Single-Particle Tracking of Proteins in Living Bacteria: From Single Cells to a Mixed Community by Chanrith Siv A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biophysics) in the University of Michigan 2017 Doctoral Committee: Assistant Professor Julie S. Biteen, Chair Associate Professor Matthew R. Chapman Professor Victor J. DiRita, Michigan State University Professor Ari Gafni Assistant Professor Sarah L. Veatch Chanrith Siv To Grandma and my parents, whose sacrifices gave me an opportunity to come to America to have a chance at the American dream ii Acknowledgements This graduate school journey has been one of the most challenging experiences I have encountered thus far in my life. Though there were many hurdles to overcome, the rewards at the end of it all definitely outweighed all of the late nights spent in lab doing experiments. This academic pursuit was not something I would have ever imagined myself doing growing up, but I am truly grateful for all of the opportunities that I have received throughout my academic career. Everything that I have accomplished so far would not have been possible without the support of some important people in my life. I would like to express my sincerest gratitude towards everyone who have played a role in shaping my journey. Finishing this dissertation would not have been possible if it wasn’t for my amazing advisors, Professors Julie Biteen and Victor DiRita. To Julie, thank you for trusting and believing in me to take on risky, scary challenges in my projects and in my career endeavors. I would not have been able to receive the necessary tools to land this dream job if it wasn’t for you supporting and allowing me to explore different, non-traditional avenues in graduate school. To Vic, thank you for letting me be a part of your lab and teaching me how to be more confident in science and in life. Thank you for the pep talks and thoughtful advice you have given me throughout the years. You two have always been there—even in the midst of your busy schedules—to reassure me that everything will be all right in the end. I am sorry that I will not be pursuing an academic career in the future, but I promise to make you both proud in some other ways. I will not let your many years of training me go to waste. iii Next, I would like to thank my committee members, Professors Sarah Veatch, Ari Gafni, Kim Seed and Matt Chapman. Thank you for coming to all of the meetings and providing wonderful feedback. Though you were all hard on me at times, I appreciate the constructive feedback because it made me a better scientist in the end. I am also indebted to the lab members who I have worked with during graduate school. You guys have taught me so much about science and about perseverance. You guys have provided me with wonderful work environments where I was allowed to be myself. I am definitely going to miss such a positive work environment. Thank you for all of the fun we have shared throughout the years; you guys definitely made work a lot easier. I especially want to thank Yi who have helped me tremendously in the past five years in the lab. To the members of the Seed and Chapman labs, I want to thank you all for welcoming me and allowing me to do experiments in your lab space. I would also like to thank my closest friends. I’m sorry that I am not listing specific names of people, but I am sure you guys all know who you are. You guys have put up with my rants about life and have always kept me in check. If it wasn’t for you guys listening and helping me get through this journey, I probably would’ve had more meltdowns. Last but not least, I want to thank my family. To Mom and Dad, thank you for your unconditional love and support. It has been over 9 years since I first left home, but I have never once felt far away from you guys. To my brother Sotheara, thank you for everything you have done for me. Though I may be tough on you, just know that it is all coming from a very good place in my heart. To my older brother Sothearak and his family, thanks for the enjoyable memories for when I visited home. Though you all may not always understand everything that I have gone through to get this PhD, you guys were always there for me. iv I am only where I am today because of the many people who have contributed to my success. Chanrith Siv v Table of Contents Dedication………………………………………………………………………………………....ii Acknowledgements…………………………………………………………………………..