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Bacillus Subtilis Biofilm Formation Under Extreme Terrestrial and Simulated Extraterrestrial Conditions Dissertation

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Bacillus Subtilis Biofilm Formation Under Extreme Terrestrial and Simulated Extraterrestrial Conditions Dissertation Bacillus subtilis biofilm formation under extreme terrestrial and simulated extraterrestrial conditions Dissertation to obtain the Ph. D. degree "Doctor rerum naturalium" (Dr. rer. nat.) at the Faculty of Biology and Psychology at the Georg-August-University Göttingen, Germany in frame of the doctoral program Georg-August University School of Science (GAUSS) - Biology submitted by Felix Matthias Fuchs Born in Münchberg on 10th November 1988 Göttingen, February 2020 II Thesis Advisory Committee Prof. Dr. Jörg Stülke Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University of Göttingen Prof. Dr. Ralf Möller Space Microbiology Research Group, Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center Cologne Prof. Dr. Fabian M. Commichau Synthetic Biology, Institute of Biotechnology, Brandenburg University of Technology Cottbus - Senftenberg Members of the examination board First reviewer: Prof. Dr. Jörg Stülke Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University of Göttingen Second reviewer: Prof. Dr. Ralf Möller Space Microbiology Research Group, Radiation Biology Department, Institute of Aerospace Medicine, German Aerospace Center Cologne Additional members of the examination board: Prof. Dr. Stefanie Pöggeler Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University of Göttingen Prof. Dr. Ivo Feussner Department of Plant Biochemistry, Albrecht von Haller Institute, Georg-August University of Göttingen PD Dr. Michael Hoppert Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University of Göttingen Dr. Heiko Liesegang Department of Genomic and Applied Microbiology, Institute of Microbiology and Genetics, Georg-August University of Göttingen Date of Disputation: 17th April 2020 ___________________________________________________________ III B. subtilis biofilms - a matter of the heart For biofilm formation, 1x106 spores of B. subtilis NCIB 3610 were inoculated on SSM-medium. Scale bar represents 5 mm. - It's the little things that make you happy - IV “Now my own suspicion is that the Universe is not only queerer than we suppose, but queerer than we can suppose.” -John Burdon Sanderson Haldane (1927) For my parents V Acknowledgements First of all, I would like to express my deep gratitude to Prof. Dr. Ralf Möller for his supervision, friendship and for the unbelievably versatile possibilities that I was offered as a PhD-student at the DLR. All the conferences, workshops, courses, travels and collaborations added up to a unique experience, for which I am grateful. I kindly would like to thank Prof. Dr. Jörg Stülke, who supervised me as external student and introduced me to the University in Göttingen despite the distance to Cologne. I want to thank PD Dr. Michael Hoppert, Prof. Dr. Pöggeler, Dr. Heiko Liesegang and Prof. Dr. Feussner for examining my thesis as well as Prof. Dr. Fabian Commichau, which supervised me during my TAC-Meetings. While being in Göttingen during an internship course, I really enjoyed the collegial atmosphere and the inclusion to the team. Futhermore, I would like to thank PD Dr. Christine Hellweg and Dr. Günter Reitz, who always supported me as department leaders. A special thanks goes to the DLR & Helmholtz Research School SpaceLife, which provided me a SpaceLife scholarship and therewith the possibility to study in the field of space microbiology. Furthermore, I would like to thank PD Dr. Ruth Hemmersbach as my mentor and for her support from the gravitational biology as well as her colleagues Christian Liemersdorf, Yannick Lichterfeld and Timo Frett. From the radiation biology department, I would like to thank Claudia Schmitz & Sebastian Feles for excellent support, Dr. Sebastian Diegeler for being a good friend and comrade, André Parpart & Carina Fink for our discussions, Dr. Petra Rettberg for her support, Thomas Urlings for his informatics expertise, Dr. Corinna Panitz for our discussions as well as Andrea Schröder, who helped me with thousands of plates, incubation and so many other lab-things - thank you all. I really loved and enjoyed working in the space microbiology working group and I want to thank all of you, whether alumni or present, namely Katharina Siems, Erika Muratov, Stella Koch, Dr. Katja Nagler, Dr. Marina Pyc, Marny Hoef-Emden, Nikea Ulrich, Bahar Djouiai, Aram de Haas, Tim Erler, Florian Rosenbaum and Marta Cortesão (+Tiago Monteiro) for the great time we shared all around Europe. I also want to thank Britta Rowehl, Dagmar Koschnitzki, Claudia Hahn, Dr. Karel Marsalek, Bartos Przybyla, Dr. Kai Schenetten and all of my students