A metE mutant of Chlamydomonas reinhardtii provides new perspectives on the evolution of vitamin B12 auxotrophy Frederick Xavier de St Pierre Bunbury Emmanuel College University of Cambridge This dissertation is submitted for the Degree of Doctor of Philosophy September 2018 Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. It does not exceed the prescribed word limit for the relevant Degree Committee. Signed: .................................................................................................................................. Date: ...................................................................................................................................... i Acknowledgments First of all, I must thank the sources of funding that made this research possible. The Biotechnology and Biological Sciences Research Council provided my stipend and funds to cover the cost of research consumables. Emmanuel College and the Cambridge Philosophical Society both provided grants that allowed me to attend and present at international conferences. Professor Alison Smith has been a better supervisor than I could have hoped for. That Alison seemed to have more enthusiasm than even I myself had on some occasions for my project, while also giving me the reins to shape it as I saw fit, was very much appreciated. Alison’s extensive knowledge of algal metabolism and attention to detail was also invaluable in guiding this work, and I hope that we might publish together soon! My second supervisor, Dr. Uta Paszkowski, introduced me to more sophisticated symbiotic systems that initiate in the plant rhizosphere, and helped me refocus the aims from my first-year report so that they would be more achievable. Dr. Katherine Helliwell produced the metE7 strain, which was the focal point of most of my research, and has maintained an interest in my findings as well as making helpful suggestions for future work. Dr. Elena Kazamia performed many coculture experiments with a similar system that informed my experiments. In my first year in the lab Dr. Vaibhav Bhardwaj was a fantastic mentor, introducing me to many of the techniques I would later go on to use countless times. Dr. Payam Mehrshahi has provided great encouragement and helped me to better understand many of my results. Similarly, I have had many interesting conversations with Dr. Maria Huete-Ortega, who provided lots of useful advice. Dr. Katrin Geisler is one of the most hard-working people I have met and has often gone out of her way to ensure that I could conduct my experiments easily and to a high standard. Sue Aspinall has also made my work much easier and safer by keeping a busy lab running smoothly. Dr. Matthew Davey taught me how to perform lipid analysis of algal samples, and it was a pleasure to co- supervise an undergraduate student with him. Both of my undergraduate students, Sam Fitzsimmons and Bradford Loh, were capable scientists who were willing to learn and enthusiastic about their projects, and whose results helped guide some of my own work. Dr. Deborah Salmon performed the amino acid and methionine cycle metabolite analysis that is presented here. I had several interesting conversations with Dr. Ottavio Croze, Dr. Francois Peaudecerf and Hannah Laeverenz Schlogelhofer relating to the mathematical modelling of algal-bacterial cocultures. Andy Sayer and Andre Holzer will continue with work most closely related to my own, and it has been very rewarding to be able to discuss our findings together. Dr. Matthew Cooper, Dr. Mark Scaife, Dr. Louisa Norman, Dr. Christian Ridley, Dr. Alex Litvinenko, Dr Johan Kudahl, Aleix Gorchs-Rovira, Iain Bower, Patrick Hickland, Marcel Llavero-Pasquina, Stefan Grossfurthner, Sam Coffin, and Dr. Gonzalo Mendoza have been great people to share a lab with and all of them have helped me along the way. ii Kshitij Sabnis and Anna Gibbons did so much to make my role as Men’s Captain of the Pentathlon team as easy as possible, and therefore allowed me to have a very productive final year in Cambridge both in and out of the lab. My housemate Alice Rees has gone through the same undergraduate and graduate courses as myself over the last seven years in Cambridge and it has been great to have someone to talk to who has shared so many experiences. My brother Fabian has recently taken an increasing interest in evolutionary biology, and it has been very satisfying to have long conversations with a kindred spirit. My sisters, Madeleine and Jemima, and particularly my parents, Michael and Lotty, have been incredibly supportive, and it is still their advice that I trust more than anyone else’s. Having parents that are not only unconditionally supportive, but also show a genuine interest in my work has been a real source of pride. Thinking back to over a decade ago I can now remember my father saying how interesting it was that so many animals had evolved to become dependent on vitamins. Perhaps that was the seed that set me on this journey in the first place. iii Summary Vitamin B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Roughly half of all algae require vitamin B12, and the phylogenetic distribution of this trait suggests that it has evolved on multiple occasions. Previous work using artificial evolution generated a metE mutant of Chlamydomonas reinhardtii (hereafter metE7) that requires B12 for growth. Here, I use metE7 to investigate how a newly-evolved B12 auxotroph might cope with B12 limitation and interact with B12-producing bacteria. Compared to the closely related natural B12 auxotroph Lobomonas rostrata, metE7 has a higher requirement and lower binding affinity for B12. B12 deprivation of metE7 caused an increase in cell diameter, indicative of a decreased rate of cell division relative to growth. Other responses included an accumulation of starch and triacylglycerides at the expense of polar lipids and free fatty acids, and a decrease in photosynthetic pigments, proteins and free amino acids. This is reminiscent of nitrogen deprivation, but closer investigation revealed that B12 deprivation caused a substantial increase in reactive oxygen species, which preceded a rapid decline in cell viability. This might be explained by the observation that there was no induction of non-photochemical quenching, unlike under nitrogen deprivation. The metabolite S-adenosyl homocysteine, a potent inhibitor of methylation, increased substantially during B12 deprivation, as did the transcripts for several enzymes of one-carbon metabolism. The rhizobial bacterium Mesorhizobium loti formed a commensal relationship with wild-type C. reinhardtii, benefiting from the alga’s photosynthate. When co-cultured with metE7 this interaction should be considered a mutualism as the alga was dependent on bacterial B12. Adding B12 or glycerol to the coculture increased the cell density of metE7 and M. loti respectively, revealing that the rate of nutrient transfer between species was the factor limiting growth. It was unsurprising therefore that when grown in triculture, the wild type soon outcompeted metE7, even when M. loti growth and B12 production were fuelled by added glycerol. B12 release was also shown to be critical for mutualism: a mutant of E. coli that released more B12 into the media was, in contrast to its parental strain, able to support metE7. The mutual support of metE7 and M. loti over 6 months during an artificial evolution experiment illustrates that newly-evolved B12 auxotrophs could survive in the presence of B12- producers, but it was also demonstrated that M. loti could not produce sufficient B12 to favour the evolution of B12 dependence in C. reinhardtii. In summary, this work has provided an insight into the challenges of evolving B12 auxotrophy making it all the more remarkable that it is such a common trait. iv Table of contents Declaration ............................................................................................................................................... i Acknowledgments ................................................................................................................................... ii Summary ................................................................................................................................................ iv Table of contents .................................................................................................................................... v Table of figures .................................................................................................................................... viii List of abbreviations ............................................................................................................................... ix Chapter 1: Introduction .........................................................................................................................