MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Tomislav Ticak Candidate for the Degree Doctor of Philosophy _________________________________________ Director Dr. Donald J. Ferguson _________________________________________ Reader Dr. Gary R. Janssen _________________________________________ Reader Dr. Natosha L. Finley _________________________________________ Dr. Annette Bollmann _________________________________________ Graduate School Representative Dr. Carole Dabney-Smith ABSTRACT ANOXIC QUATERNARY AMINE UTILIZATION BY ARCHAEA AND BACTERIA THROUGH A NON-L-PYRROLYSINE METHYLTRANSFERASE; INSIGHTS INTO GLOBAL ECOLOGY, HUMAN HEALTH, AND EVOLUTION OF ANAEROBIC SYSTEMS by Tomislav Ticak Quaternary amines are compounds which are important for every domain of life and play roles as carbon and nitrogen sources but also are known to act as osmoregulants. One quaternary amine, glycine betaine, is considered a key osmoregulatory compound due to its chemical nature and is often the main intersection of choline and carnitine metabolism, both aerobically and anaerobically. Many organisms have the capability of degrading glycine betaine through oxygenases or dehydrogenases aerobically, but there is little literature related to the fate of glycine betaine in anaerobic systems. Many of the reported anaerobic systems for glycine betaine involve a reductase pathway that leads to the formation of trimethylamine and acetate, which are well established methanogenic precursor compounds in anaerobic environments. However, there exist a few reports of acetogens and methanogens with the capability of converting glycine betaine to dimethylglycine, which is a strict deviation from the aformentioned reductase pathway. This suggests a pathway exists for anaerobic glycine betaine metabolism that has largely gone uncharacterized. We used a series of bioinformatic, biochemical, and physiological experiments to examine carbon metabolism in Desulfitobacterium hafniense strain Y51 and demonstrated its ability to perform this novel mechanism of glycine betaine metabolism. We proposed that non-L-pyrrolysine trimethylamine methyltransferases may act as quaternary amine methyltransferases. As a result of this study; we discovered a theoretical key in explaining the evolution of the glycine betaine and trimethylamine methyltransferases regarding incorporation of L-pyrrolysine. The fact that a quaternary amine (e.g., glycine betaine) may bind into the near identical location of the proposed trimethylamine-pyrrolysine adducts may help us to better understand this widespread superfamily of methyltransferases. By using our knowledge of the glycine betaine methyltransferase, we began to investigate anaerobic communities for the presence of these methyltransferase genes by enrichments with quaternary amines resulting in the discovery of methanogens capable of glycine betaine, choline, and tetramethylammonium metabolism. Genomic analysis of these organisms revealed the presence of glycine betaine and trimethylamine methyltransferase-like genes supporting the hypothesis of quaternary amine demethylation by non-L-pyrrolysine methyltransferases. Our future work now points toward the examination of microbial distribution and physiology for anaerobic quaternary amine utilization in human systems, marine and freshwater environments to determine the evolutionary pressure(s) that may have selected for the advent of L-pyrrolysine. ANOXIC QUATERNARY AMINE UTILIZATION BY ARCHAEA AND BACTERIA THROUGH A NON-L-PYRROLYSINE METHYLTRANSFERASE; INSIGHTS INTO GLOBAL ECOLOGY, HUMAN HEALTH, AND EVOLUTION OF ANAEROBIC SYSTEMS A Dissertation Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Tomislav Ticak Miami University Oxford, OH 2015 Dissertation Director: Donald J. Ferguson, Ph.D. TABLE OF CONTENTS LIST OF TABLES iii LIST OF FIGURES iv LIST OF COMMON ABBREVIATIONS vii DEDICATION ix INTRODUCTION 1 CHAPTER 1. A nonpyrrolysine member of the widely distributed trimethylamine 32 methyltransferase family is a glycine betaine methyltransferase CHAPTER 2. Isolation and characterization of a tetramethylammonium-degrading 78 Methanococcoides strain and a novel glycine betaine-utilizing Methanolobus strain CHAPTER 3. Analysis of the function of L-Pyrrolysine within the trimethylamine 125 methyltransferase superfamily (COG5598) by comparison to the glycine betaine methyltransferase APPENDIX A. Cloning and expression of auxiliary genes predicted to play roles 147 in quaternary amine-dependent methylotrophy in Desulfitobacterium hafniense Y51 CONCLUDING REMARKS AND FUTURE DIRECTIONS 153 REFERENCES 164 ii LIST OF TABLES Table Page 1 Gene-specific qRT-PCR and cloning primers used in this study 42 2 