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The Halobacterium Salinarum Taxis Signal Transduction Network: a Protein-Protein Interaction Study

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The Halobacterium Salinarum Taxis Signal Transduction Network: a Protein-Protein Interaction Study Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München The Halobacterium salinarum Taxis Signal Transduction Network: a Protein-Protein Interaction Study Matthias Schlesner aus Kiel 2008 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe angefertigt. Martinsried, am 07.10.2008 Matthias Schlesner Dissertation eingereicht am: 14.10.2008 1. Gutachter: Prof. Dr. Dieter Oesterhelt 2. Gutachter: Prof. Dr. Wolfgang Marwan Mündliche Prüfung am: 9.12.2008 Meiner Familie Contents Summary xvii 1 Background 1 1.1 H. salinarum, an archaeal model organism ................ 1 1.1.1 Halobacterium salinarum ...................... 1 1.1.2 Archaea ............................... 2 1.1.3 Halophiles and their ecology .................... 3 1.1.4 Adaptation to hypersaline environments ............. 4 1.1.5 Bioenergetics ............................ 5 1.2 Signal transduction and taxis in prokaryotes ............... 7 1.2.1 Two-component systems ...................... 8 1.2.2 The principles of prokaryotic taxis ................ 9 1.3 Protein-protein interaction analysis .................... 10 1.4 Objectives .................................. 12 2 Materials and methods 15 2.1 General materials .............................. 15 2.1.1 Instruments ............................. 15 2.1.2 Chemicals and Kits ......................... 15 2.1.3 Enzymes ............................... 15 2.1.4 Strains ................................ 17 2.1.5 Software ............................... 17 2.2 General methods .............................. 17 2.2.1 Growth and storage of E. coli ................... 17 2.2.2 Growth and storage of H. salinarum ................ 18 2.2.3 Separation of DNA fragments by agarose gel electrophoresis .. 18 2.2.4 Purification of DNA fragments ................... 18 2.2.5 Analytical and preparative restriction digestion ......... 18 2.2.6 Dephosphorylation of linearised plasmids ............. 19 2.2.7 Ligation ............................... 20 2.2.8 In-Fusion™ cloning ......................... 20 2.2.9 Gateway™ cloning ......................... 20 2.2.10 Transformation of E. coli ...................... 22 2.2.11 Transformation of H. salinarum .................. 23 2.2.12 Polymerase chain reaction (PCR) ................. 24 vii Contents 2.2.13 DNA sequencing .......................... 24 2.2.14 Isolation of plasmid DNA ..................... 25 2.2.15 Protein precipitation with TCA .................. 25 2.2.16 SDS PAGE ............................. 25 2.2.17 Coomassie staining of protein gels ................. 25 2.2.18 Silver staining of protein gels ................... 25 2.2.19 Western blot ............................ 26 2.2.20 Preparation of genomic DNA ................... 27 2.3 Materials and methods for yeast two-hybrid screening .......... 28 2.3.1 Growth and storage of S. cerevisiae ................ 28 2.3.2 Construction of two-hybrid expression plasmids ......... 28 2.3.3 Transformation of yeast ...................... 28 2.3.4 Protein interaction assay ...................... 31 2.4 Materials and methods for AP-MS of halobacterial protein complexes . 32 2.4.1 Construction of vectors ....................... 32 2.4.2 Generation of bait expression and control strains ........ 34 2.4.3 Establishing the affinity purification procedure .......... 35 2.4.3.1 Purification from E. coli ................. 35 2.4.3.2 Purification from H. salinarum ............. 36 2.4.4 Affinity purification of CBD-tagged proteins ........... 37 2.4.5 CBD-AP and SILAC: Direct bait fishing ............. 38 2.4.6 CBD-AP and SILAC: Indirect bait fishing ............ 38 2.4.7 Mass spectrometry: Sample preparation ............. 39 2.4.8 Mass spectrometry: Data acquisition ............... 41 2.4.9 Mass spectrometry: Data processing ............... 42 2.4.10 Determination of SILAC ratios .................. 42 2.4.11 Thresholds and statistics ...................... 44 2.5 Materials and methods for the chemotaxis protein interaction network . 44 2.5.1 Generation of expression and control strains ........... 44 2.5.2 Bait fishing, mass spectrometry, data analysis .......... 44 2.5.3 Che protein interactions in other organisms ........... 45 2.6 Materials and methods for identification of archaea-specific Che proteins 48 2.6.1 Construction of in frame deletion mutations ........... 48 2.6.2 Southern blot analysis ....................... 49 2.6.3 Complementation of deletions ................... 50 2.6.4 Swarm plates ............................ 50 2.6.5 Computerised cell tracking (Motion analysis) ........... 