Discoveries in the Biology of Oxidized Chlorine By Tyler P Barnum 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 John D. Coates, Chair Professor Jillian Banfield Professor Kara Nelson Fall 2019 © Copyright 2020 Tyler Barnum All rights reserved Abstract Discoveries in the Biology of Oxidized Chlorine by Tyler Patrick Barnum Doctor of Philosophy in Microbiology University of California, Berkeley Professor John D. Coates, Chair Chlorine participates in a biogeochemical cycle that rivals that of elements like nitrogen and sulfur in chemical diversity. Nature produces a variety of organic and inorganic chlorine-containing molecules that participate in different biological, chemical, and geological processes. The most defining feature of chlorine is its high electronegativity, perhaps best illustrated by the very high reduction potential of molecules in which - chlorine is at a higher oxidation state, such as hypochlorous acid (HOCl), chlorite (ClO2 - - ), chlorate (ClO3 ), and perchlorate (ClO4 ). Compared to research on the biogeochemical cycles for other elements, the mechanisms by which oxidized chlorine molecules are produced and consumed are relatively understudied. One reason is that descriptions of the biogeochemical chlorine cycle have been incomplete, omitting important processes involving inorganic chlorine molecules. Another reason is that the microbiology of oxidized chlorine has been studied almost exclusively through a reductionist approach that can preclude the discovery of ecological interactions. Yet another reason is that the genes known to be involved in the metabolism of oxidized chlorine have yet to be used to find new organisms and processes that metabolize oxidized chlorine. Here, a more holistic approach, enabled by improvements in genome sequencing, is used to better describe the biology of oxidized chlorine across several research projects. The first chapter of this dissertation provides the first review of the entire biogeochemical chlorine cycle, emphasizing connections between the various biological, chemical, and geological processes that interconvert chlorine between different chemical forms. The second chapter of this dissertation is a published research article describing the reduction of perchlorate in microbial communities for the first time by using bioinformatics techniques to obtain genomes from metagenomes. Instead of being dominated by the specific bacteria known to respire perchlorate, perchlorate-reducing communities contain diverse organisms that interact via the chemical intermediates of dissimilatory perchlorate reduction. The third chapter of this dissertation is a published research article investigating the mechanism of one such interaction between perchlorate-reducing bacteria and chlorate- 1 reducing bacteria. A combination of genomics, strain isolation, genetics, metabolite measurements, and theoretical modeling are used to learn that these two metabolisms, which have been studied separately for several decades, have a conserved interaction due to the accumulation of chlorate by perchlorate-reducing bacteria. The fourth chapter of this dissertation is a brief report characterizing a possible perchlorate reductase or chlorate reductase first identified in perchlorate-reducing communities. An organism with this reductase, which is always found in genomes adjacent to the chlorite-degrading enzyme chlorite dismutase (Cld), is capable of chlorate reduction but not perchlorate reduction, indicating the enzyme is a chlorate reductase. The fifth chapter of this dissertation extends the above comparative genomics analysis to identify any gene or organism linked to the enzyme Cld. Because Cld is biomarker for chlorite and other chlorine oxyanions, this approach was able to expand the environments, organisms, and processes known to participate in oxidized chlorine biology beyond the organisms and genes described above. Specifically, more was learned about the reduction of perchlorate and chlorate in the environment; the potential oxidation of chloride beyond hypochlorous acid by chemical or biological activity; and the connection between chlorite and reactive chlorine stress response. Together, this research has answered important questions about the reduction of chlorine while opening new questions about the oxidation of chlorine and the role of oxidized chlorine species in the environment. 2 Dedication To the next generation of scientists, who cannot perform science for their own curiosity but must perform science for the selfless betterment of our planet. i Acknowledgements I would like to thank those who made this research possible. The National Science Foundation provided financial support for this work through its Graduate Research Fellowship Program. My development as a scientist and research was buoyed by dedicated staff in the Department of Plant & Microbial Biology and by broader programming available at U.C. Berkeley for better teaching, science, and programming. Such institutional support greatly enabled better scientific inquiry. My thesis committee helped me prioritize research projects and charitably made themselves available to help me. Dr. Kara Nelson, my outside committee member, volunteered her time and attention to make sure my degree and research were advancing appropriately. Dr. Jill Banfield was the reason I applied to U.C. Berkeley and is a role model for using genomics techniques with scientific rigor. Dr. John Coates, my advisor, provided invaluable expertise in microbial physiology while learning new methodologies to help me pursue questions in different areas of microbiology. Participants in this research from the John Coates’s lab were many. Lauren Lucas, Dr. Charlotte Carlström, and Anna Engelbrektson performed the enrichments and isolations that initiated chapters two and three of this dissertation. Dr. Israel Figueroa helped design some of the bioinformatics methods used in chapter two. Kaisle Hill helped me plan and execute experiments in chapter three. An outside collaborator, Yiwei Cheng, provided expertise in modeling for chapter three. Other help came from my graduate student peers, Dr. Ouwei Wang, Dr. Maggie Stoeva, Victor Reyes-Umaña, Sophia Ewens, and Dr. Matthew Olm, who provided feedback on many chapters and general support. Yi Liu ensured a functioning laboratory for conducting research. Finally, in helping me critically think through different experiments and conclusions, Dr. Hans Carlson acted as a de facto fourth committee member. Everyone has something to teach others, and the scientific and personal role models that I learned from have been too many to list here. Many people have the potential to do what I have done but did not reach this stage. For that, I owe a stable early childhood in which my parents, family, and friends allowed my curiosity to blossom. I received the benefit of state-supported advanced curriculum in excellent public elementary, middle, and high schools staffed by many wonderful, caring, dutiful teachers. Johns Hopkins University accepted this boring Indiana boy, unlike other elite institutions, and offered me need-based tuition plus additional support through a Bloomberg Scholarship when I needed it most. I owe my intellectual heritage to my educators and peers at Johns Hopkins, who are truly world-class in character. In particular, Dr. Jocelyne DiRuggiero gave me the confidence and preparation for graduate school that every undergraduate student hopes for. I acknowledge these unearned advantages and promise to help others throughout my life. ii Table of Contents Preface 1 Chapter 1 2 Unifying the biogeochemical chlorine cycle Chapter 2 18 Genome-resolved metagenomics identifies genetic mobility, metabolic interactions, and unexpected diversity in perchlorate-reducing communities Chapter 3 37 Identification of a respiratory symbiosis in the biogeochemical chlorine cycle Chapter 4 53 An uncharacterized clade in the DMSO reductase family of molybdenum oxidoreductases is a new type of chlorate reductase Chapter 5 59 Insights into the chlorine cycle through comparative genomics of chlorite dimutase Chapter 6 85 Future Directions Chapter 7 87 Conclusions iii Preface Because the first chapter of this dissertation does not serve as a traditional introduction, I would like to explain the motivation behind my research. Perchlorate and chlorate have long been identified as pollutants addressed through biological remediation. When I began this research, how bacteria degrade perchlorate and chlorate through metabolic respiratory pathways had been adequately explained. Only a few questions remained about how it occurs: how other organisms in communities may affect the behavior of perchlorate-reducing bacteria and chlorate-reducing bacteria, and if there are a greater variety of organisms and enzymes that participate in this process than currently understood. Answering these questions motivated chapters two, three, and four of this dissertation and could only be enabled by recently developed metagenomic approaches to peer into microbial communities and predict metabolic activity in different organisms. Underlying that research, though, were broader questions about the context of perchlorate and chlorate reduction. How do these metabolisms occur in the natural environment? How do they fit into the global biogeochemical cycle of chlorine? How are the enzymes