TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Mikrobiologie Elucidation of the Cell Division Mechanism and Characterization of Tubulins in the Bacterial Phylum Verrucomicrobia Martin Pilhofer Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. W. Höll Prüfer der Dissertation: 1. Univ.-Prof. (i. R.) Dr. K.-H. Schleifer 2. Univ.-Prof. Dr. W. Liebl 3. Priv.-Doz. Dr. G. Petroni (Università di Pisa, Italy) Die Dissertation wurde am 15.04.2008 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 29.05.2008 angenommen. meiner Familie Contents CONTENTS Abbreviations............................................................................................................ 7 Original Publications................................................................................................ 9 A General Introduction.................................................................................... 11 A.1 The Cytoskeleton: Eukaryotes versus Bacteria.......................................... 13 A.2 Bacterial Cell Division................................................................................. 21 A.3 The Bacterial Phylum Verrucomicrobia ...................................................... 25 A.4 Aims of This Study ..................................................................................... 33 B Results and Discussion............................................................................... 35 B.1 Development of the Two-Step Gene Walking Method................................ 37 B.2 23S rRNA Phylogeny of Verrucomicrobia : Inter- and Intraphylum Relationships ............................................................................................. 39 B.3 Detection of Tubulin Genes in Verrucomicrobia ......................................... 41 B.4 Cell Division in the PVC Superphylum ....................................................... 42 B.5 Characterization of Bacterial Tubulins........................................................ 50 B.6 Origin of Bacterial Tubulins ........................................................................ 71 C Summary....................................................................................................... 75 C.1 Summary.................................................................................................... 77 C.2 Zusammenfassung..................................................................................... 79 D References.................................................................................................... 81 Appendices ............................................................................................................. 97 Appendix A............................................................................................................ 99 Appendix B.......................................................................................................... 119 Appendix C ......................................................................................................... 131 Appendix D ......................................................................................................... 149 Appendix E.......................................................................................................... 177 Acknowledgments................................................................................................ 187 Curriculum Vitae................................................................................................... 191 Figures FIGURES Figure 1. The structure of a eukaryotic microtubule and its subunit [Alberts et al. (2002), modified]...................................................................................... 14 Figure 2. Structure comparison of tubulin homologs (Michie and Lowe 2006) ......... 16 Figure 3. Model of the septum and order of protein recruitment (Margolin 2005)..... 22 Figure 4. dcw gene cluster in different bacteria (Mingorance and Tamames 2004). 24 Figure 5. Epixenosomes and their host (Petroni et al. 2000).................................... 26 Figure 6. Stalked bacteria from Lake Alexander (Henrici and Johnson 1935).......... 28 Figure 7. Prosthecobacter debontii ........................................................................... 29 Figure 8. 16S rRNA tree showing the sister-phyla Verrucomicrobia and Lentisphaerae .......................................................................................... 31 Figure 9. SDS gel showing cultures overexpressing different btub -operon genes.... 54 Figure 10. Western hybridizations using antibodies specific for btub -operon gene products................................................................................................... 57 Figure 11. Determination of specificity for immunofluorescence staining ................. 59 Figure 12. Immunofluorescence staining of recombinant E. coli expressing btubA - btubB -bklc ................................................................................................ 61 Figure 13. Intracellular rod-like structure in recombinant E. coli expressing btubA - btubB -bklc ................................................................................................ 62 Figure 14. TEM micrographs of recombinant E. coli cells expressing the btub -operon ................................................................................................................. 64 Figure 15. TEM micrographs of P. debontii .............................................................. 66 Figure 16. Immunogoldstaining of Prosthecobacter cells ......................................... 68 Abbreviations ABBREVIATIONS AMP adenosine monophosphate ATP adenosine triphosphate bklc bacterial kinesin light chain bp basepair BSA bovine serum albumin btub bacterial tubulin Ca Candidatus cDNA complementary DNA CODEHOP consensus degenerate hybrid oligonucleotide primer Da dalton dcw division and cell wall ddl d-alanine d-alanine ligase DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen °C degree Celsius DNA deoxyribonucleic acid DTT dithiothreitol FRAP fluorescence recovery after photo bleaching fts filamentous temperature sensitive GDP guanosine diphosphate GFP green fluorescent protein GTP guanosine triphosphate h hour His histidine IF intermediate filament IgG immunoglobulin G IMG Integrated Microbial Genomes IPTG isopropyl-beta-D-thiogalactopyranosid kb kilobases kDa kilodaton klc kinesin light chain kV kilovolt 7 Abbreviations M molar µF microfarad µg microgram µm micrometer µM micromolar MT microtubule MTOC microtubule organizing center mg milligram ml milliliter min minute ng nanogram Ni nickel nm nanometer OD optical density PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction Pde Prosthecobacter debontii Pdj Prosthecobacter dejongeii rpm rounds per minute Pva Prosthecobacter vanneervenii PVC Planctomycetes-Verrucomicrobia-Chlamydiae PVDF polyvinylidene fluoride RNA ribonucleic acid rRNA ribosomal RNA RT reverse transcription / real time SDS sodiumdodecylsulphate TBS tris buffered saline TEM transmission electron microscopy TPR tetratricopeptide repeat V volt v/v volume/volume Vsp Verrucomicrobium spinosum w/v weight/volume 8 List of Original Publications ORIGINAL PUBLICATIONS Some main results of this study and the corresponding discussion, conclusions and materials and methods are described in detail in the publications listed below. The original articles and the corresponding author contributions can be found in the section Appendix A-D. Also, the study contains unpublished data for which additional materials and methods are specified in section Appendix E. The symbol at the beginning of a chapter designates that the chapter content was published as part of the indicated appendix / publication. Appendix A Pilhofer M, Rosati G, Ludwig W, Schleifer KH, Petroni G. Coexistence of tubulins and ftsZ in different Prosthecobacter species. Molecular Biology and Evolution 2007 Jul;24(7):1439-42. Appendix B Pilhofer M, Bauer AP, Schrallhammer M, Richter L, Ludwig W, Schleifer KH, Petroni G. Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method. Nucleic Acids Research 2007 Nov;35(20):e135. Appendix C Pilhofer M, Rappl K, Eckl C, Bauer AP, Ludwig W, Schleifer KH, Petroni G. Characterization and evolution of cell division and cell wall synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, Chlamydiae and Planctomycetes and phylogenetic comparison with rRNA genes. Journal of Bacteriology 2008 doi:10.1128/JB.01797-07. Appendix D Pilhofer M, Ludwig W, Schleifer KH, Petroni G. Phylogenetic relationships of bacterial tubulins within the Tubulin/FtsZ superfamily: Implications on their evolutionary origin Prepared for submission. 9 10 General Introduction A GENERAL INTRODUCTION 11 General Introduction 12 General Introduction A.1 The Cytoskeleton: Eukaryotes versus Bacteria In the last 15 years, our view of the bacterial cytoskeleton has basically changed. Before 1990, the cytoskeleton was thought to have evolved only in Eukaryotes. The “tea-bag model”, considering the bacterial cytoplasm unorganized and its constituents randomly