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A Journey to the Center of a Bacterial Organelle by Onur Erbilgin A

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A Journey to the Center of a Bacterial Organelle by Onur Erbilgin A A Journey to the Center of a Bacterial Organelle By Onur Erbilgin A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Cheryl A. Kerfeld, Co-Chair Professor Arash Komeili, Co-Chair Professor Kathleen R. Ryan Professor David F. Savage Spring 2015 Copyright 2015 Onur Erbilgin All rights reserved Abstract A Journey to the Center of a Bacterial Organelle by Onur Erbilgin Doctor of Philosophy in Microbiology University of California, Berkeley Professors Cheryl A. Kerfeld and Arash Komeili, Co-Chairs Cellular compartmentalization is a fundamental strategy through which complex tasks can be carried out, where each compartment carries out a specialized function. Compartmentalization strategies in the bacteria have (relatively) recently been discovered and are not present across the entire domain; they instead seem to be tied to niche specialization of the host species. For example, carboxysomes are necessary for all cyanobacteria to fix carbon dioxide in present-day atmospheric conditions. Carboxysomes are part of a larger family of bacterial organelles termed bacterial microcompartments (BMCs), which are all related by the proteinaceous shell that bounds the compartment. Many different metabolic functions have been attributed to BMCs, such as carbon fixation, propanediol metabolism, and ethanolamine metabolism, and cursory genome gazing had hinted that there may be several more functional types of these organelles in a vast number of bacteria. In order to survey the functional and phylogenetic diversity of BMCs, a bioinformatic algorithm was developed to predict and categorize genetic loci that encode genes that can construct a complete organelle. Our analyses result in a taxonomy of BMCs that identifies several candidate loci and individual genes for further investigation. We predicted many of these loci to encode for BMCs with novel functions, and examined one locus apparently isolated to the Planctomycetes and Verrucomicrobia phyla. By genetically manipulating Planctomyces limnophilus, we identified that this locus encodes a fully-functional organelle that is involved in degrading fucose and rhamnose, 1 which is likely critical to the niche specialization of many Planctomycetes that associate with algae. We also found the widespread usage of a novel phosphotransacetylase enzyme in the BMCs. By analyzing the primary, secondary, and quaternary structures of this novel protein, we identify well-conserved motifs within distinct domains of the protein and identify a peptide that is involved in modulating the quaternary structure. One of the many uses of fundamental research is to provide the foundation for engineering synthetic constructs. Finally, we describe two bioengineering strategies geared towards the goal of improving the efficiency of the carbon fixation step of photosynthesis that are based on our knowledge of the cyanobacterial carbon concentrating mechanism, including the carboxysome. The first strategy focuses on the input for the carboxysome, where inorganic carbon must be transported into the cytosol. The second strategy utilizes a carboxysome assembly factor as a scaffolding mechanism. 2 Dedication I would like to dedicate this dissertation to my loving family, both living and passed, who shaped me as a person for my 22 years prior to attending graduate school and truly made this body of work possible before it had even started. My paternal grandmother, Mesrure Erbilgin*, for feeding me delicious “brain food.” My maternal grandfather, Mustafa Yücetürk*, for never failing to make me laugh and appreciate life. My paternal grandfather Ibrahim Erbilgin, whom I have to thank for teaching me the lesser-known “Einstein’s formula:” x + y + z = success + happiness, where x = working hard, y is playing hard, and z is realizing that what you say cannot be taken back (Coincidentally, x and y happened to be the mantra of my department, so I knew I was in the right place!). My maternal grandmother, Hayriye Yücetürk, for showing me how strong the human will can be in times of trouble when you truly are in the abyss. My mother, Süheyla Erbilgin, for instilling in me my “work before play” mindset, and pushing me to perform my best at everything I attempted. My father, Bülent Erbilgin, for teaching me that in order to solve a problem, you must first understand the fundamentals behind said problem. And last but not least, my sister Ayça Erbilgin- Köstem, for inspiring me to study biology, and doing her damnedest to teach me how to be socially adept and to live a life outside of scholarship. *Deceased i Table of Contents Abstract 1 Dedication i Table of Contents ii Acknowledgements iii Preface A one-page summary of what to expect in this dissertation v Chapter 1: Bacterial microcompartments and the modular