ASPECTS OF SULFUR METABOLISM OF METHANE-PRODUCING ARCHAEON METHANOCOCCUS MARIPALUDIS by FENG LONG (Under the Direction of William B. Whitman) ABSTRACT Sulfur is vital for the growth of all known organisms and is present in a wide variety of metabolites with different physiological functions. Consistent with their obligate anaerobic habitats, most methanogenic Archaea only assimilate sulfide and elemental sulfur as sulfur sources, whereas sulfate and other oxidized sulfur compounds are rarely utilized. The methanogenic lifestyle may have evolved from ~ 3.5 billion years ago, and contemporary methanogens may have preserved some of the metabolic relics which were common in the early Earth anaerobic lifestyles. Recent studies have revealed multiple novel traits of sulfur assimilation in methanogens. However, many aspects of the sulfur assimilation processes and their regulations remain to be investigated. Thereby, the study of the physiology and biochemistry of the sulfur metabolism in methanogens may provide new insights into the biology of ancient microbial life. Methanococcus maripaludis is unable to assimilate sulfate as a sulfur sole and does not produce sulfate when grew with sulfide as the sole sulfur source. Nevertheless, Methanococcus maripaludis possesses homologs of proteins involved in the sulfate assimilatory reduction pathway. None of these proteins was functional in the Escherichia coli mutant strains deficient in sulfate assimilation metabolism. These results indicated that the assimilatory sulfate reduction pathway is most unlikely to be present in Methanococcus maripaludis. When grown with elemental sulfur as the sole sulfur source, Methanococcus maripaludis produced sulfide at about 6 mmol per g cell dry weight per hour. Moreover, adenylyl-sulfate reductase (MMP1681), an enzyme that contains an iron-sulfur cluster, was found to be required for elemental sulfur assimilation. Furthermore, proteomics data indicated that the expression of this protein increased three-fold during growth with elemental sulfur in comparison to growth with sulfide. Together with bioinformatics analysis, a different physiological role of MMP1681 in elemental sulfur assimilation, in addition to its in vitro catalytic function as an adenylyl-sulfate reductase was demonstrated. Although the explicit mechanism by which MMP1681 participating in the elemental sulfur incorporation process remains to be elucidated, this evidence advances our understanding of how methanogen possessing MMP1681 assimilate elemental sulfur into key sulfur intermediates. INDEX WORDS: Archaea, methanogens, Methanococcus maripaludis, sulfur metabolism, elemental sulfur, sulfide, sulfate, assimilatory sulfate reduction, iron-sulfur clusters. ASPECTS OF SULFUR METABOLISM IN METHANE-PRODUCING ARCHAEON METHANOCOCCUS MARIPALUDIS by FENG LONG BS, Nanjing Forestry University, China, 2009 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2017 © 2017 Feng Long All Rights Reserved ASPECTS OF SULFUR METABOLISM IN METHANE-PRODUCING ARCHAEON METHANOCOCCUS MARIPALUDIS by FENG LONG Major Professor: William B. Whitman Committee: Diana Downs Jorge C. Escalante-Semerena Ellen L. Neidle Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia December 2017 DEDICATION To my grandparents, Long Yongqiu and Liu Donge, my parents, Long Jianming and Ouyang Mei, and my husband, Wu Yinghui, for their endless love, patience and support every step of the way. I am grateful for every sacrifice you have made to provide me with the best life possible. I love you more than words can describe. iv ACKNOWLEDGEMENTS First, I would like to thank my advisor Dr. William B. Whitman, who brought me into the Microbiology department and coached me on how to be a better scientist. He introduced me to the field of Archaea research and taught me the principles of research. His talent, passion, self- disciple and dedication to his work always inspired me when performing my research. Without his invaluable guidance, support and encouragement both in research and life, I would not have been able to make it this far. I would like to thank my doctoral committee; Dr. Diana Downs, Dr. Jorge C. Escalante-Semerena, and Dr. Ellen L. Neidle for their constructive suggestions all these years. My special thanks go to Dr. Yuchen Liu, she was always there to share her valuable research experiences, informative discussions, and great ideas! Many thanks to Dr. Hannah Bullock for her company as a great lab colleague and best friend at UGA. I would like to thank Dr. Zhe Lyu for teaching me many of the techniques used in the laboratory and