ENVIRONMENTAL EFFECTS ON BACTERIAL COMMUNICATION AND COMMUNAL BEHAVIOR by Eric Ka-On Chu A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland January 2019 © 2019 Eric Ka-On Chu All Rights Reserved Abstract Despite their autonomous nature, bacteria can often reside within complex, multicellular communities. One benefit of communal organization is the protection it offers from hazardous environments around the cells, which can come in the form of physical shielding or collective adaptive behaviors that arise from cell aggregation. This dissertation explores how environmental conditions itself might modulate or trigger these collective cell behaviors. We first explored how the environment can affect the active coordination of collective cell behavior, which involves cell-to-cell communication mechanisms such as quorum sensing (QS). Using a microfluidic platform to modulate the environment, we showed that existing explanations of environmental dependence pertaining to modulation of signal retention alone were inadequate in explaining the response. Instead, a dynamics- based analysis coupled with a mathematical model revealed a regulatory mechanism that is defined by the growth-mediated balance between synthesis and dilution of the signaling machinery proteins. This mechanism is able to account for the temporal and spatial properties observed during the onset and propagation of the collective response. These properties culminated in a cell education strategy that effectively combines response diversification with cell signaling to accelerate the onset of the collective cell behavior, which can have tremendous implications for the fitness of the cells that can exhibit this behavior. In addition, we also examined the effects of direct environmental cues, such as mechanical cues, on the emergence of collective cell behaviors. We found that physical confinement of bacterial colonies can lead to a buildup of self-imposed mechanical stress, which can elicit a biological stress response and the secretion of biofilm-related extracellular materials. We demonstrate that this renders the colony biofilm-like, with the ii associated functional consequence of increased antibiotic tolerance. Across these studies, we combined engineering approaches with experimentation and computational modeling to explore the relationship between bacterial colonies and its surrounding environment and found a high degree of dependence, most often reflected in spatial dependences of responses. As the appreciation for the importance of the microenvironment and its influence on bacterial colonies grow, we anticipate that the interdisciplinary approaches presented here will prove to be valuable tools in helping us understand the workings of bacterial collective cell behavior. Advisor: Andre Levchenko, PhD Readers: Andre Levchenko, PhD; Jie Xiao, PhD Thesis Committee: Andre Levchenko, PhD; Winston Timp, PhD; Jie Xiao, PhD iii Acknowledgements First and foremost, I wish to thank my advisor, Prof. Andre Levchenko, for his mentorship throughout my Ph.D. studies. He gave me the freedom and opportunity to explore on my own, which allowed me to make and learn from my own mistakes, and in the process, become a better scientist. For that I am very grateful. I would also like to thank my thesis committee members, Profs. Jie Xiao and Winston Timp, for their guidance and numerous helpful suggestions over the years that significantly strengthened the work in this thesis. I also wish to express gratitude to Dr. Onur Kilic, who taught me the fundamentals of microfabrication that laid the foundations for much of the work presented in this thesis. I must also thank Prof. Alex Groisman for sharing with me his best microfluidic ideas and helpful critique. I also wish to acknowledge Dr. Hojung Cho for laying down the groundworks for the biofilm study. I want to express my heartfelt thanks to all members of the Levchenko Lab, past and present, who have taken part in this amazing journey with me. It has been an honor and a privilege to have had the chance to know every one of you, and to be able to learn a little bit about this mysterious world together. Nevertheless, I wish to highlight a few people who have been particularly important to me during my time in the lab as a Ph.D. student. Sincere thanks to Dr. Hao Chang for letting me pick his brain whenever I was stuck on a problem, and also for bringing me on frequent Costco runs to buy pizza, which I am sure fueled more than half of the work done in this thesis. Dr. Tae-Yun Kang never fails to amaze me with her depth and breadth of knowledge, and I thoroughly enjoyed each and iv every one of our talks, whether it was about 3D printing or the latest Korean trends. I’ve also been very fortunate to have met Rebecca LaCroix, who shares the same passion for food as I and with whom I have shared numerous wonderful meals as we explored the New Haven restaurant scene. In no particular order, I also wish to thank Ben, Jinseok, Sunghoon, Kiran, Patrick, Kshitiz, Rita, Masha, and Archer for all of their help and advice, and for making Levchenko lab such a fun place to work. I will always cherish our friendships and remember the numerous fun times we’ve spent together. I also wish to thank Hong, Naomi, and Meredith for all of their administrative help. Finally, I wish to thank my family. I thank my parents, Cheuk Wai and Im Ngan, who have both sacrificed so much to give me the chance to succeed, and for all of their love and support, I will always be grateful. I am also extremely thankful for all of the love and prayers from my sister, Jenny, brother-in-law, Jeremy, and nephew, Timothy, which helped tremendously when my cells were not showing me any love. Most important of all, I thank Roxane, my soulmate and best friend, for her unconditional love and boundless patience throughout this long journey. Without her unwavering support and joyous optimism that helped me through the trying times during my studies, this thesis would not have been possible. Thank you. v Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents ............................................................................................................... vi List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x Chapter 1. Introduction ........................................................................................................1 1.1 Bacterial Communities ...............................................................................................1 1.2 Quorum sensing ..........................................................................................................2 1.3 Biofilms ......................................................................................................................4 1.4 Microfluidics for studying bacteria ............................................................................6 1.5 Aims and significance of this thesis research .............................................................9 Chapter 2. Influence of the environment on the quorum sensing response .......................12 2.1 Introduction ..............................................................................................................12 2.2 Results ......................................................................................................................14 2.2.1 Development of microfluidic platform ..............................................................14 2.2.2 Characterization of differential diffusive properties .........................................16 2.2.3 Characterization of differential growth rates .....................................................19 2.2.4 Neither classical quorum sensing nor diffusion sensing can explain QS response variation .......................................................................................................22 2.2.5 Response dynamics is reflective of environmental conditions..........................26 2.2.6 Response dynamics is modulated by balance of synthesis and dilution ...........29 2.2.7 Analysis of the balance of synthesis and dilution to understand QS dynamics 32 2.2.8 Mathematical model of QS dynamics ...............................................................35 2.2.9 Medium composition changes synthesis and dilution rates ..............................36 2.2.10 Temperature changes synthesis and dilution rates ..........................................40 2.2.11 Changes in synthesis and dilution affects QS protein accumulation ...............43 2.2.12 Information theoretic analysis of QS sensitivity to environmental conditions ...................................................................................................................48 2.3 Discussion ................................................................................................................49 vi 2.4 Experimental methods ..............................................................................................52 2.4.1 Bacterial strain