Proteomics and Microbiology Faculty of Sciences University of Mons, UMONS Mons, Belgium Deciphering metal-impacts on synthetic and natural microbial communities Dissertation submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Biological Sciences by Valentine Cyriaque This work was promoted and funded by the Fond National de Recherche Scientifique (F.R.S.-FNRS) Jury: Pr. Ruddy Wattiez –UMONS Director of Thesis Pr. David C. Gillan– UMONS Co- Director of Thesis Pr. Patrick Flammang – UMONS Academic President of the Jury Pr. Søren Sørensen – University of Copenhagen External Reviewer Pr. Dirk Springael – KU Leuven External Reviewer Pr. Isabelle George – ULB External Reviewer Cover: Tyson Dudley “Don't waste your hate Rather gather and create Be of service, be a sensible person Use your words and don't be nervous You can do this, you've got purpose Find your medicine and use it” Nahko, Manisfesto “Sono pessimista con l'intelligenza, ma ottimista per la volontà.” “Pessimiste par l’intelligence mais optimiste par la volonté.” “Pessimistic by intelligence but optimistic by the will.” Antonio Gramsci, Prison letter (1929) Summary Metal contamination poses biotoxicity and bioaccumulation issues, affecting both abiotic conditions and biological activity in ecosystems. The use of metals and metalloids as raw materials, in industries and technologies drastically increased from the industrial revolution and urbanization of the XVIIIth century. For 100 years (1893-2003), the MetalEurop foundry released zinc, copper, cadmium and lead directly into the river “la Deûle”, resulting in up to 30- fold increase in metal concentrations in downstream sediments. We used an integrative approach coupling in-situ 16Sr RNA sequencing from both DNA and RNA extracts, microcosm supervision and Horizontal Gene Transfer (HGT) monitoring in order to fully understand the mechanisms driving community resilience to metal pollution. We applied the ecological concept of Functional Response Groups (FRGs) to decipher the adaptive tolerance range of the in-situ sediment communities through characterization of microbial strategists, revealing differences in diversity and composition. Furthermore, in-vitro microcosm analysis with upstream non-polluted sediments challenged with metals and daily supplied of fresh river water, allowed to monitor the short-term impact of metal pollution on the microbial community over 6 months, in controlled conditions. We used qPCR and 16S rRNA gene amplicon sequencing with the ecological concept of Treatment Response Groups (TRGs). Both in-situ and microcosm studies reinforced the notion that mechanisms are taking place at the community level to face the metal pollution, such as facilitation processes and microbial community coalescence leading to an unexpectly high microbial diversity. However, if in-situ results suggest HGT as a key process in the long-term resilience of the community, the monitoring of IncP plasmid by qPCR in microcosms revealed a negative effect of metals on the short-term in comparison with control microcosms. Deeper HGT analysis using qPCR revealed the presence of an enriched native pool of conjugative IncP plasmids in the MetalEurop polluted sediments, confirming their importance for long-term adaptation of the community facing metal contamination. Furthermore, in-vitro conjugation assays coupled to Fluorescence Activated Cell Sorting (FACS) allowed to assess the plasmid transfer rate and permissiveness in Férin and MetalEurop communities showing sediments as hotspot for plasmid transfer. To understand the direct metal-impact on plasmid persistence in a microbial community, we used a consortium conjugative assay to monitor the dispersion efficiency of the plasmid in the recipient strain population by flow-cytometry and link it to the metaproteomic profile of the consortium by SWATH quantitative proteomics. I Acknowledgements When time comes to write acknowledgements, the thesis can be considered with the benefit of hindsight. When I attended my first class in biology, I’d never thought I would now dive into the “micro”. Happy thing, I have now realized how bacteria are fascinating (and I hope this work will convince you about that). This PhD was the continuity of 5 years of permanent exploration and celebration of life in all its forms. Nine years in the scientific community, ups and downs were deeply rewarding, scientifically and humanely. Then, first and foremost, I would express my deep gratitude to my supervisor, the Pr. Ruddy Wattiez, “Chef”, who gave me the opportunity of doing this PhD. Thank you so much for your support and your precious time during these 4 years. I would thank my co-supervisor, Pr. David Gillan for his very careful readings. Of course, I would thank the