A big role for small molecules in mediating Emiliania huxleyi – Roseobacter interactions by Leen Labeeuw A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology Department of Biological Sciences University of Alberta © Leen Labeeuw, 2016 Abstract Microalgae are a diverse group of photosynthetic microorganisms that have complex relationships with their surrounding bacteria, which are often controlled by the exchange of bioactive molecules. Emiliania huxleyi is a ubiquitous marine microalga, forming massive blooms, driving the marine carbon pump and biogeochemical cycles like the sulphur cycle. Bacterial symbionts of this alga include an abundant group of α-proteobacteria known as roseobacters. These bacteria are known to containing a variety of secondary metabolites that may benefit an algal symbiont; for example, certain members produce a growth-promoting plant hormone, indole-3-acetic acid (IAA) or tropodithietic acid (TDA) that acts to chemically defend their host from further bacterial colonisation. However, at least one species of the roseobacters can switch to produce a pathogenic effect as the alga enters late-stage growth, secreting algaecides called roseobacticides. These are formed in response to a lignin intermediate, p- coumaric acid (pCA). This duality of mutualism and pathogenicity seems to be controlled by the various bioactive molecules released by the algae and bacteria. This thesis seeks to investigate the interactions between algae and their selected members of their microbiome using controlled laboratory conditions and by doing so, further our understanding of their natural interactions and find novel methods to enhance algae processing for industrial purposes, such as biofuels. Although pCA has been shown to be linked to aging microalgae, the role it plays in the physiology and/or ecology of these organisms is unclear, as is the molecular pathway used to create this compound. Lignin, one of the innovations of land plants, has been found in various algae, raising questions about the evolution of the lignin biosynthetic pathway. To determine the taxonomic distribution of the lignin biosynthesis genes, all publicly available genomes of algae were screened. Genes associated with p-coumaryl alcohol (H-monolignol) biosynthesis were found widely present in algae, and therefore postulated to have evolved long before the transition of photosynthetic eukaryotes ii to land. The original function of this lignin precursor is therefore unlikely to have been related to water transport. Lignin intermediates are shown to have an antimicrobial action against common marine bacteria, suggesting an early role in the biological defence of some unicellular and multicellular algae. Roseobacticides production by the roseobacter Phaeobacter gallaeciensis was stimulated after addition of a lignin intermediate, pCA. However, pCA depressed the levels of the antimicrobial tropodithietic acid (TDA) that is normally produced by the bacterium, thereby providing less protection to the alga and P. gallaeciensis itself against other bacteria. P. gallaeciensis accelerates senescence and selectively kills one cell type from E. huxleyi, the coccolith producer, while leaving the bald strain alive. Indole-3-acetic acid (IAA) is an important auxin influencing plant development, but the production in algae has been contentious. Screening the tryptophan dependent pathway revealed that the biosynthetic potential for IAA is present in various algal groups, especially in E. huxleyi. Addition of L- tryptophan to E. huxleyi stimulated IAA production, but only in the coccolith-bearing strain. Conversely, addition of exogenous IAA only elicited a physiological response in the bald cell type. A roseobacter Ruegeria sp. R11, previously shown to produce IAA, co-cultured with L-tryptophan and both cell types of E. huxleyi produced less IAA than the axenic coccolith cell type culture similarly induced. This suggests that IAA plays a novel role signalling between different E. huxleyi cell types, rather than between a bacteria and its algal host. In order to determine a possible commercial application of these findings, the growth and lipid yield of these bacteria and bioactives were measured against E. huxleyi, another haptophyte Isochrysis sp., as well as the chlorophyte Dunaliella tertiolecta. Only R11 showed early promise in stimulating the lipid content of the green alga, leading to potential industrial applications. Despite their small size, unicellular organisms such as the microalga E. huxleyi are capable of a complex set chemical interactions, both interspecies (e.g. E. huxleyi – roseobacter) and even intraspecies iii (e.g. E. huxleyi coccolith bearing – bald cell types). These interactions and the bioactives that mediate them could be important drivers