Characterization of different types of radiation- and pressure-induced DNA damage in Bacillus subtilis spores and their global transcriptional response during spore germination Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Ralf Möller aus Suhl / Deutschland Characterization of different types of radiation- and pressure-induced DNA damage in Bacillus subtilis spores and their global transcriptional response during spore germination Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Ralf Möller aus Suhl 1. Referent: Prof. Dr. Dieter Jahn 2. Referent: Prof. Dr. Erko Stackebrandt eingereicht am: 26.09.2007 mündliche Prüfung (Disputation) am: 18.12.2007 Druckjahr 2008 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: PUBLIKATIONEN Moeller, R., G. Horneck, R. Facius, and E. Stackebrandt. 2005. Role of pigmentation in protecting Bacillus sp. endospores against environmental UV radiation. FEMS Microbiol. Ecol. 51:231-236. Moeller, R., G. Horneck, P. Rettberg, H.-J. Mollenkopf, E. Stackebrandt, and W. L. Nicholson. 2006. A method for extracting RNA from dormant and germinating Bacillus subtilis strain 168 endospores. Curr. Microbiol. 53:227-231. Moeller, R., G. Horneck, U. Pogoda de la Vega, P. Rettberg, E. Stackebrandt, G. Reitz, R. Okayasu, and T. Berger. 2006. DNA damaging effects of heavy ion and X-ray irradiation of Bacillus subtilis spores. Int. J. Astrobiol. 5:87 (abstract). Moeller, R., E. Stackebrandt, G. Reitz, T. Berger, P. Rettberg, A. J. Doherty, G. Horneck, and W. L. Nicholson. 2007. Role of DNA repair by non-homologous end-joining (NHEJ) in Bacillus subtilis spore resistance to extreme dryness, mono- and polychromatic UV and ionizing radiation. J. Bacteriol. 189:3306-3311. Moeller, R., T. Douki, J. Cadet, E. Stackebrandt, W. L. Nicholson, P. Rettberg, G. Reitz, and G. Horneck. 2007. UV radiation induced formation of DNA bipyrimidine photoproducts in Bacillus subtilis endospores and their repair during germination. Int. Microbiol. 10:39-46. Moeller, R., E. Stackebrandt, T. Douki, J. Cadet, P. Rettberg, H.-J. Mollenkopf, G. Reitz, and G. Horneck. 2007. DNA bipyrimidine photoproduct repair and transcriptional response of UV-C irradiated Bacillus subtilis. Arch. Microbiol. (Epub ahead of print; DOI: 10.1007/s00203-007-0263-4). Stöffler, D., G. Horneck, S. Ott, U. Hornemann, C. S. Cockell, R. Moeller, C. Meyer, J. P. de Vera, J. Fritz, and N. A. Artemieva. 2007. Experimental evidence for the potential impact ejection of viable microorganisms from Mars and Mars-like planets. Icarus 186:585-588. Edwards, H. G. M., R. Moeller, S. E. Jorge Villar, G. Horneck, and E. Stackebrandt. 2006. Raman spectroscopic study of the photoprotection of extremophilic microbes against ultraviolet radiation. Int. J. Astrobiol. 5:313-318. Wassmann, M., R. Moeller, J. Nellen, G. Reitz, E. Rabbow, and P. Rettberg. 2007. Bacillus subtilis’ ability to adapt to extreme UV stress. Int. J. Astrobiol. 6:71 (abstract). Horneck, G., D. Stöffler, S. Ott, U. Hornemann, C. S. Cockell, R. Moeller, C. Meyer, J. P. de Vera, J. Fritz, S. Schade, and N. A. Artemieva. 2007. Microbial Rock Inhabitants Survive Hypervelocity Impacts on Mars-like Host Planets: First Phase of Lithopanspermia Experimentally Tested. Astrobiology (AST-2007-0134.R1; accepted for publication). TAGUNGSBEITRÄGE Moeller, R., G. Horneck, E. Stackebrandt, H. G. M. Edwards, and S. E. Jorge Villar. 2003. Do endogenous pigments protect Bacillus spores against UV-radiation? 3rd European Workshop on Exo-/Astrobiology, Mars: The search for Life, ESA SP-545:241-243 (abstract). Moeller, R., E. Stackebrandt, H. G. M. Edwards, and G. Horneck. 2004. Bacterial endospores stretch the limits of survivability. EMBO conference on Molecular Microbiology, Heidelberg, Germany, Topic: Exploring prokaryotic diversity, p. 97 (abstract). Moeller, R., and G. Horneck. 2004. Bacillus endospores - an ideal exobiological tool. Abstracts of the 35th Committee on Space Research (COSPAR) Scientific Assembly, Paris, France (COSPAR04-A-02596) (abstract). Moeller, R., G. Horneck, E. Stackebrandt, G. Reitz, P. Rettberg, U. Pogoda de la Vega, R. Okayasu, and T. Berger. 2005. DNA damaging effects of heavy ion and X-ray irradiation of Bacillus subtilis spores. 5th European Workshop on Exo-/Astrobiology, Budapest, Hungary (poster). Moeller, R., T. Berger, G. Reitz, and R. Okayasu. 2006. Gene activation of heavy ion treated Bacillus subtilis 168 (DSM 402) endospores during germination involved DNA-repair - first results. Annual Report of the Research Project with Heavy Ions at NIRS-HIMAC 2005/2006 (abstract). Moeller, R., G. Horneck, P. Rettberg, and E. Stackebrandt. 