GENOMIC AND MECHANISTIC STUDIES OF (HYPER)THERMOPHILIC DISSIMILATORY IRON - REDUCING BACTERIA AND ARCHAEA By MICHAEL P. MANZELLA A DISSERTATION Submitted to Michigan State University in partial fulfillment of th e requirements for the degree of Microbiology and Molecular Genetics Doctor of Philosophy 2015 PUBLIC ABSTRACT GENOMIC AND MECHANISTIC STUDIES OF (HYPER)THERMOPHILIC DISSIMILATORY IRON - REDUCING BACTERIA AND A RCHAEA By MICHAEL P. MANZELLA The majority of life on our planet is thanks to the sun. From trees to microscopic algae, all because they are at the base of the food chain. As you proceed up the food chain life does not rely directly on the sun and instead feeds off of these primary producers. Yet, sites exist on our planet where life exists without any influence from the sun. D eep - sea environments, called hydrothermal vents, are one such example. core bubbles up to the sea floor and supports a community of microbes and larger organisms , some of which can grow at extremely high temperatures . These communities are often found thousands o f meters below the ocean surface, and they too rely on primary producers. These primary producers do not rely on the sun and instead get food and energy from the fluids that come out of the vents. The microorganisms at these sites are thought to be ancient , and thus studying them can help us understand how life originated on our pl anet. I chose one microorganism from each of the other Eukarya , do not belong the Bacteria and the Archaea . I se quenced their DNA and searched for tools they use to survive at insoluble iron oxides, rust. This rust is a challenge to breathe, as it cannot enter the ir cells like oxygen can. Fortunately, I found a number of tools which could allow them to use iron for growth. Most interestingly among the tools I found wer e microscopic hairs on the outside of the cell that work like metal wires and allow the cells to p ass electricity to the iron, thereby breathing it at a distance temperatures and within the Archaea . ABSTRACT GENOMIC AND MECHANISTIC STUDIES OF (HYPER)THERMOPHILIC DISSIMILATORY IRON - REDUCING BACTERIA AND ARCHAEA By MICHAEL P. MANZELLA Some members of the Bacteria and the Archaea have the rare ability to transfer electrons beyond the surface of their cells. This extracellular electro n transfer permits the reduction of otherwise inaccessible electron acceptors such as insolub le Fe(III) oxides. T he mechanisms that enable this ability have direct implications for geochemical cycles today and for life on early Earth. The physical settings present on early Earth, hot and influenced primarily by hydrothermal activity, can be found in rare sites on modern Earth. These sites most often surround deep - sea hydrothermal vents . Organisms present at these sites thrive, in most cases, absent of the s un influence . Instead of primary production from photosynthetic microorganisms, these communities rest on the shoulders of chemoautolithotrophic bacteria and archa ea. Two of these organisms, the thermophilic bacterium Geothermobacter ehrlichii and the hy perthermophilic archaeon Geoglobus ahangari were selected to undergo genome and physiological characterization to determine how they interact with the abundant insoluble Fe(III) oxides found at hydrothermal vents . Both genomes were sequenced and, while onl y the genome of G. ahangari was complete, this permitted identification of critical components for iron respiration. In addition, mechanistic studies were performed on G. ahangari to elucidate a direct - contact mechanism of iron reduct ion. Finally, the extr acellular filaments from these microorganisms were characterized and the more abundant filaments, in both organisms , were found to be conductive. These are the first examples of nanowires discovered outside of the mesophilic bacteria . In addition, the phyl ogenetic and geographic diversity between these isolates suggest s that microbial nanowires are more widespread than previously thought. Copyright by MICHAEL P. MANZELLA 201 5 iv This dissertation is dedicated to my family. My m other, Lynn, for always being there and y father, David, and his wife, Mary, for always asking and for being an inspiration (especially towards the end) to never give up. My sister, Jennifer , whose constant worrying (about everything) ensu red that I never had, or wanted to. And last but not least, my brother, Kevin, for never ceasing to be a wise - [guy] and making sure I never took myself too seriously. Dad, I will miss you for the rest of ou were proud of me finishing my Ph.D. , it meant the world to me. I love you dad. v ACKOWLEDGEMENTS I would like to first thank both Dr. Gemma R eguera and Dr. Kazem Kashefi for the advice and supp ort I have received while at Michigan State University graduate student and, while there were some growing pains on all of our parts, I have benefited a great deal f rom having not one, but two wonderful mentors. The flow of the project did not proceed as any of us imagined at the start, but in the end I believe we ended up exactly where it needed to. Thank you both, for everything you have done. I gratefully acknowled ge all of the resources at Michigan State University. The support I received from Dr. Tracy K. Teal was essential to my success with the genome work (Chapters 2 and 3) and it could not have been done without the support from the Research Technology Support Facility and the Hypercomputing Center at Michigan State University, the Deep Sequencing Core Facility at the University of Massachusetts Medical School, and the Genomics Resource lab at the University of Massachusetts - Amherst. I would also like to thank Dr. Alicia Withrow and Dr. Abigail Vanderberg at the Center for Advanced Microscopy at Michigan State for help with transmission and scanning electron microscopy , respectively . Dr. Sanela Lampa - Pastirk and Krista Cosert, from the Reguera lab, are also ackn owledged for their assistance with the Atomic Force Microscopy work. I also thank Kelly Nevin for advice to ma ke the entrapped Fe(III) oxides . M y two undergraduate mentees, Michael Paxhia and Lucas Demey, are also acknowledged for their assistance with m y work and for being the (often) willing subjects of my unconventional and experimental mentoring techniques . Finally, I would like to acknowledge Dr. research er and as an instructor while at MSU. Teaching is my future, and I owe that to you. This work was supported with funds from a Strategic Partnership Grant from the MSU Foundation, a continuation fellowship from the College of Natural Sciences, a GAANN fello wship by the Department of Education, a DuVall Award, and an award from the Rudolph Hugh vi fellowship . The Microbiology and Molecular Genetics department and the College of Natural Science has taken great care of me since my admission, and I hope I can make you all proud. In addition, scientific and personal support from my friends and (past and present) lab - mates (particularly Rebecca Steidl) is acknowledged. While the support you all have given me with my science helped me get my work done, the social suppo rt you all provided helped to ensure I stayed happy and saw this through to the end . Lastly, I would like to thank Megan Goodall for . you all. Thank you. vii TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ...................... x LIST OF FIGURES ................................ ................................ ................................ .................... xi KEY TO ABBREVIATIONS ................................ ................................ ................................ ..... xiii CHA PTER 1. HYDROTHERMAL VENTS AND MICROBIOLOGICAL ISOLATES AS MODEL SYSTEMS TO STUDY THE ORIGINS OF IRON RESPIRATION AND LIFE ON EARTH ......... 1 Life on early Earth ................................ ................................ ................................ ........... 1 Hydrothermal vents as analogous sites for life on Early Earth ................................ ......... 4 Hydrothermal vent community physiology, metabolism, and ecology ............................... 6 From phylogenetics and physiology to metagenomics ................................ ..................... 8 ................................ ................................ 9 Mechanisms for iron oxide respiration in mesophilic environments ................................ . 10 Identification of model iron - reducing organisms from hydrothermal vents ....................... 13 1.7.1 Geothermobacter ehrlichii as a model organism for the thermophilic bacteria isolated from a diffuse - flow mid - ocean vent system ................................ ................................ ...... 14 1.7.2 Geoglobus ahangari as a model organism for the hyperther mophilic archaea isolated from a coastal hydrothermal vent chimney ................................ ................................ ....... 15 Mechanistic studies of (hyper)thermophilic iron reduction and the need for additional model systems ................................ ................................ ................................ ................................ 16 Dissertation Ou tline ................................ ................................ ................................ ........ 17 1.9.1 The high - quality draft genome sequence of the thermophilic Fe(III) - reducing bacterium Geothermobacter ehrlichii strain SS015 ................................ .......................... 