University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2004 Using a Bioluminescent Bacterial Bioreporter to Assess Iron Bioavailability in the Oceans Cecile Elise Mioni University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Microbiology Commons Recommended Citation Mioni, Cecile Elise, "Using a Bioluminescent Bacterial Bioreporter to Assess Iron Bioavailability in the Oceans. " PhD diss., University of Tennessee, 2004. https://trace.tennessee.edu/utk_graddiss/2302 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Cecile Elise Mioni entitled "Using a Bioluminescent Bacterial Bioreporter to Assess Iron Bioavailability in the Oceans." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Microbiology. Stephen W. Wilhelm, Major Professor We have read this dissertation and recommend its acceptance: Lee W. Cooper, Gary S. Sayler, Robern N. Moore Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Cécile Élise Mioni entitled “Using a bioluminescent bacterial bioreporter to assess iron bioavailability in the Oceans.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Microbiology. Steven W. Wilhelm Major Professor We have read this dissertation and recommend its acceptance: Lee W. Cooper Gary S. Sayler Robert N. Moore Accepted for the Council: Anne Mayhew Vice Chancellor and Dean of Graduate Studies (Original signatures are on file with official student records) USING A BIOLUMINESCENT BACTERIAL BIOREPORTER TO ASSESS IRON BIOAVAILABILITY IN THE OCEANS A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Cécile Élise Mioni December 2004 i Copyright © (2004) by Cécile Élise Mioni All right reserved ii DEDICATION This dissertation is dedicated to my parents, Charles Mioni and Dolorès Mioni; my sister, Amandine Mioni; my fiancé, Lawson Worrell IV, and the rest of my family, for their love and continuous support, for their encouragements and infinite patience, for sharing my sacrifices, and because they have never left the boat when the fire drill was going off. “The sea is a mirror which confronts us with our own ignorance.” Anita Conti iii ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Steven W. Wilhelm, for his guidance and patience, as well as for giving me the opportunity to work on such a beautiful, although challenging, project. His enthusiasm about Sciences and his contagious motivation were inspiring throughout this work. I would like to thank also Dr. Gary S. Sayler, for his generosity and his advice all along my Ph.D. research. I would like to thank as well the other members of my committee, Dr. L. Cooper, Dr. D.A. Hutchins, and Dr. R.N. Moore, for their advice and feedback on this research project. I am also very grateful to my friends Dr. M. Allen and Amy Allen, for their help, for their practical advices and for sharing their tips throughout this work. I would like to thank as well all my friends and colleagues at the Wilhelm’s lab and at the Center for Environmental Biotechnology, who helped me in many ways. The list is not exhaustive but I would like to thank especially, Matt Carberry, Amanda Dean, Melanie Eldridge, Shannon Efteland, Julie Higgins, Lynn Neal, Andy Ouellette, Leo Poorvin, Renhui Li, Casey Rentz, Johanna Rinta-Kanto, Janet Rowe and Matt Smith. I have learned a lot from you guys and you really made me feel home since the very beginning. I would like to thank also Dr. H. Dionisi, Dr. S. Ripp, Dr. J. Sanseverino, A. Fraley, A. Ozen, L. Mitchell, V. Garrett, L. Saidak, D. Williams and his wife Cathy, who were all very supportive and always ready to help. I have never felt bad to ask them questions, they have always taken the time to reply to my questions with kindness, I am really indebted to all of them. I would like to thank as well everybody at the Microbiology Department. I would like to thank especially my friends Steve Minkin, Shannon Efteland, Judson Hervey and Heather Connelly for their priceless friendship and continuous support. I would like to thank also to my friends Johanna Rinta-Kanto, Melanie Eldridge and Chris Pack, they have always been here, even during the down times. I would not have made it through without their help; they always anticipated my needs without waiting for me to ask. They iv were all very caring and supportive when I became sick and I will always be indebted to them. I would like to thank the Center for Environmental Biotechnology, the International Women’s Fishing Association 2003, and Daniel Jouvance to provide fundings during this PhD project. I would like to thank as well some exceptional instructors and professors who has the truly rare gift to transmit their passion for atmospheric and marine sciences and to inspire their students. I thank Dr. P.-J. Labourg and Dr. X. de Montaudoüin (Marine Station of Arcachon) for initiating me to marine ecology and encouraging me to pursue in oceanography. I thank Dr. P. Castaing (University of Bordeaux I) for being such a wonderful and supportive professor; through his encouragements and advice, I eventually found my way to graduate school. I would like to thank especially Pr. P. Tréguer and Dr. S. Blain who initiated me and guided my first steps in the research on iron limitation in oceans while I was at the European Institute of Marine Studies. They transmitted me their enthusiasm for this field and are still a great source of inspiration for me. I would like to thank Dr. A. Milligan and Pr. F.M. Morel for helping me in pursuing my research work in the iron field. I would have not been here without the encouragements and the help of my friend Dr. A. Milligan, I am really grateful for his support and advices. Lastly, I would like also to thank Dr. V. Garçon, D. Thouron and J. Sudre (LEGOS) for introducing me to other aspects of oceanography. I would like to thank my dear friend Lucile Storti who, despite the distance, was always here for me. She helped me in many ways and has shared so much with me, I am so glad to have her as a friend for more than a decade now. I would like to thank my parents, Charles and Dolorès Mioni, my sister, Amandine Mioni, my grand-parents, Santiago and Josepha Martinez, and Antonio and Anna Mioni, as well as the rest of my family. Since the very beginning, their encouragements, their moral and material support, greatly help me through it all. They are my roots that keep my feet on Earth. I would like to thank as well my fiancé, Lawson Worrell IV, I could not imagine life without him; he just made it so much more beautiful. His love and support helped me a lot to go through this both at sea and on land. v ABSTRACT Recent improvements in modern analytical methods have considerably increased our understanding of Fe biogeochemistry in the Ocean. Compiled data have shown that Fe concentrations are low in most open ocean surface waters and that the bioavailability of this Fe is influenced by organic complexation. Of presumed biotic origin, the importance of this organic complexation to Fe availability remains to be elucidated. Unfortunately, current analytical tools do not allow for a linkage to be established between Fe speciation to Fe bioavailability. To supplement chemical analyses, we have developed a bioanalytical tool: a heterotrophic bacterial bioluminescent reporter system that responds quantitatively to bioavailable Fe concentrations. Data collected during one field study in a freshwater system as well as three independent field studies in marine systems demonstrate that these whole-cell biosensors are a powerful tool for environmental monitoring, providing both qualitative and quantitative insights on Fe biogeochemistry. To complement this work, laboratory studies have been carried out to characterize the influence of various sources of Fe-complexing organic ligands, including synthetic chelators, bacterial and fungal siderophores and virus-mediated bacterial lysis products. The results of this study will be presented in the context of attempting to clarify the role that the organic complexation of Fe may play in regulating Fe bioavailability to the diverse planktonic components of the aquatic microbial community. To improve our abilities to standardize this tool in the field, a green fluorescent protein gene expressed under control of a constitutive promoter has been introduced in the genomic DNA of P. putida FeLux. The resulting dual bioreporter will allow for the enumeration of bioreporter cells in environmental samples in parallel to the detection the bioluminescent signal in response to changes in Fe bioavailability. vi TABLE OF CONTENT SECTIONS