2006. Induced DNA damage by simulated space conditions in Bacillus subtilis endospores. DFG workshop “Viability in Space” Abstract book, p. 52-53, Cologne, Germany (abstract). Moeller, R., G. Horneck, P. Rettberg, and E. Stackebrandt. 2006. Gene activation of Bacillus subtilis after UV irradiation”, BIOspektrum (special edition, 2006), p. 77, Annual VAAM Meeting, Jena, Germany (abstract). Moeller, R., T. Berger, G. Reitz, M. Okada, and R. Okayasu. 2006 and 2007. Gene activation of heavy ion treated Bacillus subtilis 168 endospores during germination involved DNA- repair (17B-463). HIMAC Heavy Ion Researcher Investigator Meeting at NIRS, Inage, Japan (poster). Moeller, R., G. Horneck, D. Stöffler, S. Ott, U. Hornemann, C. Meyer, J. P. de Vera, and J. Fritz. 2006. Could microorganisms survive an impact ejection? Deutsche Physikalische Gesellschaft - Annual meeting in Heidelberg, Germany (abstract). Moeller, R., T. Berger, G. Reitz, and R. Okayasu. 2007. Gene activation of heavy ion treated Bacillus subtilis 168 (DSM 402) endospores during germination involved DNA-repair - site specific mutagenesis and oxidative DNA damage induction. Annual Report of the Research Project with Heavy Ions at NIRS-HIMAC 2006/2007 (abstract). Moeller, R., E. Stackebrandt, G. Horneck, T. Berger, P. Rettberg, R. Okayasu, and G. Reitz. 2007. NHEJ - a DNA strand breaks mechanism of Bacillus subtilis spores. BIOspektrum (special edition, 2007), p. 83; Annual VAAM Meeting, Osnabrück, Germany (abstract). “All truths are easy to understand once they are discovered; the point is to discover them.” (Galileo Galilei, 1564 - 1642) This thesis is dedicate to my mother (Marion Möller, 1949-2006). TABLE OF CONTENTS INTRODUCTION 1 1. Introduction 1 1.1 Bacterial endospores stretch the limits of life 1 1.2 Impact of sporulation on the life cycle of Bacillus subtilis 2 1.3 Factors contributing to spore resistance 5 1.4 Role of germination in spore’s survival 10 1.5 DNA repair systems 12 1.6 Aim of this thesis 19 MATERIAL AND METHODS 21 2. Material and methods 21 2.1 Cultivation of Bacillus subtilis and nucleic acids isolation 21 2.1.1 Media 21 2.1.2 Bacillus subtilis strains 22 2.1.3 Cultivation of vegetative cells 23 2.1.4 Sporulation, spore purification and storage 23 2.1.5 Assay for colony formation ability 23 2.1.6 Spore germination 24 2.1.7 DNA extraction 24 2.1.8 RNA isolation and quantification 25 2.2 Experimental conditions and exposure facilities 26 2.2.1 Experimental strategy for studying DNA repair processes in B. subtilis spores 27 2.2.2 Sample preparation 27 2.2.2.1 Air-dried spore (mono-)layers and sample recovery via “PVA-stripping” 27 2.2.2.2 Spores in aqueous suspension 28 2.2.3 UV irradiation 29 2.2.3.1 UV-C irradiation of B. subtilis spores 29 2.2.3.2 Polychromatic UV irradiation of B. subtilis spores 30 2.2.4 Exposure to ionizing radiation 31 2.2.4.1 X-rays 32 TABLE OF CONTENTS 2.2.4.2 Heavy ions irradiation 32 2.2.5 High vacuum exposure 33 2.2.6 Shock pressure experiments 34 2.2.6.1 Sample preparation and recovery of the shocked host rock and soil material 36 2.3 Characterization of induced DNA damage 39 2.3.1 Quantitative analysis of inter- (and intrastrand) bipyrimidine photoproducts 39 2.3.2 Determination of nucleobase modifications as oxidative DNA damage 42 2.3.3 Assays for mutation induction to rifampicin resistance 43 2.3.4 Sample requirements for studying the transcriptional profile 44 2.4 Gene expression analysis via cDNA microarray technology 44 2.4.1 Microarray fabrication and construction 45 2.4.2 Preparation of fluorescent-labeled cDNA 47 2.4.3 Microarray hybridization and washing 47 2.4.4 DNA microarray scanning and data processing 48 2.4.5 Statistical significance data analysis of differential gene expression 50 2.4.6 Microarray data interpretation 51 2.4.7 Comparative evaluation of the mRNA pool via quantitative RT-PCR 53 2.5 Numerical and statistical analysis 54 2.5.1 Survival curve analysis 55 2.5.2 Numerical analysis of the DNA photoproduct status 58 RESULTS 59 3. Results 59 3.1 Setting up the experiment protocol: spore germination and RNA isolation 59 3.1.1 Kinetics of spore germination of different Bacillus sp. strains 59 3.1.2 Quality control of the standard experimental protocol 62 3.1.3 RNA isolation from dormant, germinating and outgrowing B. subtilis spores 63 3.2 Responses to UV-C radiation 68 3.2.1 Survival and induction of DNA bipyrimidine photoproducts upon UV-C irradiation 68 3.2.2 Repair of DNA (UV-C) photolesions during spore germination 72 TABLE OF CONTENTS 3.2.3 Transcriptional profile of UV-C irradiated germinating B. subtilis spores 75 3.3 Responses to UV-A radiation 89 3.3.1 Survival of UV-A irradiated B.