17 1.9.2 The complete genome sequence and emendation of the hyperthermophilic, obligate iron - Geoglobus ahangari T ................................ ............... 18 1.9.3 Extracellular electron transfer to Fe(III) oxides by the hyperthermophilic archaeon Geoglobus ahangari via a direct contact mecha nism ................................ ....................... 18 1.9.4 Microbial Nanowires: A conserved mechanism for extracellular electron transfer .... 18 CHAPTER 2. THE HIGH - QUALITY DRAFT GENOME SEQUENCE OF THE THERMOPHILIC FE(III) - REDUCING BACTERIUM GEO BACTER EHRLICHII STRAIN SS015 .......................... 20 Abstract ................................ ................................ ................................ .......................... 20 Keywords ................................ ................................ ................................ ....................... 20 Introduction ................................ ................................ ................................ ..................... 21 Organism information ................................ ................................ ................................ ..... 22 2.4.1 Classification and features ................................ ................................ ...................... 22 Genome sequencing and annotation ................................ ................................ .............. 24 2.5.1 Genome project history ................................ ................................ ........................... 24 2.5.2 Growth conditions and DNA isolation ................................ ................................ ...... 24 2.5.3 Genome sequencing and assembly ................................ ................................ ........ 25 2.5.4 Genome annotation ................................ ................................ ................................ 26 Genome properties ................................ ................................ ................................ ......... 27 Insights from the genome ................................ ................................ ............................... 28 2.7.1 Thermophilic adaptations ................................ ................................ ........................ 28 2.7.2 Central metabolism ................................ ................................ ................................ . 30 viii 2.7.3 Utilization of sulfur - containing compounds ................................ .............................. 34 2.7.4 Nitrogen compounds as electron ac ceptors ................................ ............................ 35 2.7.5 Motility and chemotaxis ................................ ................................ ........................... 36 2.7.6 Fe(III) as electron acceptor ................................ ................................ ..................... 37 2.7.7 Extracellular polymeric substances ................................ ................................ ......... 40 Conclu sions ................................ ................................ ................................ .................... 40 CHAPTER 3. THE COMPLETE GENOME SEQUENCE AND EMENDATION OF THE HYPERTHERMOPHILIC, OBLIGATE IRON - REDUCING ARCHAEON GEOGLOBUS AHANGARI STRAIN 234 T ................................ ................................ ................................ ........ 42 Abstract ................................ ................................ ................................ .......................... 42 Keywords ................................ ................................ ................................ ....................... 42 Introduction ................................ ................................ ................................ ..................... 42 Organism information ................................ ................................ ................................ ..... 44 3.4.1 Classification and features ................................ ................................ ...................... 44 Genome sequencing and ann otation ................................ ................................ .............. 45 3.5.1 Genome project history ................................ ................................ ........................... 45 Growth conditions and genomic DNA preparation ................................ .......................... 46 Genome sequencing and assembly ................................ ................................ ................ 46 3. 7.1 Genome annotation ................................ ................................ ................................ 48 Genome properties ................................ ................................ ................................ ......... 49 Insights from the genome ................................ ................................ ............................... 51 3.9.1 Autotrophic growth with H 2 as electron donor ................................ .......................... 51 3.9.2 Central metabolism ................................ ................................ ................................ . 52 3.9.3 Fatty acids as electron donors ................................ ................................ ................ 55 3.9.4 Degradation of aromatic compounds and n - alkanes ................................ ............... 57 3.9.5 Nitrogen compounds as electron acce ptors ................................ ............................ 58 3.9.6 Sulfur compounds as electron acceptors ................................ ................................ 59 3.9.7 Fe(III) as the sole electron acceptor for respiration ................................ ................. 60 Conclusions ................................ ................................ ................................ .................. 65 Taxonomic note ................................ ................................ ................................ ............ 66 Emended description of Geoglobus Kashefi et al. 2002 ................................ ................ 66 Emended description of Geoglobus ahangari Kashefi et al. 2002 ................................ . 66 CHAPTER 4. EXTRACELLULAR ELECTRON TRANSFER TO FE(III) OXIDES BY THE HYPERTHERMOPHILIC ARCHAEON GEOGLOBUS AHANGARI VIA A DIRECT CONTACT MECHANISM ................................ ................................ ................................ ............................ 67 Abstract ................................ ................................ ................................ .......................... 67 Introduction ................................ ................................ ................................ ..................... 68 Materials and Methods ................................ ................................ ................................ ... 70 4.3.1 Bacterial strains, culture conditions and mineral characterization ............................ 70 4.3.2 Assays for endogenous mediators ................................ ................................ .......... 70 4.3.3 Micr oscopy ................................ ................................ ................................ .............. 72 4.3.4 Denaturing Polyacrylamide Gel Electrophoresis (SDS - PAGE) and heme staining .. 72 4.3.5 Effect of mechanical shearing of outer surface proteins on Fe(III) reduction ........... 73 Results and discussion ................................ ................................ ................................ ... 74 4.4.1 Stimulation of Fe(III) reduction by exogenous mediators ................................ ......... 74 4.4.2 G. ahangari does not secrete endogenous mediators for Fe(III) reduction. ............. 76 4.4.3 Cellular components involved in Fe(III) reduction in G. ahangari ............................. 77 Conclusions ................................ ................................ ................................ .................... 79 ix CHAPTER 5. MICROBIAL NANOWIRES: A CONSERVED MECHANISM OF EXTRACELLULAR ELECTRO N TRANSFER ................................ ................................ .......... 81 Abstract ................................ ................................ ................................ .......................... 81 Introduction ................................ ................................ ................................ ..................... 81 Materials and Methods ................................ ................................ ................................ ... 85 5.3.1 Bacterial strains and culture conditions ................................ ................................ ... 85 5.3.2 Isolation and purification of extracellular filaments ................................ .................. 85 5.3.3 Thermostability measurements by thermal shift assay ................................ ............ 86 5.3.4 Conductive probe atomic force microscopy ................................ .......................... 87 5.3.5 Elemental analysis of purified filament preparations ................................ ................ 87 5.3.6 Examination of the curli - like nature of G. ahangari nanowires ................................ . 88 Results and discussion ................................ ................................ ................................ ... 89 5.4.1 Isolation and purification of filaments ................................ ................................ ...... 