50 2.6.6 Dark-field microscopy ........................ 53 2.6.7 Bioinformatic analysis ....................... 53 viii Contents 3 Yeast two-hybrid analysis of halobacterial proteins 57 3.1 Introduction ................................. 57 3.2 Results and Discussion ........................... 58 3.2.1 Analysis of a test set of proteins .................. 58 3.2.2 Rescreening with higher stringency ................ 59 3.2.3 Halobacterial proteins and yeast transcriptional activation ... 60 3.3 Conclusions ................................. 61 4 Affinity purification and mass spectrometry of halobacterial protein com- plexes 63 4.1 Introduction ................................. 63 4.2 Results and Discussion ........................... 66 4.2.1 Construction of vectors ....................... 66 4.2.2 The purification procedure ..................... 68 4.2.2.1 Expression in E. coli ................... 68 4.2.2.2 Expression in H. salinarum ............... 70 4.2.3 Identification by mass spectrometry ................ 72 4.2.3.1 Identification by MALDI TOF PMF .......... 72 4.2.3.2 Identification by LC MS/MS .............. 74 4.2.4 SILAC: Discrimination of interaction partners from background 75 4.2.4.1 Direct bait fishing .................... 75 4.2.4.2 The exchange problem .................. 76 4.2.4.3 Indirect bait fishing ................... 78 4.2.4.4 Thresholds ........................ 79 4.3 Conclusions ................................. 80 5 The bioinformatics environment 83 5.1 Introduction ................................. 83 5.2 The databases ................................ 83 5.2.1 Bait DB ............................... 83 5.2.2 Experiment DB ........................... 85 5.2.3 Result DB .............................. 86 5.3 The applications .............................. 86 5.3.1 Bait management .......................... 87 5.3.2 Experiment management ...................... 88 5.3.3 Result evaluation .......................... 89 5.4 Conclusions ................................. 90 6 Chemotaxis protein interaction network 91 6.1 Introduction ................................. 91 6.1.1 The Che system: a specialised two-component system for taxis signalling .............................. 91 ix Contents 6.1.1.1 Signal reception and transduction ............ 91 6.1.1.2 Excitation ......................... 92 6.1.1.3 Adaptation ........................ 94 6.1.1.4 Signal termination .................... 95 6.1.1.5 Fumarate as switch factor ................ 95 6.1.2 The components of the Che system of H. salinarum ....... 96 6.2 Results and Discussion ........................... 99 6.2.1 Evaluation of experimental results ................. 100 6.2.1.1 Contaminants ....................... 100 6.2.1.2 Reproducibility ...................... 101 6.2.1.3 How to interprete the results .............. 102 6.2.2 The interaction network ...................... 103 6.2.2.1 The core: CheA, CheY, CheW1 ............. 103 6.2.2.2 Different interactions of the two CheW proteins .... 108 6.2.2.3 The transducers exhibit nonuniform interaction patterns110 6.2.2.4 Other Che Proteins .................... 112 6.2.2.5 Connectors: Hubs or sticky background ........ 113 6.2.2.6 Unexpected interactors .................. 115 6.2.2.7 Not connected: ParA1 .................. 116 6.2.3 Comparison with data from other organisms ........... 117 6.3 Conclusions ................................. 121 7 Identification of archaea-specific chemotaxis proteins 123 7.1 Introduction ................................. 123 7.1.1 The archaeal and bacterial flagellum are distinct structures ... 124 7.1.1.1 The bacterial flagellum .................. 124 7.1.1.2 The archaeal flagellum .................. 124 7.1.2 The flagellar motor switch is the target of CheY-P ........ 128 7.2 Results and Discussion ........................... 128 7.2.1 Interaction analysis revealed connectors of Che and Fla proteins 128 7.2.2 Construction of in-frame deletion mutants ............ 130 7.2.3 OE2401F and OE2402F are essential for chemotaxis and photo- taxis ................................. 131 7.2.4 ∆1, ∆2, and the double deletion ∆2-4 show almost 100% CW rotational bias ............................ 133 7.2.5 Interpretation of deletion phenotypes ............... 134 7.2.6 Complementation of deletions reverted their phenotype to that of wildtype ............................. 137 7.2.7 Bioinformatic analysis ....................... 137 7.2.7.1 Occurrence of che and fla genes in archaeal genomes . 138 7.2.7.2 Only few findings for OE2401F ............. 139 x Contents 7.2.7.3 OE2402F and OE2404R belong to a family of unique archaeal Che proteins .................. 140 7.3 Conclusions ................................. 145 8 Concluding remarks 147 9 Supplementary material 149 Bibliography 161 Appendix
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