Interaction of the Marine Bacterium Marinobacter adhaerens HP15 with the Diatom Thalassiosira weissflogii Analyzed by Proteomics Approaches by Antje Stahl A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Microbiology Dissertation Committee Prof. Dr. Matthias Ullrich Jacobs University Bremen Prof. Dr. Frank Oliver Glöckner Jacobs University Bremen Prof. Dr. Jens Harder Max-Planck-Institute for Marine Microbiology Date of Defense: December 8, 2015 Contents Contents Acknowledgements ............................................................................................................................ III Summary ............................................................................................................................................... V List of Abbreviations ......................................................................................................................... VII 1. Introduction ................................................................................................................................ 1 1.1. Oceanic carbon cycling ........................................................................................................... 1 1.1.1. Carbon fluxes and the biological carbon pump ............................................................... 4 1.1.2. Features of marine aggregates and marine snow ............................................................. 4 1.1.3. Transparent exopolymer particles trigger aggregate formation ....................................... 6 1.2. Bacteria-diatom interactions .................................................................................................... 8 1.2.1. Diatoms are colonized by bacteria................................................................................... 8 1.2.2. Bacteria-diatom interactions and the global carbon cycling ............................................ 9 1.2.3. Bacterial chemotaxis promotes the encountering of nutritional hot spots ....................... 9 1.2.4. Bacteria-diatom interaction on a molecular level .......................................................... 11 1.2.5. Marinobacter adhaerens HP15 – Thalassiosira weissflogii : A model system ................. to understand bacteria-diatom interactions .................................................................... 14 1.3. Heavy metal resistance in bacteria ........................................................................................ 17 1.3.1. Bacterial strategies to cope with environmental heavy metal stress .............................. 17 1.3.2. Marinobacter – a genus inhabiting environments, enriched in heavy metals? ............. 19 2. Aims of the Present Study ...................................................................................................... 21 3. Results ......................................................................................................................................... 23 3.1. Proteomics analysis focusing the interaction of the marine bacterium Marinobacter adhaerens HP15 with the diatom Thalassiosira weissflogii .......................... 25 3.2. Stereo-tracking of chemosensing-deficient and motility impaired Marinobacter adhaerens HP15 strains during marine particle colonization – a novel methodical approach ............... 51 3.3. Marinobacter adhaerens HP15 harbors two CzcCBA efflux pumps involved in zinc detoxification ................................................................................................................. 69 4. Discussion and Future Scope ......................................................................................... 93 4.1. Understanding Marinobacter adhaerens HP15 – Thalassiosira weissflogii interaction and suggestions on methodical improvements ...................................................................... 93 4.2. Chemotaxis and the colonization of marine particles ............................................................ 99 4.3. Heavy metals resistance in Marinobacter adhaerens HP15 and further genus members ... 101 5. References ..................................................................................................................... 103 6. Supplementary – Tables and Figures ......................................................................... 115 Statutory Declaration .............................................................................................................. 127 I II Acknowledgements Acknowledgements I would like to thank my main supervisor Prof. D. Matthias Ullrich as well as my thesis committee members Dr. Katja Metfies, Prof. Dr. Frank Oliver Glöckner, and Prof. Dr. Jens Harder for continuous guidance, recommendations, and reflection of my work during the last three years. I also thank all present and former members of the Ullrich Lab for sharing the last years in a very friendly and diverse working atmosphere. Particularly I would like to mention my ‘PhD student batch’ Khaled Abdallah and Amna Mehmood, furthermore Mariann Árkosi, Anja Kamp, and Daniel Pletzer as well as our lab technicians Nina Böttcher und Maike Last whose effort allows us lab members to fully focus on our work. The ‘Fritten-Freitag-Gang‘ is thanked for sharing many fried lunches, conversations apart from the usual PhD business, and simply good times on campus. I further express my gratitude to the ‘Graduate School for Polar and Marine Research’ (POLMAR) which supported me financially over the last three years. In particular I would like to emphasize the travel options I gained due to POLMAR, enabling outstanding trips to a variety of conferences and events. Here I would also like to mention Dr. Claudia Hanfland and Dr. Claudia Sprengel as coordinators of the graduate school as well as their associated colleagues. Last but highly important I thank my family, friends, and boyfriend for guidance, support, and understanding over the last years. III IV Summary Summary Bacteria and microalgae conduct close mutualistic interactions that might impact metabolic and physiological features in both partners. As microalgae play major roles in global primary production and carbon cycling in marine systems, knowledge about such interactions is directly linked to an understanding of large-scale carbon and nutrient turnover processes. The impact of bacteria that colonize microalgae has been the focus of a number of studies. However, an understanding of the basis of such interactions at an advanced molecular level, including the identification of ‘traded items’ between the interacting partners, remained scarce. In the present study, we analyzed a specific beneficial bacteria-diatom interaction by application of proteomics approaches. A bilateral model system formerly established for the analysis of such interactions was used, consisting of the marine γ-proteobacterium Marinobacter adhaerens HP15 and the ubiquitously occurring centric diatom Thalassiosira weissflogii. The present study focused on the proteome of M. adhaerens HP15. Alterations in the bacterial proteome during co-cultivation with the diatom in comparison to a reference treatment were identified. Results imply that free amino acids are probable those traded items provided by the diatom, taken up by the bacterium, and potentially used as carbon and nitrogen source. Furthermore, proteomic results indicated a favorable supply with nutrients, particularly with nitrogen. For prospective studies, we suggest including the analysis of the diatom’s proteome and recommend the application of proteomics in combination with metabolomic approaches. For bacteria encountering and attaching to microalgae and other marine particles, the ability of chemotaxis, combining chemosensing and motility is beneficial. Existing mutants of M. adhaerens HP15 lacking chemotaxis features like motility response regulators or flagella were used. As part of the present thesis, we generated transformants that allow stereo-detection via differential fluorescence emission, in order to distinguish wild type and chemotaxis-impaired mutants in comparative studies. This advanced tool allows an easy analysis and quantification of effects of chemotaxis features on particle colonization. As a further topic of this thesis, features of heavy metal resistance in M. adhaerens HP15 and other members of the genus Marinobacter were analyzed. The genus Marinobacter has only recently been suggested as a bacterial taxon inhabiting metal-enriched environments and potentially impacting biogeochemical cycling of metals. M. adhaerens HP15 encodes two CzcCBA efflux pumps assumed to facilitate resistance towards cadmium, zinc, and cobalt. Single and double knock-out mutants of the representative genes were generated and V Summary phenotypically characterized. Results showed that both pumps play a role in zinc tolerance but not in resistance towards cadmium and cobalt, respectively. In some but not all of the analyzed Marinobacter genomes czcCBA clusters were identified, substantiating the hypothesis of an association with environments containing increased levels of heavy metals. The actual role of the genus Marinobacter in biogeochemical cycling and its feature to inhabit heavy metal enriched environments remains to be determined in future studies. VI List of Abbreviations List of Abbreviations 5%-Suc 5% sucrose 5-ALA 5-aminolevulinic acid Ap ampicillin ASW artificial sea water bp base pairs BLAST Basic Local Alignment Search Tool BSA bovine serum albumin