89 5.4.2 G. ehrlichii expresses multiple filaments while only the Geopili are conductive ....... 91 5.4.3 G. ahangari expresses multiple filaments while only the smaller, pilus - like filaments are conductive ................................ ................................ ................................ ................. 92 5.4.4 Thermostability of soluble cell extracts and thermophilic nanowires ........................ 93 5.4.5 Pilus - like filaments from G. ah angari demonstrate curli - like behavior ...................... 95 Conclusions ................................ ................................ ................................ .................... 96 CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS ................................ ................. 100 Review of project ................................ ................................ ................................ .......... 100 6. 1.1 Review of Geothermobacter ehrlichii genome studies ................................ ........... 101 6.1.2 Review of Geoglobus ahangari genome studies ................................ ................... 102 6.1.3 Review of Geoglobus ahangari iron - respiration mechanism ................................ .. 104 6.1.4 Review of (hyper)thermophilic microbial nanowires ................................ ............... 105 Future directions ................................ ................................ ................................ ........... 106 6.2.1 Geoglobus ahangari ................................ ................................ .............................. 106 6.2.2 Geothermobacter ehrlichii ................................ ................................ ..................... 107 Looking forward ................................ ................................ ................................ ............ 108 APPENDICES ................................ ................................ ................................ ......................... 110 APPENDIX A T A B L E S ................................ ................................ ................................ ....... 111 APPENDIX B F I G U R E S ................................ ................................ ................................ ..... 136 REFERENCES ................................ ................................ ................................ ....................... 160 x LIST OF TABLES Table 2.1. Classification and general features of G. ehrlichii ................................ ................... 112 Table 2.2. Genome sequencin g project information for the genome of G. ehrlichii .................. 114 Table 2.3. Assembly reconciliation statistics for the G. ehrlichii genome ................................ . 115 Table 2.4. Primers used for gap closure of the G. ehrlichii genome ................................ ......... 116 Table 2.5. Nucleotide content and gene count levels of the G. ehrlichii genome ..................... 118 Table 2.6. Number of genes within G. ehrlichii associated with the 27 subsystem categories in RAST ................................ ................................ ................................ ................................ ...... 119 Table 2.7. Putative c - type cytochromes identified in th e genome of G. ehrlichii ...................... 121 Table 3.1. Classification and general features of G. ahangari ................................ ................. 123 Table 3.2. Genome sequencing proj ect information of G. ahangari ................................ ......... 125 Table 3.3. Nucleotide content and gene count levels of the G. ahangari genome ................... 126 Table 3.4. Number of genes in G. ahangari associated with the 25 general COG functional categories ................................ ................................ ................................ ............................... 127 Table 3.5. Terminal electron acceptors in the Archaeoglobales ................................ ............... 129 Table 3.6. Putative c - type cytochromes in G. ahangari ................................ ........................... 130 Table 3.7. Uniquinone and menaquinone biosynthesis proteins in G. ahangari ....................... 132 Table 3.8. Fe - S binding domain proteins and ferredoxins within the genome of G. ahangari ... 133 xi LIST OF FIGURES Figure 1.1 . Geologic timescale of the Precambrian Supereon ................................ ................. 132 Figure 1.2. Location of the Axial Seamount and the layout of the Axial Seamount caldera ...... 133 Figure 1.3. Location of the Guaymas Basin ................................ ................................ ............. 134 Figure 2.1. Phylogenetic tree of G. ehrlichii ................................ ................................ ............. 135 Figure 2.2. Tra nsmission electron micrograph of G. ehrlichii ................................ ................... 136 Figure 2.3. Amino acid usage of G. ehrlichii and G. electrodiphilus in comparison to mesophilic members of the Geobacteraceae ................................ ................................ ............................ 137 Figure 2.4. Central metabolism in G. ehrlichii ................................ ................................ .......... 138 Figure 2.5. Sequence relatedness of the pilin subunit protein to members of the Desulfuro monadales ................................ ................................ ................................ ............... 140 Figure 2.6. SDS - PAGE gel of sheared proteins stained with the TMBZ protocol ..................... 142 Figure 3.1. Phylogenetic tree of G . ahangari ................................ ................................ ........... 143 Figure 3.2 . Scanning electron micrograph of cells of G. ahangari ................................ ........... 144 Figure 3.3. Graphical circular map of the G. ahangari genome ................................ ............... 145 Figure 3.4. Central metabolism in G. ahangari ................................ ................................ ........ 146 Figure 4.1. Effect of electron shuttles or metal chelator s on the reduction of Fe(III) oxides by G. ahangari ................................ ................................ ................................ ................................ .. 147 Figure 4.2. G. ahangari lacks the ability to produce endogenous electron shuttles .................. 148 Figure 4.3 TEM micrographs of negatively - stained cells of G. ahangari ................................ .. 149 Figure 4.4. Surface - exposed c - type cytochromes are essential for the reduction of insoluble Fe(III) oxid es ................................ ................................ ................................ ........................... 150 Figure 5.1. Transmission electron micrographs of G. ahangari filaments ................................ 151 Figure 5.2. Transmission electron micrographs of G. ehrlichii filaments ................................ .. 152 Figure 5.3. Preliminary AFM data on G. ehrlichii ................................ ................................ ..... 153 Figure 5.4. Atomic force microscopy on G. ehr lichii purified pili ................................ ............... 154 xii Figure 5.5 Preliminary AFM data on G. ahangari ................................ ................................ .... 155 Figure 5.6. Atomic force microscopy on G. ahangari p urified pilus - like filaments ..................... 156 Figure 5.7. Sypro Orange thermal shift assays on soluble protein extracts ............................. 157 Figure 5.8. Failu re of the Sypro Orange thermal shift assay to characterize the T m of prep - cell purified filaments ................................ ................................ ................................ ..................... 158 Figure 5.9. Effect of 72 h incubation on the pilus - like filaments of G. ahangari ........................ 159 xiii KEY TO ABBREVIATIONS APS - phosphosulfate 23G 23 gauge AFM atomic force microscopy AQDS anthraquinone - 2,6 - disulfonate ATCC American Type Culture Collection BLAST basic local alignment search tool CDS coding seq uence COG clusters of orthologous group CP - AFM conductive probe - atomic force microscopy DHAP dihydroxyacetone phosphate DNA deoxyribonucleic acid DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen EB elution buffer EC enzyme commission EDTA ethy lenediaminetetraacetic acid EET extracellular electron transfer EPS extracellular polysaccharide Fe - S iron - sulfur GAPOR glyceraldehyde - 3 - phosphate:ferredoxin oxidoreductase GDH glutamate dehydrogenase gDNA genomic deoxyribonucleic acid GS - GOGAT glutamine s ynthetase - glutamate synthase Gya gigayears ago xiv Gyr gigayears HOPG highly ordered pyrolytic graphite HPLC high performance liquid chromatography ICP - AES inductively coupled plasma atomic emission spectroscopy IEB iron extraction buffer IGS Institute for Gen ome Sciences IMG - ER Integrated Microbial Genomes Expert Review KEGG Kyoto Encyclopedia of Genes and Genomes LSU large subunit ribosomal RNA MEC microbial electrochemical cell MM marine medium MMWB modified marine wash buffer Mya million years ago Myr meg ayears N 50 50% of the assembly is contained in contigs or scaffolds equal to or larger than this value NR non - redundant NTA nitrilotriacetic acid ORB origin recognition box PacBio Pacific Biosciences PAPS 3' - phosphoadenylyl sulfate PCR polymerase chain re action PRPP 5 - phosphoribosyl