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View This Section Focuses on the Genomic and Proteomic Analyses That Were Performed on Methanolobus Vulcani B1d MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Adam John Creighbaum Candidate for the Degree Doctor of Philosophy ______________________________________ Dr. Donald J. Ferguson Jr, Director ______________________________________ Dr. Annette Bollmann, Reader ______________________________________ Dr. Xin Wang, Reader ______________________________________ Dr. Rachael Morgan-Kiss ______________________________________ Dr. Richard Page, Graduate School Representative ABSTRACT EXAMINATION AND RECONSTITUTION OF THE GLYCINE BETAINE- DEPENDENT METHANOGENESIS PATHWAY FROM THE OBLIGATE METHYLOTROPHIC METHANOGEN METHANOLOBUS VULCANI B1D by Adam J. Creighbaum Recent studies indicate that environmentally abundant quaternary amines (QAs) are a primary source for methanogenesis, yet the catabolic enzymes are unknown. We hypothesized that the methanogenic archaeon Methanolobus vulcani B1d metabolizes glycine betaine through a corrinoid-dependent glycine betaine:coenzyme M (CoM) methyl transfer pathway. The draft genome sequence of M. vulcani B1d revealed a gene encoding a predicted non- pyrrolysine MttB homolog (MV8460) with high sequence similarity to the glycine betaine methyltransferase encoded by Desulfitobacterium hafniense Y51. MV8460 catalyzes glycine betaine-dependent methylation of free cob(I)alamin indicating it is an authentic MtgB enzyme. Proteomic analysis revealed that MV8460 and a corrinoid binding protein (MV8465) were highly abundant when M. vulcani B1d was grown on glycine betaine relative to growth on trimethylamine. The abundance of a corrinoid reductive activation enzyme (MV10335) and a methylcorrinoid:CoM methyltransferase (MV10360) were significantly higher in GB-grown B1d lysates compared to other homologs. The glycine betaine:CoM pathway was fully reconstituted in vitro using recombinant MV8460, MV8465, MV10335, and MV10360. Demonstration of the complete glycine betaine:CoM pathway expands the knowledge of direct QA-dependent methylotrophy and establishes a model to identify additional ecologically relevant anaerobic quaternary amine pathways. EXAMINATION AND RECONSTITUTION OF THE GLYCINE BETAINE- DEPENDENT METHANOGENESIS PATHWAY FROM THE OBLIGATE METHYLOTROPHIC METHANOGEN METHANOLOBUS VULCANI B1D A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Adam J. Creighbaum The Graduate School Miami University Oxford, Ohio 2020 Dissertation Director: Donald J. Ferguson Jr., Ph. D. © Adam John Creighbaum 2020 TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v LIST OF COMMON ABBREVIATIONS viii DEDICATION ACKNOWLEDGEMENTS INTRODUCTION 1 CHAPTER 1. Examination of the glycine betaine-dependent 30 methylotrophic methanogenesis pathway: insights into anaerobic quaternary amine methylotrophy Chapter 1.1. Genomic and proteomic analysis of Methanolobus 31 vulcani B1d Chapter 1.2. Screening the function of MV8460, MV8465, 57 MV10335, and MV10360 from Methanolobus vulcani B1d Chapter 1.3. In vitro reconstruction of the glycine betaine:CoM 86 methylotrophic pathway from Methanolobus vulcani B1d APPENDIX I. Analyzing the interchangeability of the MtaA and RamM 105 with homologs from Methanococcoides methylutens Q3c, Methanosarcina acetivorans WWM73, Methanosarcina barkeri Fusaro, Methanomethylovorans hollandica to reconstruct the glycine betaine:CoM methyl transfer pathway from Methanolobus vulcani B1d. REFERENCES 135 iii LIST OF TABLES Table Title Page 1 Methylotrophy-associated proteins encoded 35 within Methanolobus vulcani B1d 2 Primers and plasmids 59 3 Proteins selected from M. barkeri Fusaro based on 113 transcriptomic data (López Muñoz et al., 2015) 4 Proteins selected from M. acetivorans WWM73 based on 114 transcriptomic data (Peterson et al., 2016) 5 Proteins selected from M. hollandica based on shotgun 115 Proteomic data and genomic analysis (Chen et al., 2017) 6 Primer sequences of the MtxAs and Rams 117 7 Current status of cloning, production, and activity testing 118 of the proteins selected for this study iv LIST OF FIGURES Figure Page 1 Schematic pathway of the three methanogenesis pathways 2 2 Three component system depicting demethylation 7 of a substrate 3 Interaction of MtaB and MtaC with the methyl 11 donor methanol 4 Phylogenetic tree of the COG5598 superfamily of enzymes 15 5 Proposed mechanism utilized by Desulfitobacterium 17 hafniense Y51 to demethylate glycine betaine and methylate tetrahydrafolate 6 Schematic depicting the Stickland reaction on glycine, 20 sarcosine, and betaine 7 Growth curve of Methanolobus vulcani B1d 25 8 Hypothetical methanogenesis pathways for the breakdown 27 quaternary amines 9 The genome of M. vulcani B1d encodes a