Iron As an Integral Constituent of Ancient Metabolism and Biochemistry

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Iron As an Integral Constituent of Ancient Metabolism and Biochemistry IRON AS AN INTEGRAL CONSTITUENT OF ANCIENT METABOLISM AND BIOCHEMISTRY A Dissertation Presented to The Academic Faculty by Marcus Salvatore Bray In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Biology Georgia Institute of Technology August 2019 COPYRIGHT © MARCUS SALVATORE BRAY 2019 IRON AS AN INTEGRAL CONSTITUENT OF ANCIENT METABOLISM AND BIOCHEMISTRY Approved by: Dr. Jennifer B. Glass, Advisor Dr. Loren Dean Williams, Co-Advisor School of Earth & Atmospheric Sciences School of Chemistry & Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Joel E. Kostka Dr. Kostas T. Konstantinidis School of Biological Sciences, School of School of Civil & Environmental Earth & Atmospheric Sciences Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Frank J. Stewart School of Biological Sciences Georgia Institute of Technology Date Approved: April 25th, 2019 “Into the desert, with only a head and some crossing wires to trace. Here's the world - the one that kills our fathers, and that births our children. May we forever wind, crawl and stumble below the stars, barely living before we shed our mind to teach ourselves anew.” -anonymous ACKNOWLEDGEMENTS I would first like to express my deep gratitude to both of my thesis advisors, Dr. Jennifer B. Glass and Dr. Loren Dean Williams for their continued guidance, support, and mentorship. I would also like to thank all the members of my thesis committee: Dr. Frank Stewart, Dr. Joel Kostka, and Dr. Kostas Konstantinidis for their service and support. I would like to thank members of both the Glass and Williams labs, with whom I had the pleasure of working closely during my time at Tech. I would especially like to thank Dr. Nadia Szeinbaum, and Jessica Bowman, for helpful discussion on experimental design and scientific theory. I would like to thank the amazing undergraduate students with whom I worked including Miles Mobley, Claudia Montllor Albalate, and Bianca Costa, for all their help with my research. I would also like to thank members from other labs, including the Kostka Lab, the Stewart Lab including Dr. Cory Padilla and Dr. Anthony Bertagnolli, the DiChristina lab including Ben Reed and Yael Toporek, and Lieberman lab, for helpful discussion and graciously allowing me access to their equipment. I would also like to thank Dr. Amit Reddi for helpful discussion and collaboration with Total Reflective X-ray Spectroscopy, as well as Dr. Roger Wartell for helpful discussion and troubleshooting of experiments. I would like to thank Sean Crowe from the University of British Columbia for helpful discussion and providing us with Lake Matano samples as well as members of his lab including Dr. Rachel Simister and Kate Thompson for help with experiments. I would also like to thank Dr. Corinna Tuckey from New England Biolabs for many helpful discussions. The research presented in this thesis was funded by NASA Exobiology grants NNX14AJ87G and NNX16AJ28G. Finally, I would like to sincerely thank my wife, iv Kristina L. Leyva, and my parents, Jeffrey and Anna Bray for their love, support, and most importantly sacrifice, without which none of this would have been possible. v TABLE OF CONTENTS ACKNOWLEDGEMENTS iv LIST OF TABLES x LIST OF FIGURES xii LIST OF SYMBOLS AND ABBREVIATIONS xv SUMMARY xvii CHAPTER 1. Introduction: Iron and the ancient biosphere 1 1.1 The Archean eon 1 1.2 Sources and availability of iron in the Archean 2 1.3 Iron in ancient biochemistry 5 1.4 Iron in ancient metabolism 7 CHAPTER 2. Multiple prebiotic metals mediate translation 9 2.1 Abstract 9 2.2 Introduction 9 2.3 Experimental Methods 14 2.3.1 rRNA SHAPE reactions. 14 2.3.2 In vitro translation. 14 2.3.3 Translation reaction buffer. 15 2.3.4 Translation experimental conditions. 17 2.3.5 mRNA template. 17 2.3.6 Protein activity assay. 17 2.3.7 Ribosome metal content. 18 2.3.8 Quantum mechanical calculations. 19 2.4 Results 21 2.4.1 Fe2+ and Mn2+ fold LSU rRNA to a near-native state. 21 2.4.2 Fe2+ and Mn2+ mediate translation. 24 2.4.3 Iron and Mn associate extensively with the ribosome. 29 2.5 Discussion 31 2.5.1 Fe2+ and Mn2+ can replace Mg2+ as the primary ribosomal cofactor. 31 2.5.2 Physiological relevance of these results. 32 2.6 Conclusions 33 CHAPTER 3. Functional iron-containing ribosomes under low oxygen, high iron conditions 34 3.1 Abstract 34 3.2 Introduction 35 3.3 Experimental Methods 36 3.3.1 Cell culture and harvesting. 36 3.3.2 Ribosome purification. 37 vi 3.3.3 Ribosomal iron content. 