bioinformatic tools. I would also like to thank Dr. Michael Adams for helping me with data analysis and providing a great deal of insights in sulfur metabolism through my research. Furthermore, I thank many of the past and present members of Whitman lab including Felipe Sarmiento, Joseph S. Wirth, Liangliang Wang, Warren Crabb, Nana Shao, Tao Wang, Taiwo Akinyemi, Hao Shi, Qiuyuan Huang, Yixuan Zhu, Suet Yee Chong, Peiying Chang, Ghazal Motakef, Dahyun Ji, Hirel B. Patel, Lola Osiefa, Courtney Ellison and Courtney Grant Jr. for their help, friendship, and encouragement. I thank Dr. Wendy A. Dustman, Dr. Anna C. Glasgow Karls and Dr. Jennifer Walker, for their support in my teaching certificate application. I also thank my classmates Ajay Arya, Nicole Laniohan, v Christopher Abin, Julie Stoudenmire, Christopher Cotter and the rest of the Microbiology Department. Lastly, I would like to thank my family and all my friends for being a constant source of encouragement, support, and friendship all the way along. Thank you all for being the best memories in my life! vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 2 A FLEXIBLE SYSTEM FOR CULTIVATION OF METHANOCOCCUS AND OTHER FORMATE-UTILIZING METHANOGENS ...............................................50 3 AN ADENYLYL-SULFATE REDUCTASE IN METHANOCOCCUS MARIPALUDIS, CONTAINS AN IRON-SULFUR CLUSTER, AND IS REQUIRED FOR ELEMENTAL SULFUR ASSIMILATION .......................................................71 4 EXPLORING THE USE OF GENOME COMPARISONS OF WILD-TYPE AND RESISTANT MUTANTS OF METHANOCOCCUS MARIPALUDIS TO IDENTIFY POTENTIAL TARGETS OF INHIBITORY COMPOUNDS ..................................129 5 CONCLUSIONS........................................................................................................156 APPENDICES A CHAPTER 2 SUPPLEMENTARY INFORMATION ..............................................159 B CHAPTER 3 SUPPLEMENTARY INFORMATION ..............................................179 C CHAPTER 4 SUPPLEMENTARY INFORMATION ..............................................214 vii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Introduction Methanoarchaea, or methanogens are strictly anaerobic microorganisms that form methane as a major product of their energy metabolism (1). They serve as key agents catalyzing transformations of organic carbon on earth and are responsible for producing most of the methane found in the Earth’s atmosphere (1). Methanogens originate from the early, anoxic earth and share several unique features (1, 2): (i) they are all Archaea; (ii) they produce methane as the end-product in their anaerobic respiration-methanogenesis, which provides all or most of the energy for supporting their growth; (iii) they utilize a restricted number of substrates for methanogenesis: mainly CO2 + H2 and/or formate (hydrogenotrophic methanogens), methyl-containing C-1 compounds (methylotrophic methanogens) and acetate (aceticlastic methanogens). They do not use sugars, amino acids, or most other common organic substrates; (iv) they can be found in a diverse range of anaerobic environments, including marine sediments, flooded soils, gastrointestinal tracts, anaerobic digesters, landfills, geothermal systems, and heartwood of trees. Methanogenesis may represent one of the most ancient metabolisms and likely contributed a significant role in the evolution of the Earth’s atmosphere. Although it is unknown when methanogens first appeared on Earth, the oldest evidence of their emergence was 3.5 billion years ago (Ga), 1.1 Ga before oxygen appeared (3). Several lines of evidence have suggested that methanogens retain unique metabolic features that may have been common in the anoxic early Earth. Frist, methanogenic archaea may have a unique origin. The methanogenesis pathway of 1 producing methane requires at least 25 core genes and more than 20 biochemically characterized proteins involved in the biosynthesis of coenzymes. Genes encoding different subunits of an enzyme are usually clustered together in the genome, but these clusters and genes are scattered around the methanogen genome (4). Phylogenetic analysis has found that these core methanogenic enzymes likely originated (as a whole) from the last ancestor of all methanogens and do not seem to have been horizontally transmitted to other organisms or between the methanogenic classes (5). Nonetheless, part of the pathways involved in methanogenesis, as well as single genes, may have been transferred across diverse phylogenic lineages (6). Second, several methanogenic and biosynthetic enzymes are O2-sensitive, such as methyl-coenzyme