diphosphate RAST Rapid Annotations using Subsystem Technology RNA ribonucleic acid rRNA ribosomal ribonucleic acid xv ROK r epressor protein, o pen reading frame, sugar k inase RT room temperature RuMP ribulose monophosphate SDS sodi um dodecyl sulfate SDS - PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SSU small subunit (16S) ribosomal ribonucleic acid TCA tricarboxylic acid TE Tris - ethylenediaminetetraacetic acid ThT thioflavin T T m melting temperature TMBZ 3,3',5 ,5' - tetramethylbenzidine T opt optimum temperature 1 CHAPTER 1. HYDROTHERMAL VENT S AND MICROBIOLOGICAL ISOLATES AS MODEL SYSTEMS TO STUDY THE ORIGINS OF IRON RESPIRATION AND LIFE ON EARTH Life on early Earth The formation of planet Earth some 4.55 gigayears ago (Gya) marks the beginning of the Precambrian Super eon (4.55 0.54 Gya ) (Figure 1.1) . The early periods of Earth logic history can be further divided chronologically into the Hadean Eon (4.55 3.9 Gya), where the Earth was cooling and r ecovering from the formative process, the early (3.9 2.9 Gya) and late (2.9 2.5 Gya) Archaean Eon, and the Proterozoic Eon (2.5 0.54 Gya). Geological evidence (discussed later, and reviewed in (1) ) supports the notion that life was already present on our planet 3.5 Gya, and likely e merged 3.8 Gya or earlier. Thus, even with the most restrictive estimates, life existed on our planet only 950 megayears (Myr) after its formation, and only 1,050 Myr after the formation of our solar system (2) . The study of life, an d of its origins, on early Earth is challenging because organisms present at the time, including the ancestors of all extant organisms, seldom, if ever, left a fossil record (3 5) . This contrasts with the relative abundance of micro - and macro - fossils identified that date back to the Proterozoic Eon (3) . The lack of a fossil record in earlier periods can be attributed in part to the meta morphosis or tectonic submission of strata from that age, effectively erasing the majority of the fossil record (4, 6, 7) . Based on evidence discussed later, researchers th eorize that life originated during the early Archaean, although it would not emerge until after the development of the theorized pre - cellular systems (8, 9) . Some microscopic structures from the early Archaean do exist in the fossil record, mainly ( 4) - grained, thinly layered, mounded sedimentary structures which are presumed to have been created primarily by mat - building microbial communities of mucilage - secreting photoautotrophs (3, 4, 7) . Some of the most 2 ancient fossil records in existence, including those within the 3.5 Gya Warrawoona Group (Western Australia) (10, 11) and the 3.5 3.3 Gya Swaziland Sequence (South Africa) (12) remain intact and some have undergone morphotypes or species identification (4) . These morphotypes were compi led in 2006, by J. William Schopf, who tallied 40 microorganism - like morphotypes residing within 6 distinctive classes all identified in Archaean - age deposits (reference (4) Table 2). Yet, physical fossils are not the sole evidence of when life began on Earth. Living systems tend to utilize, and therefore enrich for, lighter isotopes of carbon. Thus, examination of the 12 C/ 13 C ratio in Archaean age (and more ancient) rocks compared to abiogenic carbon can lend further evidence to th e origin of life in this age (7, 13) . Archaean - age carbonate, for example, is isotopically similar to carbonates produced in modern systems, where light carbon isotopes are sequestered by living systems and heavier isotopes are left to precipitate out as carbonate (14) . While researchers may have a reasonably accurate guess regarding the time period at which life originated, a variety of theories exist for the origin of life on Earth and where it bega n. One theory hypothesis (14) in which life originated in a warm, if not hot, marine environment. The hypothesis that life originated in a warm or hot environment was first proposed by Darwin in 1871 in a letter to J. D. Hooker, and was again proposed in 1924 by R. B. Harvey (15) and speculation has continued to this da y. Initial speculations were based on high temperature substitution of enzymatic hydrolysis (15) , while current proponents of t he hypothesis point to the environmental conditions at the time and the conditions required for the pre - biotic synthesis and use of RNA as a biomolecule. produced nucl eotide precursors self - assembling into ribonucleic acid (RNA) (9) . While RNA and deoxyribonucleic acid (DNA) are both able to store genetic information, only RNA is able to catalyze chemical reactions, including the synthesis of additional RNA, the basis of self - replication 3 (8, 14) . Thus, the utilization of a single molecule able to serve both a hereditary and a functional role is believed to have opened the proverbial flood - gates for life on Earth (9) . Some critics argue stability of the nucleotide precursors and RNA itself is minimal at high temperatures (6) . Yet pre - biotically synthesized oligonucleotides have been known to form under Archaean - age atmospheres, hydrothermal vent systems, and meteorites (6, 16) . Furthermore, the early biomolecules were likely functional only immediately following their synthesis (6) or were contained within a low water activity environme nt to maintain functionality (7) . Additionally, it is well known that reaction rates increase as temperatures increases (17) . Thus, these RNA enzymes, or the proteinaceous enzymes contained within early microorganisms, could have been relatively inefficient when compared to modern enzymes while still maintaining reaction rates suitable to sustain life (6, 15) . Evidence in the form of the molecular record also support s the notion that the cellular ancestor of all extant organisms was thermophilic. Alignments and phylogenetic trees derived from the commonly used phylogenetic timepiece, the small subunit (16S) ribosomal RNA (18, 19) , place thermophiles at the base of the tree of life (20) . However, the position of these same thermophilic organisms at the base is challenged when other molecular markers, such as DNA - dependent RNA polymerases (21, 22) and DNA topoisomerases (22) , are used instead. The widespread presence of heat - shock proteins in nearly a ll modern organisms has also le d to the proposal that a (hyper)thermophile was the cellular ancestor of all extant microorganisms (6) . Modern heat - shock proteins play critical roles in stress responses in the cell (6, 23) and, along with other housekeeping proteins, could have evolved from a (hyper)th ermophilic ancestor (14) . Alternatively, (hyper)thermophiles could have outcompeted earlier mesophilic organisms due to their increased ability to deal with a range of stressors (6) . Furthermore, the bombardment of early Earth by extra - planetary bodies could have super - heat ed the shallow oceans present at the time to over 100 °C, thereby creating a hot - ocean bottleneck (14) and the extinction of any early 4 mesophiles (6, 14) . Whether (hyper)thermophiles were the earliest cellular forms or emerged later, scientists do agree on the fact that these organisms played a critical role in the evolut ion of early life. Thus, critical insights about the ancestral microorganisms can be gained by studying extant (hyper)thermophiles and extrapolating backwards. Hydrothermal vents as analogous sites for life on Early Earth Evidence to date indicates that th e ecology of the Archaean eon was solely microbial and was likely less productive than the systems in place today (24) . Despite their reduced productivity, early microorganisms are believed to have carried out the same biogeochemical processes as on modern Earth (14, 24) . Life on early Earth relied on th e availability of electron donors and acceptors, as well as carbon sources generated independently of photosynthetic primary producers. Dark sites were especially important on early Earth as the lack of an ozone layer permitted extreme amounts (1000x curre nt values) of ultraviolet radiation to reach the ocean surface and permeate into the shallow seas (25, 26) . Sim ilar dark environments, though rare on modern Earth, do exist in deep - sea hydrothermal vent systems (5, 14, 24, 27) . Wherea s photosynthetic primary producers drive life on the surface of our planet, elemental gradients drive life in and around hydrothermal vents, be they diffuse - flow sites where superheated water flows gently from the subsurface or in chimney - sites where the flow is concentrated and flows from, often, a single chimney - like outflow (5, 14, 24, 27) . The geochemistry of hydrotherma l vent fluids can be affected by magmatic intrusions, tidal processes, and seismic activity (28) . For this reason, the chemi stry of individual vents from the same hydrothermal system is often distinct, though relatively constant over time (28) . Sea water seeps down through faults in the seafloor, becoming superheated as it gets closer to the sub - seafloor magma chambers. The heat strips chemicals such as sulfate (SO 4 2 - ) and manganese (Mn) from the seawater (5) . However, once discharged from the vents, the fluids become enriched in compounds leeched from the oceanic crust such as CH 4 , CO 2 , CO, H 2 , H 2 S, and various metals 5 including Ca, Fe, Cu, Zn, Ba, and Mn, which precipitate out of solution as soon as they interact with the cold ( ca. 2 - 4 °C (29) ) , oxygenated ( ca. 150 µmol kg - 1 (30) ) seawater (5) . In addition, the primary