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Investigations on Flagellar Biogenesis, Motility and Signal Transduction of Halobacterium Salinarum

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Investigations on Flagellar Biogenesis, Motility and Signal Transduction of Halobacterium Salinarum Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Investigations on Flagellar Biogenesis, Motility and Signal Transduction of Halobacterium salinarum Wilfried Staudinger aus Heilbronn 2007 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe angefertigt. München, am 22. April 2008 .................. Wilfried Staudinger Dissertation eingereicht am: 09.11.2007 1. Gutachter: Prof. Dr. Dieter Oesterhelt 2. Gutachter: Prof. Dr. Wolfgang Marwan Mündliche Prüfung am: 17.03.2008 This dissertation was generated at the Max Planck Institute of Biochemistry, in the De- partment of Membrane Biochemistry under the guidance of Prof. Dr. Dieter Oesterhelt. Parts of this work were published previously or are in preparation for publi- cation: Poster presentation at the Gordon Conference on Sensory Transduction In Micro- organisms, Ventura, CA, USA, January 22-27, 2006. Behavioral analysis of chemotaxis gene mutants from H. salinarum. del Rosario, R. C., Staudinger, W. F., Streif, S., Pfeiffer, F., Mendoza, E., and Oesterhelt D. (2007). Modeling the CheY(D10K,Y100W) H. salinarum mutant: sensitivity analysis al- lows choice of parameter to be modified in the phototaxis model. IET Systems Biology, 1(4):207-221. Koch, M. K., Staudinger, W. F., Siedler, F., and Oesterhelt, D. (2007). Physiological sites of deamidation and methyl esterification in sensory transducers of H. salinarum. In preparation. Streif, S., Staudinger, W. F., Joanidopoulos, K., Seel, M., Marwan, W., and Oesterhelt, D. (2007). Quantitative analysis of signal transduction in motile and phototactic archaea by computerized light stimulation and tracking. In preparation. Meinen Eltern “Die beste und sicherste Tarnung ist immer noch die blanke und nackte Wahrheit. Die glaubt niemand!” Max Frisch schweizer Schriftsteller (1911 - 1991) Contents 1 Summary 1 2 Introduction 5 2.1 The organism Halobacterium salinarum and its lifestyle . 5 2.1.1 Taxonomic classification . 5 2.1.2 The habitat of H. salinarum ..................... 6 2.1.3 Adaptation to hypersaline conditions . 7 2.1.4 Energy metabolism . 7 2.1.5 Morphology and swimming behavior of H. salinarum ....... 8 2.2 Structure and biogenesis of archaeal flagella . 10 2.2.1 Archaeal and bacterial flagella are only superficially similar . 10 2.2.2 Structure, function and assembly of bacterial flagella . 11 2.2.3 Structure and morphology of archaeal flagella . 11 2.2.4 Archaeal flagellar and type IV pili biogenesis share similarities . 13 2.3 Signal Transduction . 19 2.3.1 Two-component systems in prokaryotic signal transduction . 19 2.3.2 Bacterial chemotaxis as a paradigm of signal transduction . 20 2.3.3 Principles of prokaryotic taxis . 20 2.3.4 Chemotaxis in E. coli ........................ 21 2.3.5 Chemotaxis in B. subtilis ....................... 23 2.3.6 Chemo- and phototaxis of H. salinarum shares similarities with E. coli and B. subtilis chemotaxis . 29 2.3.7 Phototaxis in H. salinarum ..................... 30 2.3.8 A model for the H. salinarum motor switch and its photosensory control . 32 2.3.9 Bacterial and Archaeal Chemoreceptors . 33 2.3.10 Structure of transducers and transmembrane signaling . 36 2.3.11 Receptor clustering and the sensitivity paradox . 38 2.4 Objectives of the thesis . 41 3 Results and Discussion 43 3.1 Gene deletion as an approach to elucidate protein function in H. salinarum 43 3.1.1 General strategy to create in-frame deletions with subsequent com- plementation . 43 3.2 Investigations on flagellar biogenesis and motility of H. salinarum .... 48 3.2.1 Objectives . 48 3.2.2 The fla gene cluster of H. salinarum ................ 49 I Contents 3.2.3 Bioinformatic analysis of the proteins encoded in the fla gene cluster 51 3.2.4 Construction and genotypic analysis of fla gene knockout mutants 54 3.2.5 FlaH - and flaJ - mutants are devoid of flagella while flaD - and flaCE - mutants have less flagella . 55 3.2.6 Deletion of flgXXX has neither an effect on flagellar biosynthesis nor on motility . 70 3.2.7 Summary . 75 3.2.8 Conclusions and outlook . 76 3.3 Studies on phototaxis of H. salinarum wild type cells . 78 3.3.1 The response of H. salinarum cells to blue light pulses obeys the Bunsen Roscoe law of reciprocity . 78 3.4 Behavioral studies of H. salinarum cells deleted for chemotaxis genes . 82 3.4.1 The H. salinarum che operon . 82 3.4.2 Objectives . 83 3.4.3 Analysis of already available H. salinarum che mutants . 85 3.4.4 Bioinformatic analysis of the H. salinarum CheC proteins . 