i Exploration of chemical interactions between Streptomyces and eukaryotes By Louis K. Ho A thesis submitted in conformity with the requirements For the degree of Doctor of Philosophy Department of Biochemistry University of Toronto © Copyright by Louis K. Ho 2020 i Exploration of chemical interactions between Streptomyces and eukaryotes Louis K. Ho Doctor of Philosophy Department of Biochemistry University of Toronto 2020 Abstract In the environment, bacteria live in complex communities with other organisms. In this work, I characterize two new chemically-mediated ways in which bacteria and eukaryotes interact. First, I show that ingestion of Streptomyces bacteria can directly be lethal to fruit flies and that this toxicity is the result of bacterial production of insecticides. I also show that this toxicity is facilitated by airborne odors that reducesin insect progeny. Second, I show that the yeast Saccaromyces cerevisiae can trigger Streptomyces to enter a new mode of growth called ‘exploration’. I characterize ‘exploratory’ growth and identify how it is triggered by airborne chemicals and the chemical modification of the growth environment. Overall, this thesis describes two new ways in which bacteria and eukaryotes interact and highlights how chemicals play an important role in these interactions. ii ii Acknowledgements I would like to thank all past and present members of the laboratory for their support and putting up with me throughout the years. You are not only my trusted colleagues but also my friends that I hope to cherish and keep in touch with for many years to come*. I would like to thank my supervisor, Dr. Justin Nodwell for putting insurmountable faith in me from the very beginning and for shaping my view of the world. I would like to thank my committee members Dr. Craig Smibert and Dr. Leah Cowen for providing monumental guidance and grilling me at committee meetings. I would like to thank my family for their foundational support. It is through my cultural roots where I have found motivation and have profound respect towards. Finally, I would like to thank nature itself…for without it, we would have nothing to study. ~ *Sheila Marie Pimentel-Elardo, Martin Daniel-Ivad, Stephanie Tan, Jan ‘The Man’ Vincent Falguera, Vanessa Yoon-Calvelo, Glenna Kramer, Jing Li, Stefanie Mak, Scott McAuley, Krysten Joy Myer, Ali Nikdel, Daniel Socko, Tomas Gverzdys. Cyrus Savalanpour, Maxime Lefebvre. iiiiii Table of contents Acknowledgments……………………………………………………………………………….iii Table of contents…………………………………………………….……………………..……iv List of figures and tables………………………………..……...………………….…………….v Publications……………………………………………………..…...……………….…………vii Chapter 1 Chemical perturbation of eukaryotes by bacteria……………….………………...1 1.1 Abstract………………………………………………………………………………..1 1.2 Introduction…………………………………………………………...…………….…1 1.2.1 Targeting DNA synthesis: Doxorubicin…………………..……………..….6 1.2.2 Targeting fungal sterols: Amphotericin B………………..……………...….8 1.2.3 Targeting growth and development: mTOR……………………………….10 1.2.4 Targeting neurotransmission: Avermectin…………………………………13 1.2.5 Targeting nuclear export: Leptomycin B…………………………………..15 1.2.6 Targeting the proteasome: Epoxomicin……………………………………18 1.3 What is the biological function of eukaryote-active metabolites in nature?................21 1.4 Establishing a link between bacterial toxicity and insecticidal metabolites………...21 Chapter 2: Chemical entrapment and killing of insects by bacteria……….………………..24 2.1 Abstract……………………………………………………………………….……...24 2.2 Introduction…………………………………………………………………………..25 2.3 Results………………………………………………………………………………..28 2.3.1 Many streptomycetes make insecticidal metabolites………………………28 2.3.2 Toxicity of Streptomyces spores…………………………………………...30 2.3.3 Cosmomycin-D is the causative agent of killing by WAC-288…………...32 2.3.4 The mechanisms of spore-associated lethality is associated with mechanism of the toxic compound being produced…………………………………………..33 2.3.5 Chemical attraction by Streptomyces cultures and 2-methylisoborneol leads flies to spores …………………………………………………….……………...36 2.4 Discussion……………………………………………………………………………39 Chapter 3 Streptomyces exploration is triggered by fungal interactions and volatile signals……………………………………………………………………………………………42 3.1 Abstract………………………………………………………………………..……..42 3.2 Introduction……………………………...…………………………………………...43 3.3 Results………………………………..………………………………………………45 3.3.1 Physical association with yeast stimulates Streptomyces exploration……..45 3.3.2 The yeast TCA cycle must be intact to stimulate Streptomyces exploration48 3.3.3 Exploration is glucose-repressible and pH-dependent……………………..53 3.3.4 S. venezuelae exploration requires an alkaline stress response…………….57 3.3.5 S. venezuelae explorer cells alkalinize the medium using an airborne volatile organic compound………………………………………………………………..59 3.3.6 S. venezuelae exploratory cells use VOCs to induce exploration in other streptomycetes at a distance……………………………………………………...61 iv 3.3.7 The VOC trimethylamine stimulates Streptomyces exploratory behaviour………………………………………………………………….