nitrogen source in modern vent systems studied to date is, as in Archaean systems, abiotically - produced NH 3 (5) . Hence, hydrothermal vents and plumes provide vent - and plume - associated communities with a constant flow of electron donors, acceptors, carbon - , and nitrogen sources that closely mimic the geochemistry and physical cond itions of Archaean environments inhabited by the ancestral (hyper)thermophiles. While modern hydrothermal vent systems are both geochemically and geothermally similar to those on early Earth, the characteristics of the surrounding seawater are expected to be different. The Archaean atmosphere, unlike that of modern Earth, was reducing and composed primarily of methane, nitrogen gas and/or carbon dioxide, and molecular hydrogen (5, 6) . This reduc ing environment, which would persist into the shallow seas, is essential for the abiotic synthesis of amino acids (purines, pyrimidines) and sugars (16) . Furthermore, it maintained elements, gases, and other vent exudates in a reduced state that would keep them in soluti on (5) . Such a chemically - reduced environment may have been relatively stable for some time. However, decreases in hydrothermal vent activity as the Earth cool ed and increases in oxygen production from abiotic and microbial oxidative processes during the Archaean eon eventually altered the reduced atmosphere and changed conditions in the seas (5) . Although differences in the surrounding seawater can affect the thermal and chemical gradients present at the site, hydrothermal vent systems are valuable Archaean mimics based on their geological, geothermal, and geochemica l similarities (5) . For this reason, it has been suggested that microbial isolates and communities present in and around hydrothermal vent systems may be physi ologically and morphologically similar to those present during the Archaean (5, 14, 31) . 6 Hydrothermal vent community physiology, metabolism, and ecology Free - living organisms from deep - sea hydrothermal vent systems are often thought of as evolutionarily primitive, due to their position on the tree of life (32) evolutionary sense, these microorganisms are in fact physiologically, metabolically, and ecologically diverse. Complex communities have been identified in a range of hydrothermal habitats including vent chimneys (33 36) , vent fluids (35, 37, 38) , and in the microbial mats s urrounding both chimneys and diffuse - flow sites (39, 40) . Furthermore, there is great physiological diversity in the hydrothermal vent communities (34, 41 43) . Yet, all are adapted to growing at elevated temperatures, utilize geothermally - provided electron acceptors such as sulfur, molecular hydrogen, and iron for respiration; fix CO 2 to assimilate carbon; and have anaerobic metabolisms. Thus, all of these organisms must rely on chemo - rather than photo - synthetic processes for survival. Ch emotrophy dominates: methane producers (methanogens) and utilizers (methane oxidizers) as well as hydrogen producers and consumers (H 2 oxidizers) thrive in these environment. Furthermore, CO 2 is often the most abundant carbon source and can be assimilated using a variety of carbon - fixation mechanisms (41) . A number of sulfur/metal oxidizers are present in addition to sulfate - and iron - respiring bacteria and archaea (41) . Thus, these systems, though u nique in their chemistry, follow the same principle of life as on the surface of the planet: what is available for consumption is undoubtedly consumed. The remote location of hydrothermal vents in the deep sea make s sampling challenging and costly. The maj ority of subsurface studies have been obtained from sediment cores obtained from deep - sea drilling operations (44 46) or from fluids obtained exiting chimneys, diffuse - flow sites, or even at sites of recent volcanic activity (47 50) . Additional studies ( (51 53) for example) have relied on submersibles launched from sc ientific research vessels, a costly and limiting step. Even the enumeration of these deep - sea microorganisms is difficult, as these microbial 7 geo chemical, and geothe rmal analyse s of vent chimney sections show that within the vent chimney walls thermal and chemical gradients exist (54, 29) ey walls, which can have centimeter to meter scales, often have steep gradients of temperature ranging from more than 200 °C in the inner layers to 5 °C on the external surface, and drastically different mineral compositions throughout. This, along with th e permeation of vent - and ocean - derived compounds from both the interior and the exterior, leads to drastically different zones within centimeters or millimeters of each other (54, 29) . Therefore, microorganisms or microbial communities must maintain their position within the vent chimney or hydrothermal vent system or risk freezing, boiling, or starving because of a millimeter change in locale (29) . Despite this, large and active communities exist withi n these subsurface systems (55 59) . Microorganisms are even abundant in the vent water: bacterial cell counts range, for example, from 10 5 to over 10 9 cells/mL. Furthermore, microbial productivity (measure d by cell counts, GTP:ATP ratios, and uptake of 14 CO 2 ) is even greater than in the surrounding seawater (100 1000 x) and overlying surface waters (3 4 x) (5, 60) . The extreme conditions of these environments also make culture enrichments and isolations of native microorganisms complex, thus limiting the opportunities for standard culture - based screens (61) . In spite of this, several culture - based methods have been used to examine the biodiversity at hydrothermal vent sites (62, 63) . Culture - dependent studies indicate that the majority of these isolates are chemolithoautotrophs which can utilize the abundant, albeit atypical, nutrients produced by hydrothermal vents to sustain life and build biomass (64) . They are predominantly anaerobic and isolates typically utilize sulfur - or iron - containing compounds in oxidative or reductive reactions. Methane oxidation also predominates in vent systems as methan e is produced both by the vents themselves and via methanogenesis from CO 2 by archaeal methanogens (64) . T echniques have been developed, such as the use of insoluble Fe(I II) oxides as the sole electron acceptor during sample enrichment, to select for these 8 microorganisms (65) . These studies have led to the isolation of novel bacterial and archaeal clades (52, 65, 66) . Because of the challenges associated with culturing these organisms, culture - independent phylogenic approache s such as those based on 16S rRNA analysis have also been used to examine the phylogenetic diversity of the hydrothermal vent communities (see (35, 42, 57, 59, 67, 68) for examples). These approaches can be performed on whole - community samples - and species - level groups, ther efore providing a near complete picture of the community, pending bias of the conditions used to amplify and sequence the available DNA (69) . As phylogeny provides little to no information about the identify genomic indicators of microbial physiology or metabolic capabilities. These studies, termed metagenomics, can often shed additional light on poorly - studied systems. From phylogenetics and physiology to metagenomics Metagenomics extends the phylogenetic and physiologic info rmation derived from culture - independent and culture - based techniques, respectively, to provide a community - wide view of microbial proteins and pathways which could drive the functioning of an ecosystem. This is particularly important in hydrothermal vents and hydrothermal vent chimneys, where the microenvironment can change drastically over the scale of millimeters (54, 29) and many highly dive rse microbial communities may live within the short distances across the hydrothermal vent chimney walls (29, 68) . The metabolic potential of these difficult to study and spatially - distinct communities can be assessed using metagenomics by examining the presen ce or absence of genes encoding known enzymes within well - characterized metabolic pathways. Xie et. al. (41) used metagenomics to examine, for example, microbial communities from a range of hydrothermal vent sites and identified several pathways for CO 2 fixation, sulfur oxidation, and nitrogen metabolism. The presence and absence of several of these pat hways was found to 9 change based on the microenvironments present within a site. Thus, the technique not only identifies the metabolic pathways present in defined communities, but can provide valuable insights into the specific conditions and microhabitats that exist within the larger environment under examination (41) . Yet limitations in metagenomic stu dies exist in that community physiologies often do not match the capabilities predicted by metagenomic methods. Physiological studies performed within the Juan de Fuca Ridge hydrothermal vent system showed, for example, that chemoautotrophs present in the plumes of hydrothermal vent chimneys at the site were able to rapidly consume ammonium (70) , but metagenomic analysis did not detect the presence of a key enzyme, ammonia monooxygenase, required for ammonia oxidation (41) . Thus, even for well - characterized systems, metagenomics fails when novel pathways or confounding evidence is present. The limitation of culture - independent approaches to understand the metabolic potential and phylogenetic diversity of microorganisms from hydrothermal vent systems is clearly seen when studying dissimilatory ir on reduction. In contrast to other respiratory metabolisms that rely on soluble electron acceptors (such as the sulfate respiration prevalent within hydrothermal vent systems and on early Earth (14) ) , dissimilatory iron reducers respire iron (Fe(III) ) oxides, an insolubl e electron acceptor (71) . In contrast to soluble electron acceptors, whic h are typically reduced by terminal reductases in the periplasm or cytoplasm (72) , iron oxides must be reduced extracellularly. To accomplish this, a number of bacterial and archaeal clades have memb ers that have evolved mechanisms to transfer respiratory electrons to the insoluble oxides outside the cell. The best studied dissimilatory iron reducers are those within the bacterial family Geobacteraceae (73) . 