20 1.2.6.1. cAPK has a bi-lobed structure ........................................................... 21 1.2.6.2. Distinct features of cAPK that are required for its activity................. 22 1.2.6.2.1. The catalytic domain .................................................................... 22 1.2.6.2.2. The activation loop ....................................................................... 22 1.2.7. Kinases in bacteria.................................................................................... 24 1.2.7.1. Two component systems (TCS) ........................................................ 24 1.2.7.2. Phosphotransferases......................................................................... 24 1.2.7.3. Serine/threonine kinases (STKs) ...................................................... 25 1.2.7.3.1. Comparison of STKs to TCS........................................................ 25 1.2.7.3.2. Structure of bacterial STKs .......................................................... 25 1.2.7.3.3. A model for allosteric activation within STKs in bacteria ............. 26 1.2.7.3.3.1. Ligand-based dimerization .................................................... 26 1.2.7.3.3.2. Activation through transphosphorylation .............................. 27 1.2.7.3.4. C-terminal domains of STKs ........................................................ 27 1.2.7.3.5. Evolution of STKs......................................................................... 28 1.2.7.3.6. Biological roles for Ser/Thr kinases ............................................. 28 1.2.7.3.6.1. Interaction with two component systems .............................. 29 1.2.7.3.6.2. Regulation of transcriptional factors ..................................... 30 1.2.7.3.6.3. Regulation of protein synthesis............................................. 31 1.2.7.3.6.4. Roles in cell wall synthesis ................................................... 31 1.2.7.3.6.5. Roles in cell morphology, division and development ........... 32 1.2.7.3.6.6. Roles in central metabolism .................................................. 34 1.2.7.3.6.7. Roles in virulence and pathogenicity .................................... 35 1.2.7.4. Dual specificity protein kinases ......................................................... 36 Table of Contents _______________________________________________________________ 1.3. Transcription in bacteria .................................................................................. 38 1.3.1. Components of the RNA polymerase ....................................................... 38 1.3.2. Structural studies on the RNA polymerase ............................................... 39 1.4. Sigma factors................................................................................................... 40 1.4.1. Discovery and key features of σ factors ................................................... 40 1.4.2. Sigma factors are important for promoter recognition .............................. 41 1.4.3. σ54 family of σ factors................................................................................. 41 1.4.4. σ70 family of σ factors ................................................................................ 42 1.4.4.1. Classification of the σ70 family ............................................................ 42 1.4.5. The different regions of σ factors .............................................................. 43 1.4.6. Interaction of the σ factor with the RNA polymerase ................................ 44 1.4.7. Molecular dynamics enabling promoter recognition and melting by σ factors 46 1.4.8. ECF sigma factors ..................................................................................... 47 1.4.8.1. Identification of ECF σ factors ........................................................... 47 1.4.8.2. Principle roles of ECF σ factors ......................................................... 48 1.4.8.3. Interactions of the ECF σ factor with the promoter DNA and the RNA polymerase .......................................................................................................... 49 1.4.8.4. Regulation of ECF σ factors .............................................................. 49 1.4.8.4.1. Regulated proteolysis ................................................................... 50 1.4.8.4.2. Conformational changes within the anti-σ factor ......................... 52 1.4.8.4.2.1. Mechanism of inhibition of ECF σ factors by anti-σ factors .. 53 1.4.8.4.3. Regulation by interactions with other proteins ............................. 54 1.4.8.4.4. Regulation by partner switching mechanism ............................... 55 1.4.8.4.5. C-terminal extensions within the σ factor ..................................... 57 1.4.8.4.6. Through transcriptional activation ................................................ 57 1.4.8.5. Classification of ECF σ factors .......................................................... 58 1.4.8.6. Functions of ECF σ factors ................................................................ 60 1.4.8.6.1. Pathogenecity ............................................................................... 60 1.4.8.6.2. Developmental processes ............................................................ 60 1.4.8.6.3. Responses to different extracellular stresses .............................. 61 1.4.8.6.3.1. Heat shock ............................................................................. 61 1.4.8.6.3.2. Iron starvation ........................................................................ 61 1.4.8.6.3.3. Antibiotic stress...................................................................... 61 1.4.8.6.3.4. ECF σ factors often sense multiple external stresses .......... 62 1.5. Vibrio parahaemolyticus as a model organism ............................................... 63 2. Chapter 2: Aim and Scope ...................................................................................... 65 3. Chapter 3: Regulation of ECF σ factor through phosphorylation in bacteria ......... 71 3.1. Introduction ...................................................................................................... 73 3.2. Results ............................................................................................................. 75 3.2.1. ECF σ factor phosphorylation in bacteria ................................................