single 36 homologous MttB that lacks pyrrolysine, mtgB (MV8460) 10 1. Proteomic analysis of likely candidate proteins for 38 glycine betaine-dependent CoM methylation 11 2. Proteomic analysis of likely candidate proteins for 40 glycine betaine-dependent CoM methylation 12 The genome of M. vulcani B1d encodes for an entire 44 methanol pathway (MeOH1) with all the essential genes within proximity of each other 13 The genome of M. vulcani B1d has three pairs of mtmBCs 51 and two pairs of mtbBCs 14 Examples of M. barkeri MS and M. acetivorans enzymes 53 involved in methylotrophy from methylated thiols v 15 The genome of M. vulcani B1d contains a mtsD/H/F 56 (MV10015) that could encode for a functional protein that could demethylate a methylated thiol compound 16 Gene sequence of the optimized gene encoding MV10360 62 from GenScript 17 A 12% acrylamide SDS-PAGE gel followed by Coomassie 69 blue staining of purified recombinant proteins used to reconstitute the glycine betaine:CoM methyl transfer pathway 18 Active site predictions of DhMtgB and MV8460 76 19 Predicted structural model of MV10350 compared to 78 known MtaB from Methanosarcina barkeri Fusaro 20 Glycine betaine:cob(I)alamin methyl-transfer activity 80 of MV8460 21 Reductive activation of MV8465 by MV10335 82 22 Methylcob(III)alamin:CoM methyl-transfer activity 84 by MV10360 23 Approximate-maximum likelihood representation of 93 the COG5598 MttB superfamily 24 Reconstitution of glycine betaine:CoM activity in vitro 95 with purified recombinant proteins 25 Glycine betaine:CoM activity in vitro using crude extracts 98 26 Representative figure of methanogenesis assays 100 performed on M. vulcani B1d 27 Proposed model of glycine betaine-dependent 104 CoM methylation 28 Relative activities of the MtbA and MtaA enzymes with 109 MttB from M. barkeri during TMA:CoM assays 29 Methylcob(III)alamin:CoM methyl-transfer activity 125 by MV1575 and MV1695 30 Methylcob(III)alamin:CoM methyl-transfer activity 127 by MM0619 vi 31 Representative figure of reconstitution of glycine 129 betaine:CoM activity in vitro with purified recombinant proteins 32 Approximate-maximum likelihood representation of the 132 MtxA phylogenetic tree 33 Approximate-maximum likelihood representation of the 134 Ram phylogenetic tree vii LIST OF COMMON ABBREVIATIONS Name Abb. Carbon dioxide CO2 Tetramethylammonium QMA Methyl-coenzyme M reductase Mcr Coenzyme M CoM Hydrogen H2 Reduced ferredoxin Fdred Methanofuran MFR Tetrahydromethopterin H4MPT Free thiol SH Coenzyme M methyltransferase Mtr Heterodisulfide reductase Hdr Oxidized ferredoxin Fdox Coenzyme B CoB Coenzyme A CoA Tetrahydrosarcinapterin H4SPT Trimethylammonium TMA Dimethylammonium DMA Monomethylammonium MMA Methylthiol:Coenzyme M Methyltransferase MtsA Methylcorrinoid:Coenzyme M Methyltransferase MtxA x: a = methanol; t = trimethylammonium; b = dimethylammonium m = monomethylammonium; s = methylated sulfurs, g = glycine betaine; q = tetramethylammonium Oxygen O2 Dissolved inorganic carbon DIC Triosephosphate isomerase TIM viii L-Pyrrolysine Pyl L-Pyrrolysine lacking non-Pyl Tetrahydrofolate THF Choline-TMA lyase CutC CutC activation enzyme CutD Glycine betaine GB Corrinoid activation enzyme Ram Glycine betaine transporter OpuD Kildalton kDa Methanol MeOH Calf-intestinal phosphatase CIP Multiple cloning site MCS Rotations per minute RPM Isopropyl β-D-1-thiogalactopyranoside IPTG Nitrogen N2 Genomic DNA gDNA ix DEDICATION I would like to dedicate this dissertation to Grace Eib. For those of you that do not know her, she is my high school sweetheart. She has been with me every step of the way since I left for my undergraduate studies at Manchester University. When I reflect on graduate school and my research that eventually resulted in this dissertation, I feel like Grace and I were in this together. Grace has been in college for the long haul too (Child Neurologist incoming!), and I think it is safe to say we have worked together through the pains of achieving our higher degrees even though our interests and paths were different. This work presented here focuses on a unique metabolic pathway from a methanogen, and Grace listened to this topic for 6 years(!) and never complained. I am no physician, but I do not think methanogenesis has much to do with Child Neurology. Instead, she was supportive and loving the entire time. I know for a fact I would have not finished the work that went into this dissertation had she not been there for me. I guess you could say that I am a pretty lucky person to have someone so remarkable in my life. This dissertation is for you, Grace. x ACKNOWLEDGEMENTS I would like to thank Dr. Joe Krzycki for many valuable discussions. I thank Dr. Annette Bollman and Dr. Xin Wang for their
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