ProtMic team for these years spent together. To Melanie for having watched over my work during my master thesis and beyond, for having taught me the tricks! To Augustin, as a master student first, and as a colleague. To Giuseppe, for all these hours spent at the bench with me combined with joyful conversations. To Cyril, for his help at the bench and long anticipatory conversations. To Baptiste and Corentin for their help with mass spectrometry. To Catherine for making sure everything was possible! To Clotilde, Quentin, Fred, Jérémy and Samia who integrated me into the team. To Guillaume, Neha, Corentin and Kurt for the fun in the office. To Alice, Camille, Angela, Haixia, Paloma, Camille, Ghenima, Catherine, Baptiste, Vincent, Médéric, Grégoire, for the daily good mood, fun and kindness! I would also express my thankfulness to Pr. Søren Sørensen for welcoming me in his lab. I was a fresh PhD student back then, and these 6 months have been rich in experience and encounters. I would also then thank the section of microbiology, Mette, Anders, Leise, Luma, Anette, Stephan, Tim, Trine, Ines, Whenzeng, Martin, Sten, Annelise, Whenzeng, Henriette, and Jakob for having warmly welcomed me in their team. Special thanks to Jonas for mentoring me during these 6 months, to Dingrong, Shaodong, Milena, Rafa, Jakob and Urvish, colleagues and friends, and to Sam for being a mentor and taking me under his wing. I would also thank the LASIR team, especially Gabriel Billon for the help with sediments, and the GIGA, especially Laurence Fievez, for the help with the flow cytometry. Thank you both for your availability, professionalism and kindness II I would also express my gratitude to the members of the jury who carefully reviewed this work. I wish to thank the FNRS fellowship for the financial support. Pendant mon parcours universitaire, j’ai eu la chance de suivre des cours donnés par des profs passionnants. Parmi eux, je voudrais remercier le Pr. Rasmont et sa passion contagieuse pour l’écologie. Je voudrais remercier Julie et Baptiste pour les fins de rapport nocturnes, les blocus et les échanges d’insectes. Ces 5 années n’auraient pas été pareilles sans l’entraide, l’amitié et les rires. Merci à Pierre, Mathilde, Edwicka et Geoffrey. Comprendre la vie est probablement le plus beau métier du monde. “First of all, the beauty that he [the artist] sees is available to other people and to me, too, I believe, although I might not be quite as refined aesthetically as he is. But I can appreciate the beauty of a flower. At the same time, I see much more about the flower that he sees. I could imagine the cells in there, the complicated actions inside which also have a beauty. I mean, it's not just beauty at this dimension of one centimetre: there is also beauty at a smaller dimension, the inner structure... also the processes. The fact that the colours in the flower are evolved in order to attract insects to pollinate it is interesting - it means that insects can see the colour. It adds a question - does this aesthetic sense also exist in the lower forms that are... why is it aesthetic, all kinds of interesting questions which a science knowledge only adds to the excitement and mystery and the awe of a flower.” Richard Feynman, 1981. Apprendre devient alors tellement excitant. A chaque étape, des noms restent. Merci à Mme Gaëtane, Mme Dewinck, Mme Blondel, Mme Vandromme et Mme Wanuffel. A mes amis. Ils sont ces lanternes colorées sur mon chemin. Merci à Sarah, Julie, Prési, Nico, Eme, Thom, Galaad, Dilan, Do, Pierre, Simon, Théo, Camille, Anto, Matthieu, Baptiste. A Antchoiine (Viens on y va !). III Last but not least, Papa, Maman, merci pour votre soutien inestimable, votre amour et votre fierté qui me poussent en avant. Merci pour les balades en forêt, pour les livres, pour avoir cultivé ma curiosité ! A Alex pour la patience, pour les rires et les jeux. Aloha IV Table of Content Summary…………………………………………………………………………….…………... I Acknowledgements……………………………………………………………………………… II Table of content……………………………………………………………………….………… IV Publication, oral communications and poster presentation……………………………………… VII Abbreviations…………………………………………………………………….………….…... IX General Introduction 1 1. Foreword……………………………………………………………………………………… 3 2. The river ecosystem and its sediment microbiome …………………………........................... 7 3. Biofilms in river ecosystems……………………………………………………….................. 9 4. Metal contaminated environments and how bacteria deal with them………………………… 9 4.1. Metals, definition and classification…………………………………............................... 11 4.2. Metals in aquatic ecosystems……………………………………………………………. 12 4.3. Interaction with metals, and mechanisms of resistance in bacteria……………………… 19 4.3.1. Interaction with lead (Pb