Structures and functions of proteins that utilize and modify Wall Teichoic Acid Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Cengiz Koç aus Hanau / Hessen Tübingen 2016 Tag der mündlichen Qualifikation: Oktober/November 2016 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Thilo Stehle 2. Berichterstatter: PD Dr. Frank Essmann Table of Contents 1 Contents I ABBREVIATIONS .................................................................................... C II Summary (english) ..................................................................................... E III Summary (german) .................................................................................... F IV List of publications .................................................................................... H V Personal contribution .................................................................................. I 2 INTRODUCTION ................................................................... 1 2.1 Gram-positive Bacteria ............................................................. 1 2.1.1 Staphylococci ................................................................................ 2 2.1.1.1 -hemolysis and Coagulase-negative Staphylococci ......................... 2 2.1.1.2 MRSA & MSSA .............................................................................. 4 2.1.2 Actinobacteria ............................................................................... 5 2.1.3 Anaerobic Bacilli .......................................................................... 5 2.2 The Gram-Positive Cell Envelope and the Cell Wall ................ 7 2.2.1 Glycosyltransferases and biofilm ................................................... 9 2.2.2 Autolysins and Penicillin binding proteins (PBPs) ......................... 9 2.3 Wall teichoic acid (WTA) & lipoteichoic acid (LTA) .............. 12 2.3.1 WTA de novo biosynthesis .......................................................... 13 2.3.2 TarM/TarS glycosylation ............................................................. 14 2.3.3 Immune evasion via WTA alanylation ......................................... 15 2.4 Immunogenic relevance of cell surface molecules .................. 17 2.5 Mobile genetic elements (MGE) ............................................. 19 2.6 Bacteriophages ....................................................................... 21 2.6.1 Lytic cycle and lysogenic cycle ................................................... 22 2.6.2 The order Caudovirales ............................................................... 23 2.6.2.1 Siphoviridae .................................................................................. 23 2.6.2.2 Myoviridae .................................................................................... 24 2.6.3 S. aureus Pathogenicity Islands (SaPIs) ....................................... 26 2.6.4 Siphoviridial architecture ............................................................ 27 2.6.4.1 The siphoviridial tail end polymorphism ........................................ 27 2.6.4.2 Receptor binding proteins .............................................................. 29 2.6.4.3 Teichoic acids as receptors for bacterial viruses ............................. 30 2.7 Aims of Thesis ....................................................................... 31 3 RESULTS AND DISCUSSION ............................................ 32 3.1 TarM ...................................................................................... 32 3.1.1 Isolation and crystallization ......................................................... 32 A Table of Contents 3.1.2 Diffraction data collection and model refinement ........................ 32 3.1.3 Overall structure and domain organization of TarM ..................... 32 3.1.4 Structural and mutational analysis of the active site ..................... 33 3.1.5 DUF1975 .................................................................................... 34 3.2 11 Receptor Binding Protein Gp45 ....................................... 36 3.2.1 Characterization of Gp45 ............................................................ 36 3.2.2 HHPRED analyses of baseplate components ............................... 36 3.2.3 Isolation and crystallization ......................................................... 37 3.2.4 Diffraction data collection and phasing ....................................... 37 3.2.5 NCS and phase improvement ....................................................... 38 3.2.6 Gp45 structure (Rbp) .................................................................. 39 3.2.6.1 Stem domain ................................................................................. 39 3.2.6.2 Shoulder domain ........................................................................... 40 3.2.6.3 Head domain ................................................................................. 41 3.2.7 Negative staining electron microscopy of the 11 baseplate ........ 42 4 FUTURE RESEARCH .......................................................... 43 4.1 TarM ...................................................................................... 43 4.2 Gp45 ...................................................................................... 43 5 REFERENCES ...................................................................... 46 6 APPENDIX ........................................................................ 60 6.1 Gp45 crystal data and phasing statistics tables ....................... 60 6.2 Publications ........................................................................... 65 B Abbreviations I ABBREVIATIONS Amp ...................................................................................................................................................... Ampicillin ACP.................................................................................................................................. Acyl-carrier protein asu ............................................................................................................................................ Asymmetric unit CAMP ................................................................................................. Cationic anti-microbial peptide CD ........................................................................................................................................ Circular dichroism CoNS................. ............................................................................ Coagulase-negative Staphylococci cpm ............................................................................................................ Capsid morphogenesis (gene) DLS ....................................................................................................................... Dynamic light scattering DUF .......................................................................................................... Domain of unknown function EM .................................................................................................................................. Electron microscopy EOP ............................................................................................................................ Efficiency of plaquing G+C............................................................................................... Guanine+cytosine content of DNA Gal............................................................................................................................................................. Galactose Glc... .............................................................................................................................................................. Glucose GlcNAc ...................................................................................................................... N-acetylglucosamine Gp ..................................................................................................................................................... Gene product GroP... .................................................................................................................................Glycerolphosphate GT. .................................................................................................................................... Glycosyltransferase hgt............................................................................................................................ Horizontal gene transfer HMW ...................................................................................................................... High-molecular weight IMAC ..................................................................... Immobilized metal affinity chromatography LMW ........................................................................................................................ Low-molecular weight LPS ....................................................................................................................................Lipopolysaccharide LTA... ...................................................................................................................................... Lipoteichoic acid ManNAc ................................................................................................................