10 The Geobacteraceae , which are members of the Desulfuromonadales within the Deltapr oteobacteria , are obligate anaerobes and iron reducers but otherwise physiologically diverse (73) . The most commonly studied members of the family are Geobacter sulfurreducens (74) and Geobacter metallireducens (75) . Both are mesophilic, with an optimal growth temperature of ca. 30 °C, and can couple the reduction of both soluble and insoluble forms of Fe(III) in addition to the oxida tion of a range of electron acceptors (74, 75) . Many members in the family have sequenced genomes (76 80) and genetic systems are available for both G. sulfurreducens (81) and G. metallireducens (82) . Genomic studies have provided very valuable insights into the res piratory strategies of these organisms (83) but mechanistic information on iron oxide reduction is not easily deduced from genome data and often requires coupled physiological and genetic studi es. These studies have identified several strategies to transport electrons to the extracellular iron oxides, which can be grouped in two: those requiring direct contact between the cell and the oxides and indirect strategies using electron shuttles or met al chelators (84) . Mechanisms for iron oxide respiration in mesophilic environments An efficient strategy to reduce iron oxides is one in which the cell establishes direct contact with the insoluble electron acceptors. To do so, the cell m ust express a terminal iron reductase on its outer surface and be able to access and attach to the mineral surface in order to transfer the respiratory electrons. The most common iron reductases in mesophilic dissimilatory metal - respiring bacteria and arch aea are c - type cytochromes (84) . They are so named due to the presence of a heme motif related to Cytochrome C (85) , are found in numerous organisms (84) , and are especially abundant within microorganisms able to respire insoluble metal oxides (86) . These cytochromes carry a conserved heme binding motif (CXXCH) and can also carry a signal peptide that is recognized and processed prior to exporting the protein to the periplasm of gram - negative bacteria or the periplasm - like space of archaea (72, 87 89) . Cells can further extend 11 their electroactive surface anchoring c - cytochromes in an exopolysaccharide (EPS) matrix, a strategy that also allows them to build electroactive biofilms (90) . In addition to c - cytochromes, dissimilatory iron reducers in the family Geobacteraceae p roduce conductive t cell and the Fe(III) oxides (91) . The expression of protein nanowires increases the redox - active surface of the cell beyond the confines of the cell envelope, thus facilitating access to the Fe(III) oxide particles, which are often dispersed in soils and sediments (91) . The pilus nanowires produced by the Geobacteraceae also function as electronic conduit s between the cell and soluble metals, such as the uranyl cation, a respiratory strategy that also serves as a protective mechanism because the extracellular reduction of the toxic metals also prevents their permeation and re duction inside the cell (92) . Nanowire - like appendages have been reported in other bacteria, including Acidithiobacillus ferrooxidans (93) and the mesophilic iron - reducing bacterium Shewanella oneidensis (94) . However, th e of S. oneidensis have recently been shown (95) and the nanowires of A. ferro o xidans function to transfer electrons from extracellular electron donors . Hence, to date, protein nanowires have only been identified in mesophiles, and those enabling gro wth on insoluble electron acceptors have only been identified in the Geobacteraceae . In contrast to direct - contact mechanisms, some microorganisms rely on indirect strategies to reduce the insoluble electron acceptors at a distance. One such strategy is e mploying metal - chelating compounds, which can be produced by the same or other microorganisms or in abiotic reactions (96) . The chelators can therefore be exogenous compounds, such as nitrilotriacetic ac id (NTA), which is often used in laboratory studies to stimulate the growth of iron reducers (96) , or can be produced by the iron - reducing microorganism itself, as reported for the mesophilic bacterium G eothrix fermentans (97) . Whether exogenous or endogenous, ch elators solubilize the Fe(III) from the oxides and provide a soluble, chelated form of iron as an electron acceptor for respiration. This alleviates the need for the cell to establish electronic contact with the insoluble 12 electron acceptors (91, 98) . Not only can the chelators diffuse in and out from the cell, they can also diffuse through biofilms, thus passively chelating the mineral and increasing its bioavailability (96, 99) . Alternatively, some microorganisms use contact - independent mechanisms based on - bounce between the cell and the mineral surface to deliver respiratory electrons (100) . Electron shuttles are abundant in many environments where iron reduction is a significant process, primarily as hu mic substances produced during the degradation of plant matter (99) , but can also be synthesized and secreted by microbial cells (97, 100, 101) . By shuttling electrons between the cell and the extracellular minerals they stimulate extracellular electron transfer in dissimilatory iron reducers and also confer on other microorganisms the ability to respire insoluble electron acceptors (99) . As discus sed previously, geno me data do not always provide sufficient information to predict whethe r an organism participates in dissimilatory iron reduction and genetic and physiological studies are still needed to identify genetic markers for this metabolic capability. In the model representative of the family Geobacteraceae , G. sulfurreducens , for example, genetic markers include the presence of a great number (more than 100) of genes encoding c - cytochromes, including some predicted to be exported to the outer membrane (76) and a pilA gene encoding the divergent type IV pilin of the Geobacteraceae (91) . Geobacteraceae also produce the Xap EPS to anchor extr acellular c - cytochromes and reduce Fe(III) oxides (90) . Furthermore, flagellar motility , chemotaxis to Fe(II), which is the soluble product of Fe(III) oxide respiration, and type IV pili allows bacteria in the G eobacteraceae to locate and access the insoluble oxides (102) . Yet, conservation in some of the genes, most notably in those encoding c - cytochromes, is low and it is challenging to predict the r espiratory path for iron reduction from genomic data (reviewed in (103) ) . This is due to the fact that the specific roles of pili, cytochromes, and EPS in Fe(III) reduction are not yet fully understood (reviewed in (103) ). Geobacteraceae t ype IV pili, for example, are essential for Fe(III) oxide reduction (91) , yet may also anchor c - 13 cytochromes (104) . Furthermore, adaptive evolution of a pilin - de ficient mutant of G. sulfurreducens in Fe(III) oxides cultures restored its ability to reduce the insoluble electron acceptor, presumably because the c - type cytochrome PgcA was overexpressed and able to shuttle electrons between the cell and the mineral (105) . Hence, several pathways may operate, each with different components. Thus, although metagenomic, genomic, and phylogenetic studies have been successful in identifying components used in well - characterized pathways such as sulfurous and nitrogenous compound reduction, its use in the field of dissimilatory iron reduction is inherently challenging. Furthermore, mechanistic information about dissimilatory iron reduction in thermophilic and hyperthermophilic bacteria and archaea is even scarcer. The decreasing costs of genomic sequencing has increased the availability of sequenced genomes from (hyper)thermophiles (68) but there is a need to identify model organisms for mechanistic studies of metal resp iration at high temperatures. Identification of model iron - reducing organisms from hydrothermal vents Hydrothermal vent systems can be grouped into mid - ocean zones and those located in clos e proximity to landmass es . The location of mid - ocean zones far away from continental land minimizes the amount of organic material that can accumulate in the hydrothermal vent sediments and creates conditions for microbial growth that are primarily influence d by hydrothermal activity. By contrast, hydrothermal vent system s geographically close to continental land, are exposed to the coastal runoff and, therefore influenced by both the hydrothermal vent and coastal discharges. Hence, iron reducers from mid - ocean versus off - shore hydrothermal vent systems are expected to hav e evolved unique metabolic adaptations to the specific conditions of their environment. For this reason, two hydrothermal vent systems were selected for further studies that represented the two types of hydrothermal vent environments. 