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Studies on the Chemotaxis Network in Halobacterium Salinarum and Helicobacter Pylori

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Studies on the Chemotaxis Network in Halobacterium Salinarum and Helicobacter Pylori Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Studies on the Chemotaxis Network in Halobacterium salinarum and Helicobacter pylori von Andreas Schaer aus Freiburg i. Br. 2002 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 selbstständig, ohne unerlaubte Hilfe angefertigt. München, den 18. August 2002. Dissertation eingereicht am: 30. August 2002 1. Berichterstatter: Hon.-Prof. Dr. D. Oesterhelt 2. Berichterstatter: Univ.-Prof. Dr. L.-O. Essen Tag der mündlichen Prüfung: 12. Februar 2003 Danksagung Die vorliegende Arbeit wurde am Max-Planck-Institut für Biochemie unter Anleitung von Prof. Dr. D. Oesterhelt angefertigt. Mein besonderer Dank gilt Herrn Prof. Dr. D. Oesterhelt für die Überlassung des sehr interessanten Themas, für sein reges Interesse am Fortgang der Arbeit und sein stetes Bemühen, optimale Arbeitsbedingungen zu schaffen. Darüber hinaus weiß ich das in mich gesetzte Vertrauen, die mir gewährte große wissenschaftliche Freiheit und die Hilfe beim Erstellen vorliegender Arbeit sehr zu schätzen. Ganz besonders möchte ich auch Herrn Prof. Dr. L.-O. Essen danken. Während der gesamten Promotionszeit stand er mir bei kleineren und größeren Problemen als Betreuer immer zur Seite. Ihm verdanke ich viele Anregungen, fruchtbare Diskussionen und Erläuterungen und die Einführung in viele biochemischen Arbeitsmethoden. Danken möchte ich auch allen Laborkollegen und Mitgliedern der Abteilung Membranbiochemie für die fröhliche Arbeitsatmosphäre, die so manchen Fehlschlag zu verkraften half. Mein herzlicher Dank gilt auch meinen Eltern sowie meinen Freunden, die nicht zuletzt durch ihre moralische Unterstützung wesentlich zum Gelingen dieser Arbeit beigetragen haben. Für meine Eltern 1. Contents 1 1. Contents 1. Contents................................................................................... 1 2. Abbreviations ............................................................................ 5 3. Introduction .............................................................................. 7 3.1. The E. coli chemotaxis network ................................................. 7 3.2. Novel chemotaxis proteins outside the Enterobacteriaceae ...........10 3.3. Halobacterium salinarum and Helicobacter pylori chemotaxis........13 3.3.1. Chemotaxis network in Halobacterium salinarum ....................13 3.3.2. Helicobacter pylori - Chemotaxis of an important human pathogen.....................................................................................16 4. Aims of this study .....................................................................19 5. Results ....................................................................................21 5.1. Identification and expression of soluble MCP homologs from H. salinarum ....................................................................................21 5.2. Purification of Car and Htr15....................................................24 5.3. Characterization of Car by CD spectroscopy ...............................26 5.4. Heat denaturation of Car .........................................................32 5.5. Characterization of Car by 1H-NMR............................................33 5.6. Crystallization trials for Car......................................................35 5.7. Expression, purification and crystallization trials with Car fragments....................................................................................36 5.8. Expression of H. salinarum CheB and CheR in E. coli ...................37 5.9. Soluble receptor homologs in C. jejuni and H. pylori....................38 5.10. Crystallization of chemotaxis components Hp0599, Cj0448 and CheW..........................................................................................42 5.11. The chemotaxis network in H. pylori........................................46 5.11.1. Modulation of CheF autophosphoylation activity .....................46 1. Contents 2 5.11.2. Phosphotransfer reactions from CheF to response regulators....57 5.11.3. Phosphate group transfer through the Che protein network......58 5.11.4. Autophosphorylation of CheF:D729K mutant..........................60 5.11.5. Phosphotransfer reactions from CheF:D729K-Pi to response regulators....................................................................................62 5.11.6. Decay of CheY-phosphate ...................................................67 5.11.7. Reverse phosphotransfer from CheY to the kinase ..................69 5.11.8. Phosphorylation of response regulators by acetyl phosphate ....72 