38 3.3.4 Ribosomal RNA purification and electrophoresis. 38 3.3.5 Ribosomal protein electrophoresis. 39 3.3.6 In vitro translation. 39 3.3.7 Protein identification by LC-MS/MS. 40 3.4 Results 42 3.5 Discussion 49 3.5.1 Iron in pre-GOE ribosomes. 49 3.5.2 Tightly-bound ribosomal iron likely occupies dinuclear microclusters. 51 3.5.3 Iron is most available for ribosomal association under pre-GOE conditions. 52 3.5.4 Differential expression of ribosome associated proteins under pre-GOE conditions. 53 3.6 Conclusions 54 CHAPTER 4. Microbial iron and methane dynamics and their effect on early earth habitability 59 4.1 Abstract 59 4.2 Introduction 60 4.3 Experimental Methods 62 4.3.1 Sample collection and storage. 62 4.3.2 Enrichment medium and substrate synthesis. 62 4.3.3 Inoculation of enrichments and amendments. 63 2+ 3+ 2+ 4.3.4 HCl extractable Fe and Fe and soluble Fe . 64 4.3.5 Methane oxidation. 64 4.3.6 Headspace methane. 65 4.3.7 Inductively coupled plasma mass spectrometry. 65 4.3.8 16S rRNA gene amplicon sequencing. 66 4.4 Results 67 4.4.1 Iron reduction. 67 4.4.2 Trace metal concentrations. 70 4.4.3 Methane production. 71 4.4.4 Methane oxidation. 73 4.4.5 Microbial taxonomy. 76 4.5 Discussion 79 4.5.1 Fe(III) reduction rates in long-term ferruginous sediment incubations. 79 4.5.2 Fe(III) oxide mineralogy controls methane production and methanogen taxonomy. 79 4.5.3 Fe(III)-dependent CH4 oxidation. 80 4.5.4 Effect of Fe(III) oxide and carbon substrates on microbial community diversity. 82 4.5.5 Nickel sources. 83 4.5.6 Geobiological implications. 84 4.6 Conclusions 85 CHAPTER 5. Phylogenetically and structurally diverse electroactive pili from anoxic ecosystems 86 5.1 Abstract 86 vii 5.2 Introduction 87 5.3 Experimental Methods 89 5.3.1 Sampling and enrichment of Lake Matano sediment. 89 5.3.2 DNA extraction and metagenome sequencing, assembly, binning and annotation. 90 5.3.3 Pilin identification from microbial metagenomes. 91 5.3.4 Pilin sequence and phylogenetic analysis. 91 5.3.5 Phylogenetic tree construction. 92 5.3.6 Pilus modelling. 92 5.4 Results 93 5.4.1 Aromatic density distinguishes e-pilins from other type IV pilins. 93 5.4.2 Putative e-pilins can be a significant portion of type IV pilins in natural environments and are longer and more aromatically dense than characterized e-pilins. 96 5.4.3 Sediment incubations enrich for different e-pilins than those present in the environment. 98 5.4.4 Putative e-pilins are phylogenetically diverse. 100 5.4.5 Identification of pilins from Lake Matano sediment enrichment MAGs. 103 5.4.6 Structural modelling of predicted e-pili. 106 5.5 Discussion 108 5.5.1 Expanding the phylogenetic diversity of e-pili. 108 5.5.2 The diversity of pilin structure and characteristics is larger than previously thought. 109 5.5.3 Laboratory incubations enrich for truncated Deltaproteobacterial e-pilins. 109 5.5.4 PilA proteins in enrichment metagenomes. 110 5.5.5 Modelling for pilus structural and functional prediction. 110 5.5.6 Possible functions for aromatically dense pilins besides electroactivity. 112 5.6 Conclusions 113 CHAPTER 6. Iron in the past, present, and future of life on Earth 114 6.1 Expanding the role of Fe in biochemistry 114 6.2 Iron influenced planetary habitability in the Archean 115 6.3 Electroactive pili may be a diverse and important Fe reduction strategy 116 APPENDIX A. Enrichment and isolation of perchlorate reducing organisms from Lake Matano 119 A.1 Introduction 119 A.2 Experimental Methods 120 A.2.1 Samples collection and storage. 120 A.2.2 Enrichment medium. 121 A.2.3 Inoculation and incubation of sediments. 121 A.2.4 δ13C-DIC analysis. 122 A.2.5 Acetate analysis. 122 A.2.6 Isolation procedure. 122 A.3 Results 123 A.3.1 Perchlorate stimulates acetate production. 123 A.3.2 Isotopic enrichment of 13C-DIC. 124 viii A.3.3 Isolation of perchlorate-reducing organisms. 126 A.4 Discussion 127 APPENDIX B Enrichment of organisms mediating iron oxidation and nitrous oxide reduction 129 B.1 Introduction 129 B.2 Experimental Methods 129 B.2.1 Sampling and incubation of inoculum sediments. 129 B.2.2 Enrichment medium. 130 B.2.3 Inoculation and incubation of enrichment cultures. 130 B.2.4 Ferrous iron measurements. 131 B.2.5 Headspace N2O 131 B.3 Results and Discussion 131 B.3.1 Iron oxidation. 131 B.3.2 Nitrous oxide reduction. 132 B.3.3 Turbidity and colour change of cultures 135 APPENDIX C Imaging cells in Lake Matano sediment enricHments using fluorescence in situ hybridization 136 C.1 Introduction 136 C.2 Experimental Methods 136 C.2.1 Fixing of samples. 136 C.2.2 Nonselective DNA staining.
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