14 1.7.1 Geothermobacter ehrli chii as a model organism for the thermophilic bacteria isolated from a diffuse - flow mid - ocean vent system The Axial Seamount system, located off the coast of Washington (USA) within the Juan de Fuca Ridge, is a spreading site that has been well studied due to its shallow depth ( ca. 1500 m) and proximity (250 nautical miles off the coast) to several oceanography centers (Figure 1.2) (106) . The seamount itself forms a large (3 x 8 km) caldera and has repeat edly been active in the past century (28) . The caldera itself contains a number of different hydrothermal vents (28) , including the Bag City vent site in the southern region, which receives its name from the numerous gelatinous polysaccharide coagu (66) . The Bag City resides within the pre - 1987 lava flow zone and just outside of the 1998 lava flow (28) . It is a stable diffuse - flow vent system with an examined temperature range of 12.9 - 31.3 °C (28) . The vent site has relatively low concentrations (nM/J of heat) of H 2 , CH 4 , H 2 S, and Fe when compared to other vents on the Axial seamount included in the analysis (28) . This may be due to the d ifficulty in assessing vent flow from diffuse sites when compared to chimney systems. Despite the relative scarcity of these chemical compounds at the time of sampling (28) , the Bag City site and the Axial Seamount support the growth of an active vent community and, for this reason, continue to be a prime model site to examine life in off - shore spreading systems. As stated abo ve, the name Bag City originated from the discovery of numerous gelatinous microorganisms (66) . Geothermobacter ehrlichii was isolated from anoxic diffuse - flow vent fluid samples collected from the Bag City hydrothermal vent by t Fe(III) oxide enrichments and produces copious amounts of exopolysaccharide when grown under conditions of stress (66) , thus serving as a good model representative iron reducer from the Bag City environment. Phylogenetic analysis placed G. ehrlichii within the Geobacteraceae family (66) , which includes the best known mesophilic iron reducers, as disc ussed previously. However, recent studies suggest a placement within the related Desulfuromonadaceae family, 15 also within the d elta subclass of P roteobacteri a (66, 107, 108) . Unlike other cultured members of the Geobacteraceae (73) or Desulfuromonadaceae (107) , which are mesophiles or psychrophiles, G. ehrlichii is a thermophile, growing at temperatures ranging from 35 to 65 °C and optimally at 55 °C (66) . The low temperature range that supports the growth of G. ehrlichii matches well the temperature maxima (31.3 °C) recorded at the Bag City site (28) . This observation is also consistent with isolates from other hydrothermal vent sites, which often have a minimum growth te mperature that is higher than the site of isolation (48, 55) . G. ehrlichii used only Fe(III) and nitrate (which was reduced to ammonia) as terminal electron acceptors for re spiration using organic acids as electron donors (66) . But unlike m ost other Geobacteraceae , it was also able to couple the oxidization of sugars, starch, and amino acids to the reduction of Fe(III) (66) . The phylogenetic proximity of this isolate to the intensely - studied mesophilic representatives G. sulfurreducens (74) and G. metallireducens (75) , combined with its thermophilic growth range and isolation site (66) make it a prime model organism for bacterial dissimilatory iron reduction at thermophilic temperatures. 1.7.2 Geoglobus ahangari as a model organism for the hyperthermophilic archaea isolated from a coastal hydrothermal vent chimney Unlike the Axial Seamount system, the Guaymas Basin hydrothermal vent system is located close to shore (Figure 1.3) . In fact, this hydrothermal field is loc ated between the Baja Peninsula and the coast of Mexico within the Gulf of California (109) . This placement has led to the accumulation of deep sediments throughout the si te (51, 109) . Hydrothermal fluids must proceed through nearly 500 meters of organic - rich sediments before they arrive at the seafloor (51, 110) . Thus, unlike the previously mentioned Juan de Fuca ridge system, the diversity of carbon sources and electron donors is greatly increased within the Guaymas Basin (51, 110) . This abundance of compounds supports the development of thick microbial mats within and on top of the sediments in the basin and these systems need not rely solely on hydrothermally - produced compounds for growth (51) . The Guaymas Bas in can then serve as a model system to 16 examine a relatively nutrient - rich oasis when compared to the desert - like mid - ocean systems, such as the Axial Seamount discussed earlier. Geoglobus ahangari 234 T was enriched and isolated from a Guaymas Basin vent sa mple using Fe(III) oxides (52) . Phylogenetic analysis placed the strain a s a new genus within the family Archaeoglobales ; thus, G. ahangari is the type strain of the Geoglobus genus (52) . All members of the Archaeoglobales are hyperthermophiles with optimal growth temperatures ranging between 70 and 88 °C, with G. ahangari thriving at the upper limit (52) . They are closely related to the archaeal methanogens, with whom they share many metabolic similarities (111) . Unlike the thermophilic G. ehrlichii which can respire nitrate in addition to Fe(III) (66) , G. ahangari is an obligate iron reducer ( i.e., it can only use soluble or insoluble Fe(III) electron acceptors) (52) . Consistent with the abundance of organic compounds in the site of isolation, G. ahangari can oxidize a large number of electron donors during Fe(I II) respiration including short - and long - chain fatty acids, TCA cycle intermediates, and amino acids such as isoleucine, arginine, and serine (52) . Furthermore, it was the first hyperthermophile reported to use acetate, an electron donor previously thought to only support mesophilic respiratory processes (52, 112) . G. ahangari was also t he first dissimilatory Fe(III) - reducing microorganism capable of autotrophic growth with molecular hydrogen, an unsuspected metabolic capability of these organisms (52) . Thus, G. ahangari represents a model organism to represent a hyperthermophilic iron - reducing archaeon isolated from a nutrient - rich hydrothermal vent system. M echanistic studies of (hyper)thermophilic iron reduction and the need for additional model systems Models, by definition, are meant to represent a system full of uncertainties. Thus, while arguments persist regarding the origins of life and the settings in which it formed, the use of extant organisms as model systems to study early life warrants special attention. The identification of conserved mechanisms for iron respiration in (hyper)thermophilic bacteria and archaea could 17 provide valuable insights into the ancestral mechanisms that supported early forms of Fe(III) respiration. Furthermore, when compared to mesophilic mechanisms, the evolution of Fe(III) respiration and adaptations to the environment can be better understood. The availability of G. ehrl ichii and G. ahangari in pure culture and the unique physiological characteristics of these iron reducers make them particularly suitable as model systems for Fe(III) respiration in mid - ocean and off - shore hydrothermal vents. They are also phylogenetically distinct, one being a bacterium ( G. ehrlichii ) and the other, an archaeon ( G. ahangari ). Furthermore, the temperature growth range is also distinct (thermophilic and hyperthermophilic). Thus, genomic data coupled with physiological studies in these organi sms can provide valuable insights into iron respiration at high temperatures. Furthermore, when compared to the respiratory strategies of other thermophiles and hyperthermophiles (113, 114) , insights into the evolution of microbial respiration on Earth can be gained . Dissertation Outline 1.9.1 The high - quality draft genome sequence of the thermophilic Fe(III) - reducing bacterium Geothermobacter ehrlichii strain SS015 Chapter 2 describes the sequencing, assembly, and annotation of the genome of the iron - reducing bacterium Geothermobacter ehrlichii strain SS015. Annotated genes and pathways are compared to other members of the order Desulfuromonales and prediction s about the mechanism for iron respiration at thermophilic temperatures are discussed. This work was performed in collaboration with Dr. Spurbeck and Dr. Sandhu from Swift Biosciences (Ann Arbor, MI, USA), who assisted with the genome sequencing and assemb ly, and an undergraduate mentee from MSU, Lucas Demey, who assisted in the genome annotation. 18 1.9.2 The complete genome sequence and emendation of the hyperthermophilic, obligate iron - Geoglobus ahangari T Chapter 3 describes the seq uencing, assembly, and annotation of the genome of Geoglobus ahangari strain 234 T . Annotated genes and pathways are compared to other members of the family Archaeoglobaceae and predictions about the metabolic potential of the isolate and mechanism of iron respiration at hyperthermophilic temperatures are discussed. This work was (Massachusetts, USA) who assisted with the genome sequencing, assembly, analysis of metaboli c pathways, and drafting of the manuscript. 