……...63 3.3.8 TMA induces exploratory growth by raising the pH of the growth medium…………………………………………………………………………..64 3.3.9 TMA can reduce the survival of other bacteria……………………………66 3.4 Discussion……………………………………………………………………………67 Chapter 4 Concluding remarks………………………………………………………….…….71 4.1 Bacteria and their relationship with eukaryotes…………………………………...…71 4.2 Future directions……………………………………………………………….…….71 4.2.1 Behavioural responses of nematodes to live Streptomyces colonies……....71 4.2.2 More screens across multiple Domains of life……………………………..73 4.2.3 Understanding of the effect of volatile compounds on insect behaviour…..74 Materials and methods…………………………………………………………………….…...76 Materials and methods (Chapter 2)……………………………………………….……...76 Materials and methods (Chapter 3)……………………………….……………………...83 Appendix……………………………………………………………………………….…......…89 A.1 Supplementary figures & tables………………………………………………..……89 References………………………………………………………………………………...……125 Copyright acknowledgements………………………………………………………..……….159 v iv List of figures and tables Table 1.1 The eukaryotic targets of actinomycete metabolites Fig. 1.1 The eukaryotic targets of actinomycete metabolites is incredibly diverse Fig. 1.2 Doxorubicin: A potent anticancer compound that intercalates DNA Fig. 1.3 Amphotericin B: A potent antifungal compound Fig. 1.4 Rapamycin: Targeting the mammalian Target of Rapamycin (mTOR) pathway Fig. 1.5 Avermectin: Targeting neurotransmission in insects and nematodes Fig. 1.6 Leptomycin B: Inhibiting the nuclear export of protein cargo Fig. 1.7 Epoxomicin, a tripeptide that targets the eukaryotic 20S proteasome Fig. 2.1 Identifying insecticidal bioactivity in actinomycete extracts Fig. 2.2 Actinomycetes pose a threat to larval viability due to the production of insecticidal metabolites Fig. 2.3 Cell death-like activity in D. melanogaster is triggered by the consumption of cosmomycin-D producing spores. Fig. 2.4 Chemical entrapment of adult flies that are attracted to actinomycetal cultures and 2- methylisoborneol Table 2.5 Establishing a link between 2-MIB and insecticidal activity Fig. 3.1 Physical association with yeast triggers Streptomyces exploratory behaviour Fig. 3.2 Video of the leading edge of S. venezuelae explorer cells over a 17 hr time frame Fig. 3.3 S. venezuelae grown beside diverse yeast strains Fig. 3.4 Identifying yeast mutants that lack the ability to stimulate exploratory growth in S. venezuelae Fig. 3.5 Yeast stimulates S. venezuelae exploratory growth by consuming glucose and inhibits it by acidifying the medium Fig. 3.6 TCA cycle is implicated in C. albicans induction of S. venezuelae exploration Fig. 3.7 Exploratory growth in S. venezuelae is stimulated by the absence of dextrose Fig. 3.8 The alkaline stress response is associated with S. venezuelae exploratory behaviour Fig. 3.9 Volatile organic compounds released by S. venezuelae raise the medium pH and induce exploratory growth in physically separated Streptomyces Fig. 3.10 S. venezuelae VOCs inhibit the growth of other bacteria Fig. 3.11 New model for Streptomyces development Fig. 4.1 How C. elegans respond to live actinomycete colonies Table 6.1 Strains, plasmids, media and culture conditions Fig. 6.2 T-maze used to test the preference of pure 2-MIB on adult fruit flies Fig A.2 The effect of Streptomyces extracts on fruit fly larvae Fig A.3 Spores kill flies similar to the effect of the extract Fig. A.4 Purification and structural elucidation of cosmomycin-D Table A.5 Biosynthetic gene clusters in WAC-288 Fig. A.6 Analyzing the cosmomycin-D biosynthetic gene cluster viv Fig. A.7 Confirming the lack of antifungal activity of WAC-288 Fig. A.8 Visible spores in Streptomyces-fed larvae Fig A.9 Crude extract of WAC-288 has activity against human cells Fig A.10 WAC-288 is active against mosquito larvae Table A.11 Summary of activity against C. elegans Fig. A.12 Production of nonactins by Cu230555 with paralysis against C. elegans Fig. A.13 Bioactivity-guided purification of larvicidal compounds Fig. A.14 Effect of cosmomycin-d and doxorubicin on 1st instar larvae Fig. A.15 Complete genome of WAC-288 Fig. A.16 Deleting Cosmomycin-D and 2-MIB Biosynthesis Table A.17 Primers used for gene disruption and confirmation Table A.18 Preference assay parameters and raw data Fig. A.19 Biosynthetic gene cluster of 2-methylisoborneol in WAC-288 Fig. A.20 Comparing biosynthetic gene clusters of 2-methylsioborneol between actinomycetes Fig. A.21 Conservation of 2-methylisoborneol