in shaping the ecology, life history, and bloom-bust interaction of this microalga in the marine environment. iv Preface Some of the research conducted for this thesis forms part of collaborative work, listed below. The functional analysis performed in Chapter 2 was performed by Dr. Yan Boucher (Figure 2-2). A version of this chapter was published as: Labeeuw L, Martone PT, Boucher Y, Case RJ (2015). Ancient origin of the biosynthesis of lignin precursors. Biology Direct 10: 1–21. The methodology outlined for Chapters 3-4 for use of the Pulse-Amplitude-Modulated (PAM) Fluorometry was determined and optimized by myself and Anna Bramucci, and published as: Bramucci AR, Labeeuw L, Mayers TJ, Saby JA, Case RJ (2015). A small volume bioassay to assess bacterial/phytoplankton co-culture using WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry. Journal of Visualized Experiments 97: e52455. In Chapter 3, screening of the bacterium against different Emiliania huxleyi strains, as well as the microscopy images taken, was conducted by Dr. Rebecca Case and Alexis Fischer (Table 3-1 and Figure 3-1). The final data collection in Chapter 3 for algal and bacterial co-cultures (Figure 3-2) was collaboration with Anna Bramucci, with initial screening and optimization performed by me. Chapter 4 was a collaborative work, whereby Dr. Paulina de la Mata optimized and determined the GCxGC-TOFMS settings, while Joleen Khey collaborated on the algal bioinformatics (Table 4-1), and Anna Bramucci collaborated on flow cytometry analysis (Figure 4-6). It has been published as: Labeeuw L, Khey J, Bramucci AR, Atwal H, de la Mata P, Harynuk J, Case RJ (2016). Indole-3- acetic acid is produced by Emiliania huxleyi coccolith-bearing cells and triggers a physiological response in bald cells. Frontiers in Microbiology, in press. v Acknowledgements I would like to thank my supervisor Dr. Rebecca Case for accepting me, despite my initial limited background in microbiology, and taking the time to teach me the techniques that I needed to succeed. Thanks to her guidance I feel more confident in myself as a scientist, and have learned valuable lessons that I hope will aid me in my career. I would like to thank my committee members, both current and past, Dr. Janice Cooke, Dr. David Bressler, and Dr. Julia Foght for providing some great insights into my thesis and advising me when I hit a few stumbling blocks. My gratitude also goes out to my collaborators: Dr. Paulina de la Mata for taking the time for our collaboration with the GCxGC-TOFMS and making it intelligible to me, and Dr. Yan Boucher for his expertise in bioinformatics. I would also like to thank Arlene Oatway in the Microscopy lab, as well as all those working at the university Flow Cytometry unit. Thanks to Dr. Brian Lanoil and Dr. Marc Strous for being on my examining committee. A big thank you has to go to my labmates, especially Anna Bramucci and Teaghan Mayers. Apart from their academic support and listening to my monologues about what to do, they have been great friends. Thanks to Anna’s patience in teaching me to ice-climb and taking me winter camping. And thanks to Teaghan for being my travel buddy throughout Korea and hosting so many great nights with her homemade brews. I would also like to thank my fellow graduate students, past and present, on the 4th floor micro-wing, as well as “Phil” Kirchberger, Fabini Orata, and the extended lunch group (even those banished to CCIS). Thanks for listening to my rants, giving occasionally solid advice, your attention to detail and colours, and providing many an interesting discussion. Thanks to my fellow graduate students in Microbiology for their support and friendship throughout my thesis. Lastly, I would like to thank Wenjun Dai and my other roommates for the last few great years, as well as my family, for always supporting me in my pursuits. vi Table of Contents Chapter 1 ............................................................................................................................................. 1 1.1 Background ....................................................................................................................................... 1 1.2 Bacterial-algal interactions in marine systems .................................................................................... 4 1.2.1 Mutualism ........................................................................................................................................... 5 1.2.2 Commensalism .................................................................................................................................... 7 1.2.3 Parasitism and pathogenicity .............................................................................................................