construction 1 of microbial metabolism Chapter 2: A taxonomy of bacterial microcompartment loci 29 constructed by a novel scoring method Chapter 3: Characterization of a Planctomycetal Organelle: A Novel 73 Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides. Chapter 4: Characterization of PduL-like core phosphotransacetylases 110 Chapter 5: Harnessing fundamental knowledge for bioengineering 124 purposes Conclusions, and my Final Message 137 Appendix A: Supplemental Information for Chapter 2 138 Appendix B: Supplemental Information for Chapter 3 154 Appendix C: Supplemental Information for Chapter 4 169 Appendix D: Supplemental Information for Chapter 5 174 ii Acknowledgements First and foremost, I thank my advisor, Dr. Cheryl A. Kerfeld, for making it all happen; without her insight, guidance, and funding, I would never have been able to explore the fascinating and wonderful biology that is bacterial microcompartments and all of the tangents that followed. Cheryl was also helpful in my thought process regarding post- graduation and career planning; for that I am very grateful. I would like to thank all members of the Kerfeld lab for being great laboratory citizens, and training me in countless ways for countless skills, but especially Dr. Jeffrey C. Cameron, who taught me how to organize and conduct experiments and to be my own biggest critic, and Dr. Patrick M. Shih, who served as my older graduate student mentor and also my role model for the ideal scientist. Also: Seth Axen, without whom there would be no Chapter 2; Dr. Markus Sutter, for teaching me everything about protein purifications and crystallography; Susan Bernstein for keeping me well fed in the office with her delicious baked goods; Cristina Mora and Shreya Condamoor, my mentees, for helping me learn skills in mentorship and making me think about the best way to package information for the uninitiated. Dr. Kent L. McDonald and Dr. Luis R. Comolli are electron microscopists who both have such passion for their work that it always left me awestruck and constantly reminded me of why I pursued science in the first place. When I thanked Kent for helping me do the electron microscopy in Chapter 3 (which he actually did most of), he replied, “I just like looking at things nobody has seen before,” highlighting one of the purest reasons for studying the natural world. I would like to thank the members of my thesis and qualifying exam committees: Dr. Arash Komeili, Dr. Kathleen R. Ryan, Dr. John Taylor, and Dr. David F. Savage, for asking the tough questions and really getting me to think about the science, my experiments, and my data analysis. I am also very thankful and grateful for Arash stepping in to serve as a co-advisor when Cheryl moved to Michigan. I did not let that be just a formal title; Arash was one of the people I frequently went to for advice when the going got tough, grad school-wise. I have the following professors to thank for introducing me to the fantastical world of microbiology through our core courses: Dr. Kathleen Ryan, Dr. John Taylor, Dr. Steven Lindow, Dr. Arash Komeili, and Dr. John Coates. I would also like to thank the entire Plant and Microbial Biology faculty for being wonderful people and for all of the conversations we have had at retreats, coffee hours, and in the hallways. iii And finally, I would like to thank the Department of Plant and Microbial Biology, the University of California, and the National Science Foundation for providing the resources and funding to allow me to obtain my degree, and also be able to pay for food and rent while doing it. iv Preface A one-page summary of what to expect in this dissertation You may be wondering why this dissertation is titled “A Journey to the Center of a Bacterial Organelle.” Other than the fact that Jules Verne was one of my favorite authors as a child, I like how it stresses that my graduate experience was very much a journey. Chapter 1 sets the stage as a literature review and also serves as a prelude of what you will read in the following chapters, and its Figure 1 (shown here to the right) serves as a road map for the following chapters. Panels A, C, D, and E depict the topics in Chapter 2, where we begin by looking at the diversity of life, more specifically the diversity of the organelles in question, the bacterial microcompartments (BMCs), which have a range of different metabolic functions. Seth Axen and I computationally identified and classified all genetic loci that have the potential to form a BMC, based on the annotation of functional domains present in those loci, and used the resulting dataset to pull out any observed patterns. Chapter 2 leaves us with a laundry list of genetic loci to experimentally test. Panels D and E represent Chapter 3, where I delve into finding out what the function of a mysterious BMC locus is and why it exists (explaining it based on the environmental niche that the host bacterium occupies; panel A).
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