5.12. Expression and purification of the motor switch protein FliM .......74 5.13. Detection of H. pylori chemotaxis proteins by antisera ...............79 6. Discussion................................................................................83 6.0. Soluble MCP homologues in two-component cascades .................83 6.1. Recombinant production of halophilic signaling components in E. coli ......................................................................................84 6.2. The arginine receptor Car - a molten globule like receptor? ..........85 6.3. Role of eubacterial soluble receptors .........................................87 6.4. Possible role of CheV proteins ..................................................94 6.5. The H. pylori motor and its switch ............................................99 6.6. Adaptation in biological networks and chemotaxis in H. pylori ..... 103 6.7. Gene regulation and adaptation in a stable environment ............ 105 7. Materials and Methods ............................................................. 109 7.1. Bacterial strains and plasmids ................................................ 109 7.2. General molecular biological techniques................................... 110 7.2.1. Culture of E. coli XL1 blue cells for plasmid growth ................. 110 7.2.2. Purification of plasmid DNA from E. coli cells ......................... 110 7.2.3. General PCR protocol for DNA amplification ........................... 111 7.2.4. Purification of DNA after enzymatic reactions......................... 111 7.2.5. Preparation of vector DNA for ligation reactions ..................... 112 7.2.6. Preparation of PCR-derived DNA fragments for ligation reactions ................................................................................... 112 1. Contents 3 7.2.7. Ligation of DNA molecules .................................................. 113 7.2.8. Restriction analysis of plasmid DNA...................................... 113 7.2.9. Analysis of DNA by agarose gel electrophoresis...................... 113 7.2.10. Sequencing of plasmid DNA............................................... 114 7.2.11. Preparation of short synthetic dsDNA linker ......................... 115 7.2.12. Construction of mutant genes by site-directed mutagenesis ... 115 7.2.13. Construction of mutant genes by PCR ................................. 116 7.2.14. Construction of chimeric FliM ............................................. 116 7.2.15. Synthesis of H. salinarum cheR and cheB genes ................... 117 7.2.16. Construction of N- and C-terminal fragments of Car.............. 119 7.2.17. Preparation of electrocompetent E. coli cells ........................ 120 7.2.18. Transformation of E. coli cells ............................................ 121 7.2.19. Primer design for PCR and sequencing reactions................... 121 7.3. Protein chemical methods...................................................... 122 7.3.1. Test for pET28a(+) directed gene expression......................... 122 7.3.2. Isolation of inclusion bodies from E. coli cells......................... 123 7.3.3. Heterologous expression of proteins in E. coli ........................ 123 7.3.4. Purification of overexpressed proteins from E. coli.................. 124 7.3.5. SDS-PAGE analysis of proteins ............................................ 128 7.3.6. Detection of proteins after SDS-PAGE by Coomassie brilliant blue............................................................................... 129 7.3.7. Detection of proteins after SDS-PAGE by silver staining .......... 129 7.3.8. Western transfer of proteins separated by SDS-PAGE to PVDF membrane................................................................................. 130 7.3.9. Immunodetection of proteins immobilized on PVDF membanes 131 7.3.10. Production of antisera against H. pylori chemotaxis proteins in rabbits ...................................................................................... 132 7.3.11. Protein concentration assay............................................... 133 7.4. Autophosphorylation and phosphotransfer reactions .................. 133 7.4.1. Synthesis of [32P] labeled dilithium acetyl phosphate .............. 133 1. Contents 4 7.4.2. Acyl phosphate assay......................................................... 134 7.4.3. Preparation of phosphorylated CheF:D729K........................... 134 7.4.4. Phosphotransfer assay from phosphorylated CheF:D729K to response regulators .................................................................... 135 7.4.5. Phosphotransfer assay from phosphorylated response regulator CheY:K106R to CheF:D729K ........................................................ 136 7.4.6. CheF autophosphorylation
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