and interns. I would like to thank the DGE (Deutsche Gesellschaft für Elektronenmikroskopie) for the financial support to work at the Robert Koch Institute (RKI). In this regard, I would like to send out my special thanks to Dr. Michael Laue for the realization of my project, Gudrun Holland & Janett Piesker & Kazimierz Madela for their excellent technical support and our discussion as well as Dr. Christoph Schaudinn for his biofilm expertise. Many thanks to Prof. Dr. Ákos Kovács for his comments and for providing several mutant strains, as well as PD Dr. Madeleine Opitz for the support of several mutants and Samir Giri for the ΔlysA-strain. Furthermore, my special thanks go to Dr. Akira Fujimori & Hirokazu Hirakawa for the support at the HIMAC facility in Japan and JAXA for funding the research trip (STARLIFE project, 13J301, insight-REPAIR, 14J410, “Living in Space” grant) as well as Dr. Daisuke Niwa for introducing me to Japan. I would like to thank Prof. Dr. Lieleg and Carolina Falcón Garcia for realizing the biofilm surface topography studies on biofilms. A very special thanks to all those who supported me in bioinformatics and other experiments: Dr. Sevasti Filippidou, Dr. Bentley Shuster, Prof. Dr. Patrick Eichenberger, Prof. Dr. Peter Setlow, Prof. Dr. Yong-Qing Li, Dr. Anne de Jong, Dr. Jan-Wilm Lackmann, Dr. Marcel Fiebrandt and Michael Morrison. For so many other reasons I want to thank Diana and Dr. Jens Boy, all BioRockers, the Betty-Crew, Britta Osterkamp, Prof. Andrew Gibson, Maximilian Wache, David Mahlberg and Constanza & Magnus von Abercron. Also thanks to all those I forgot to mention, so many personalities have helped and supported me – partwise work-related partwise with other extremely useful and motivating things – thank you all! Further, I would like to thank my family, who supported me all my life: Thank you Mama (Andrea), Papa (Andreas), Jonas, Fiona and Madita Fuchs as well as my grandparents - for your love and dedication. Finally, I would like to thank my wife Felicitas for her infinite love and dedication and our daughter Laetitia for motivating me. - Felix Fuchs, February 2020 VI Affidavit Here I declare that my doctoral thesis entitled “Bacillus subtilis biofilm formation under extreme terrestrial and simulated extraterrestrial conditions” has been written independently with no other sources and aids than quoted. Signature _________________________ Felix Matthias Fuchs Göttingen, February 2020 VII Table of contents 1. Introduction...................................................................................................................... - 1 - Preface: Life ......................................................................................................................... - 1 - Space microbiology ............................................................................................................. - 1 - Microbial burden in space ................................................................................................... - 2 - Bacillus subtilis as model organism ..................................................................................... - 7 - Simulation of space conditions ......................................................................................... - 14 - Relevance and Hypotheses ............................................................................................... - 17 - 2. Material & Methods .........................................................................................................- 18 - Bacterial strains ................................................................................................................. - 18 - Sporulation and spore purification.................................................................................... - 25 - Cultivation of standardized biofilms.................................................................................. - 27 - Simulation of microgravity (sim-µg) .................................................................................. - 29 - Microscopy ........................................................................................................................ - 34 - Survival experiments ......................................................................................................... - 37 - Germination assays ........................................................................................................... - 39 - Molecular biology methods ............................................................................................... - 39 - Statistics, data visualization and other software .............................................................. - 47 - 3. Results .............................................................................................................................-
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