…..iii List of Tables………………………………………………………………………………….....vii List of Figures…………………………………………………………………………………...viii List of Appendices………………………………………………………………………………..xi Abstract…………………………………………………………………………………………..xii Chapter 1: Introduction……………………………………………………………………………1 Chapter 2: Differences in Labeling, Expression Systems, and Hosts Produce Concealed Subcellular Phenotypes…………………………………………………………………..36 Chapter 3: Two-Color Super-Resolution Imaging in Live Vibrio Cholerae to Probe TcpP/ToxR Interactions in the ToxR Regulon…………………………………………………...…...76 Chapter 4: Toward In-vivo Imaging of the Gut Microbiome…………………………………...105 Chapter 5: Final Conclusions and Perspectives………………………………………………...132 Appendix…………………………………………………………………………………...…...148 vi List of Tables Table 1.1 Optical properties and oligomeric states of fluorophores used in this study…………..9 Table 2.1. Strains used in this study…………………………………………………………......42 Table 2.2 Statistics for all strains in different growth conditions………………………………..56 Table 2.3 Statistics for ‘slow’ and ‘fast’ TcpP-PAmCherry for endogenous and ectopic strains in different growth conditions…………………………………………………………....…56 Table 2.4 Primers used for cloning tcpP-pamcherry………………………………………….....66 Table 2.5 Primers used for qRT-PCR analysis………………………………………………......68 Table 2.6 Raw data for qRT-PCR analysis with CT method……………………………...….68 Table 3.1 Using a 3-term diffusion model to fit to CPD…………………………………….......92 Table 4.1 Classification of resistant starches and food…………………………………………108 Table 4.2 Different methods to grow a B. theta/R. bromii biofilm……………………………..122 Table A.1 List of strains in CS strain box. ……………………………………………………..158 vii List of Figures Figure 1.1 Live Vibrio cholerae imaged using different microscopy techniques…………….......3 Figure 1.2 Model of the virulence cascade in V. cholerae……………………………………….15 Figure 2.1 The ToxR Regulon regulates gene expression of the major V. cholerae virulence factors CTX and TCP through ToxT…………………………………………………….39 Figure 2.2 In vitro characterization of the O395 V. cholerae strains reveals differences in transcription and expression levels………………………………………………………43 Figure 2.3 mRNA levels in the fusion strains relative to the wildtype strain……………………45 Figure 2.4 Transcript levels of toxT, tcpP, toxR, and aphB were determined for cultures………47 Figure 2.5 Single-molecule tracking of TcpP-PAmCherry in the endogenous and ectopic V. cholerae strains reveals differences in dynamics………………………………………...49 Figure 2.6 Immunoblot with antibodies against TcpP and TcpA for the wt, endogenous and ectopic V. cholerae strains……………………………………………………………….52 Figure 2.7 Diffusion coefficients and population weights of TcpP-PAmcherry as a function of pH and temperatures…………………………………………………………………………54 Figure 2.8 Characterization of ectopically expressed TcpP-PAmCherry from a second plasmid induced by IPTG…………………………………………………………………………58 Figure 2.9 Dynamics of plasmid-expressed TcpP-PAmCherry from a second IPTG-induced plasmid…………………………………………………………………………………...59 viii Figure 2.10 Fluorescence intensity of ectopically expressed TcpP-PAmCherry in V. cholerae and in a heterologous host…………………………………………………………………....61 Figure 3.1 Virulence signaling cascade in V. cholerae and fluorescent labeling of TcpP and ToxR……………………………………………………………………………………..79 Figure 3.2 Endogenous expressions of ToxR and TcpP protein fusions in V. cholerae…………82 Figure 3.3 Immunoblots of ToxR and of TcpP…………………………………………………..84 Figure 3.4 Immunoblot of toxT-regulated toxin coregulated pilus protein TcpA………………..85 Figure 3.5 Cholera toxin ELISA of the V. cholerae strains used in this study with and without inducers……………………………………………………………………………..……87 Figure 3.6 Coomassie stain of cell lysates grown with and without inducers…………………...88 Figure 3.7 Imaging live V. cholerae cells with high resolution……………………………...…..89 Figure 3.8 Single-molecule protein tracking in live cells……………………………………......91 Figure 3.9 Cumulative probability distributions of ToxR-mCitrine and TcpP-PAmCherry motions…………………………………………………………………………….…..92 Figure 3.10 Dual band pass filter for laser excitation utilizing a 488 nm and 561 nm lasers…..100 Figure 4.1 Model for starch catabolism by the B. thetaiotaomicron Sus…………………....…107 Figure 4.2 Imaging live anaerobic bacterial cells on a conventional benchtop microscope……111 Figure 4.3 Growth curves from B. theta, R. bromii, and B. theta/R. bromii co-culture………..113 Figure 4.4 Growth of co-cultures on a coverslip……………………………………………….114 Figure 4.5 Contamination of cultures grown in the anaerobic chamber………………………..115 Figure 4.6 Fluorescence detection of BtCreiLOV in E. coli………………………………………..116 Figure 4.7 Fluorescence excitation and emission spectra of purified CreiLOV and VafLOV from Chlamydomonas reinhardtii and Vaucheria frigida………………………………………..118 ix