Comparative analysis of several Methanolobus species to B1d 95 3 Comparative analysis of several Methanococcoides species to Q3c 96 4 Primers for the generation of expression vectors in pSpeedET and pDL05c 150 iii LIST OF FIGURES Figure Page 1 Structure of base amine molecules and structures of relevant amine compounds 3 2 Overview of pathways of Car and Cho conversion to GB by microbes 5 3 Proposed mechanism of the glycine betaine/sarcosine/glycine reductase system 8 4 Schematic of methylamine dehydrogenase or oxygenases for the formation of 13 formaldehyde prior to downstream pathways 5 Overview of methanogenic pathways 17 6 Modfied vanillate:THF C1 pathway of D. hanfiense strain DCB-2 19 showing oxidation of a CH3 group from a methylated pterin molecule 7 Proposed methylotrophic models for both archaea and bacteria 21 8 Neighbor-joining 16S rRNA phylogenetic tree of the genus Desulfitobacterium 26 9 Proposed formation of the methylamine-Pyl adducts in the methylamine 30 methyltransferase during enzymatic catalysis 10 The genomic context of mttB genes suggests a role in quaternary amine metabolism 36 11 DSY3156 and DSY3157 were purified to near-homogeneity 45 12 MtgA (DSY3157) is a methylCbl:THF methyltransferase 49 13 Growth of D. hafniense Y51 in the presence of glycine betaine 52 (GB) and either fumarate (A) or nitrate (B) 14 Thin-layer chromatographic analysis of D. hafniense culture supernatants 54 15 Hypothetical pathway for the conversion of glycine betaine 56 (GB) to dimethylglycine and CO2 by D. hafniense Y51 iv 16 DSY3156 is a glycine betaine:cob(I)alamin methyltransferase 60 17 Michaelis–Menten kinetics of recombinant DSY3156 62 18 Stoichiometric demethylation of GB to produce DMG and methylCbl 65 19 Phylogenetic tree of the COG5598 Superfamily 68 20 Proposed functional relationship between MtgB and MttB 71 21 Gel electrophoresis of 16S rRNA and mcrA products 91 22 Maximum likelihood trees showing the phylogenetic position of strains B1d 93 and Q3c in relation to the most closely related organisms, based on the partial 16S rRNA gene sequence (A) or partial McrA amino acid sequence (B) 23 Microscopic examination of strains B1d and Q3c 97 24 Effect of increasing GB or QMA concentrations on the growth of 100 Methanolobus vulcani B1d and Methanococcoides methylutens Q3c 25 Growth curves are presented showing changes in OD600 as well as quaternary 103 amine and methane concentrations over time for strains B1d (A) and Q3c (B) 26 Subsystem profile of methanogen isolates generated with RAST 105 27 Proposed quaternary amine metabolic schema for methanogens 108 28 Genomic context of putative dimethylsulfide operons for B1d (A) and Q3c (B) 110 29 Maximum-likelihood tree of the COG5589 superfamily amended with non-Pyl 112 and Pyl MttBs from strains B1d and Q3 30 Gene neighborhoods of mttiB genes in strains B1d (A) and Q3c (B). 115 31 Proposed pathway of QMA breakdown in archaea and bacteria 121 32 Proposed mechanism of MMA catalysis by MtmB. 128 33 Structural superpositioning of the MtmB and MetH crystal structures. 130 34 Structure of the Escherichia coli ProX transporter with bound GB 133 35 Structural overlay of Pyl’s location within each MtxB MT 138 v 36 Proposed positioning of GB into MtgB apo-structure based on 141 Q-Site Finder analysis 37 SDS/PAGE of heterologously expressed D. hafniense Y51 MttB enzymes 151 vi LIST OF COMMON ABBREVIATIONS Name Abb. Quaternary amines QA Trimethylamine TMA Glycine betaine GB L-pyrrolysine Pyl Methyltransferase MT Choline Cho Carnitine Car Dimethylglycine DMG Sarcosine MMG Sulfur Reducing Bacteria SRB Monomethylamine MMA One-carbon C1 Dimethylethanolamine DMEA Dimethylamine DMA Tetramethylammonium QMA 2-mercaptoethanesulfonate CoM Knock-out KO Betaine/Choline/Carnitine Transporter BCCT Amine/Polyamine/Organocation APC vii Cluster of orthologous genes COG Corrinoid-binding protein CBP Cobalamin Cbl Tetrahydrofolate THF / FH4 Horizontal gene transfer HGT Last Universal Common Ancestor LUCA Open reading frame ORF Site-directed mutagenesis SDM Gas chromatography GC Reducing equivalents [H] Brackish media BM High-salt media HM 2QNE MtgB 1NTH MtmB Amino acid aa Base pair bp Kilodalton kDa viii DEDICATION I dedicate this work to all those who supported me through these long years of my graduate career. I thank my friends, mentors, co-workers, and family for continually believing in my capabilities as a scientist and as an educator. I finally would like to dedicate this work to all those who follow after me, as I hope this work will provide you the insights to understanding the microbial one-carbon world that captivated me. ix INTRODUCTION Quaternary amine (QA) utilization by microbes is well-understood