87 3.4.5 Generation and genotypic analysis of che gene knockout mutants 93 3.4.6 Generation and genotypic analysis of a cheY D10K,Y100W double mutant 93 3.4.7 Chemotaxis proteins influence the rotational bias of the H. sali- narum flagellar motor . 93 3.4.8 Phototaxis and spontaneous motor switching of H. salinarum che mutants ................................ 98 3.4.9 Chemotaxis of H. salinarum che mutants . 102 3.4.10 H. salinarum CheR is a methyltransferase and CheB is a methylesterase and glutamine deamidase . 104 3.4.11 Comparison of the phototactic and chemotactic responses of the che mutants suggests the existence of alternative Htr-mediated sig- naling pathways . 106 3.4.12 Is H. salinarum ParA1 involved in partitioning and localization of cytoplasmic transducer clusters? . 108 3.4.13 Interpretation of the switching frequencies and rotational biases and introduction of a modified motor model . 110 3.4.14 Outlook . 115 4 Materials and Methods 119 4.1 Chemicals . 119 4.2 Kits and Enzymes . 119 4.3 Microbiological materials and methods . 120 4.3.1 Strains and culture conditions . 120 4.3.2 Plasmids . 120 4.3.3 Media and antibiotics . 122 4.3.4 Transformation of E. coli ....................... 124 4.3.5 Transformation of H. salinarum ................... 124 4.4 Molecularbiological Methods . 127 4.4.1 Preparation of unpurified (“crude”) DNA from H. salinarum ... 127 II Contents 4.4.2 Preparation of plasmid DNA from E. coli ............. 127 4.4.3 Isolation of DNA fragments from agarose gels . 127 4.4.4 Determination of DNA concentration . 128 4.4.5 Sequencing of DNA . 128 4.4.6 Generation of PCR fragments and plasmid construction . 129 4.4.7 Southern Blot analysis . 131 4.5 Behavioral studies . 135 4.5.1 Swarm plate assay . 135 4.5.2 Computerized cell tracking (Motion Analysis) . 135 4.5.3 Dark-field microscopy . 141 4.6 Electron microscopy . 142 4.6.1 Growth, concentration and washing of the cells . 142 4.6.2 Preparation of grids and electron microscopy . 142 5 Appendix 143 5.1 Oligonucleotides . 143 5.2 Abbreviations . 145 5.3 Raw data . 147 References 150 Curriculum vitae 173 Danksagungen 175 III Contents IV List of Figures 2.1 Picture of a solar saltern on Lanzarote, Spain. 6 2.2 Electron micrographs of mono and bipolarly flagellated H. salinarum cells 9 2.3 Swimming modes observed in H. salinarum cells. 10 2.4 Sketch of the bacterial flagellum . 12 2.5 Schematic speculative representation of type IV pilus and archaeal flagellum 14 2.6 Proposed model for flagellin and S-layer N-linked glycan assembly and attachment in M. voltae ........................... 16 2.7 Schematic comparison of the organization of flagella-related protein genes in H. salinarum and M. voltae ........................ 18 2.8 Swimming behavior of E. coli ........................ 21 2.9 The CheC family and related proteins . 26 2.10 Comparison of E. coli and B. subtilis chemotaxis. 28 2.11 Photocycles of SRI and SRII . 31 2.12 Petri Net representation of the H. salinarum flagellar motor switch cycle 33 2.13 Overview of H. salinarum transducers and their involvement in taxis . 35 2.14 Schematic of the three transducer classes and structure of a class I transducer 37 2.15 Receptor clustering in prokaryotic chemotaxis . 39 3.1 Principle of the overlap-extension PCR . 45 3.2 Schematic diagram of first and second crossover . 47 3.3 Organization of flagella-related protein genes in H. salinarum and M. voltae with updated gene names . 50 3.4 Genotypic analysis of fla gene knockout strains . 56 3.5 Swimming tracks of motile and immotile H. salinarum cells . 57 3.6 Dark-field microscopic snapshots of swimming H. salinarum cells and super-flagella . 58 3.7 Swimming tracks of H. salinarum fla knockout- and complemented mutants 61 3.8 Swarm plates with deletions and complementations of flaJ......... 65 3.9 Swarm plate assay with mutants deleted for flaD ............. 65 3.10 Swarm plates with deletions and complementations of flaCE........ 66 3.11 Swarm plates of fla knockout strains in comparison to wild type . 66 3.12 Electron micrographs of negatively stained H. salinarum S9∆flaH cells . 67 3.13 Electron micrographs of negatively stained H. salinarum S9∆flaJ cells and S9∆flaJ /flaJ + cells . 68 3.14 Electron micrographs of negatively stained H. salinarum S9∆flaD cells . 69 3.15 Electron micrographs of negatively stained H. salinarum S9∆flaCE cells 69 V List of Figures 3.16 Sequence alignment of the known H. salinarum flagellins and the putative flagellin FlgXXX . 71 3.17 Genotypic analysis of the flgXXX knockout strain . 73 3.18 Swarm plate assay with flgXXX knockout mutants . 74 3.19 Dose-response curves of H. salinarum cells obtained with blue light pulses of 8 and 503 ms duration . 81 3.20 Validity of the Bunsen-Roscoe law for the response of H. salinarum cells to blue light pulses . 82 3.21 Schematic view of the H. salinarum motility and signal transduction (MO- ST) gene cluster .
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