1.9.3 Extracellular electron transfer to Fe(III) oxides by the hyperthermophilic archaeon Geoglobus ahangari via a direct contact mechanism Chapter 4 describes physiological studies aimed at elucidating the mechanism e mployed by Geoglobus ahangari strain 234 T to reduce insoluble Fe(III) oxides. Assays were performed to determine if exogenous compounds such as electron shuttles and chelators could promote the growth of this strain on insoluble iron oxides and to determin e if these compounds were produced endogenously. Putative c - type cytochromes were identified among proteins separated in SDS - PAGE gels and stained for heme. Furthermore, the localization of putative c - cytochromes on the outer membrane of the cells was asse ssed by performing similar studies with proteins of insoluble, but not soluble, Fe(III) was demonstrated. A model for iron reduction by G. ahangari based on a direct - contact mechanism is presented. 1.9.4 Microbial Nanowires: A conserved m echanism for extracellular electron t ransfer Chapter 5 describes the biochemical characterization and conductive properties of protein filaments produced by the thermophilic bacteriu m G. ehrlichii strain SS015 and the hyperthermophilic archaeon G. ahangari strain 234 T for the reduction of Fe(III) oxides. Mechanical sheared samples, which contain the protein filaments as well as flagella, and protein filaments 19 purified from both organi sms were probed with a Conductive Probe Atomic Force Microscope (CP - AFM) to demonstrate their conductivity. The thermal and chemical stability of the protein filaments was also investigated using the mesophile G. metallireducens as a reference. The CP - AF M work was performed in collaboration with graduate student Krista Cosert and Dr. Sanela Lampa - Pastirk from the laboratory of Dr. Gemma Reguera . 20 CHAPTER 2. THE HIGH - QUALITY DRAFT GENOME SEQUENCE OF THE THERMOPHILIC FE(III) - REDUCING BACTERIUM GEOBACTER EHRLICHII STRA IN SS015 Abstract Geothermobacter ehrlichii strain SS015 is the only thermophilic member of the family Desulfuromonadaceae available in pure culture. It was isolated from vent fluid samples of the Bag City hydrothermal vent system, within the Juan de Fuc a Ridge, and produces copious amounts City landscape. The bacterium couples the reduction of soluble and insoluble Fe(III) oxides and of nitrate to the oxidation of a wide range of electron donors . It is the only thermophilic member of the Desulfuromonadales , a bacterial order containing the Pelobacteraceae , Desulfuromonadaceae and the Geobacteraceae families , a predominant clade in nearly all metal - reducing mesophili c subsurface communities . Hence , it is a good model system to also understand the thermophilic adaptation of Fe(III) reduction . Here I describe the sequencing and annotation of the genome of G. ehrlichii strain SS015 and discuss the metabolic capabilities predicted from genome data that are shared with other members of the Desulfuromonadales or unique to its thermophilic member, G. ehrlichii . I also discuss what physiological properties may have allowed this bacterium to grow at thermophilic temperatures , w hich are otherwise lethal to all other members of the Desu l furomonadales . The high - quality draft genome presented here contains 3,276,179 base pairs (bp) in 84 contigs containing 3,059 protein - coding genes, 53 RNA genes, and a predicted 60 missing genes. K eywords Desulfuromonadales ; Geobacteraceae ; Geobacter ; hydrothermal vent; Bag City; Fe(III) respiration; extracellular electron transfer . 21 Introduction The recovery of the thermophilic iron - reducing bacterium Geothermobacter ehrlichii in pure culture and it s initial placement within the family Geobacteraceae added thermophily to the physiological properties of this otherwise mesophilic group of iron reducers in the Deltaproteobacteria (66) . The family includes some of the best studied dissimilatory metal - reducing bacteria (73) , with Geobacter sulfurreducens and Geobacter metallireducens serving as model represent atives (reviewed in (71, 99, 103) ). Recent phylogenetic analyses now place the Geobacteraceae as a clade within the family Desu lfuromonada les , and G. ehrlichii outside the Geobacteraceae but within the Desulfuromonadaceae (107, 108) . Hence, G . ehrlichii strain SS015 is the type strain of the Geothermobacter genus and the only isolated th ermophilic member of the Desulfuromonadales (66, 108) , which also includes two psychrophil ic isolate s , Geobacter psychrophilus (115) a nd Geopsychrobacter electrodiphilus (116) . G. ehrlichii strain SS015 has a reported growth range of 35 to 65 °C and an optimum growth temperature of 55 °C (66) . Originally isolated from the Bag City site within the Axial Seamount system, G. ehrlichii produces an abundance of acidic extracellular polymeric (66) . The EPS is p roduced in response to sub - optimal temperatures and the presence of antibiotics , suggesting it plays a protective role (66) . G. ehrlichii strain SS015 shares many characteristics with members of the Geobac teraceae (73) and the Desulfuromonadacea e (108) in that it is able to couple the o xidation of a broad range of organic acids to the reduction of soluble and insoluble Fe(III) as well as nitrate (66) . Yet, unlike the Geobacteraceae , it can use amino acids, starch , and sugars as electron donors with Fe(III) serving as sole electron acceptor (66) . Genomic evidence suggests that certain Geobacter aceae species may have the capability to utilize these donors yet this abilit y has not been demonstrated in the laboratory (74, 117) . The mechanisms for Fe(III) respiration ha ve been extensively studied in the mesophilic members of the family ( (103) and reference s within), but less is known about Fe(III) reduction at 22 thermophilic temperatures. Cells of G. ehrlichii strain SS015 are motile via a single flagellum , which could allow the cell to access the Fe(III) oxides , provide d chemotaxis to Fe(II) is pres ent as in the Geobacteraceae (102) . Pilus - like filaments are also apparent in electron micrographs of G. ehrlichii cells (66) , which could be conductive and function as nanowires between the cell and the minerals as in mesophilic Geobacter species (91) . The nanowire pilins form an independent line of descent among bacterial pilins and pseudopilins (91) and are divergent in structure and amino acid composit ion to provide a protein environment optimized for electron transfer (118) . For example, unlike other b acterial t ype IV pili ns, they lack the conserved C - terminal globular domain of other bacterial type IV pilins and are predominantly an - helical peptide, a structure that promotes electronic coupling and electron transfer (118) . Hence, pilin - encoding genes can provide valuable information about the putative role of the pili in electron transfer. Another con served feature of the Geobacteraceae that is linked to their ability to reduce Fe(III) oxides is the presence of a great number of genes encoding c - type cytochromes (83) . Here, I report on the draft genome sequence of G. ehrlichii strain SS015 and summarize the physiological features that are conserved and divergent between this thermophile and other members of the Desulfuromonadales , with a focus on the features that could be involved in Fe(III ) reduction . Organism information 2.4.1 Classification and features I solated from the Bag City hydrothermal vent chimney, within the Axial Seamount system (46 °N, 130 °W), at a depth of 1,400 m, Geothermobacter ehrlichii strain SS015 wa s originally described as the only thermophilic member of the Geobacteraceae available in pure culture (66) . The sequence of the previously published (66) parti al sequence for the 16S rRNA gene (NR_042754.1) matches (92 %) to the 16S rRNA gene (rna.42) found within the genome. This rRNA gene was used to construct a new phylogenetic tree in reference to the 16S rRNA gene sequences from other members of the Desulfu romonadales , using Escherichia coli as the 23 outgroup, and allowed t he revision of the phylogenic position of G. ehrlichii in relation to the Geobacteraceae and the Desulfuromonadaceae (Figure 2.1). In this new tree, G. ehrlichii is most closely related phyl ogenetically to sequences of other Geothermobacter species not yet available in pure culture and, based on the 16S Ribosomal RNA Reference Sequence Similarity Search (RefSeq NCBI) , to several Desulfuromonas species including D. palmitatis (93.2 %) and D. thiophila (93.0 %) . However , alig nment of the 16S genes using SILVA (119) places D. palmitatiis at a distance from G. ehrlichii . The low confidence level within the Geothermobacter branch of the phylogenetic t ree indicates that there is a vast diversity of Geothermobacter species remaining to be discovered. Cells of G. ehrlichii strain SS015 are rod - shaped, 1.2 to 1.5 µm in length, can appear as single cells or in chains (Figure 2. 2) , and s pores were not observ ed (66) . Cells were motile, which was predicted to be enabled by th e single polar flagellum produced by the cell (66) . In addition, nu merous pilus structures could be seen on the surface of the cell (Figure 2. 2) (66) . Gram stain and thin section microscopy revealed G. ehrlichii to be Gram - negative (66) . When stressed, by sub - opti mal temperatures or antibiotics, cells were seen to produce an abundance of extracellular polymeric substances, as visualized by ruthenium red (66) . Growth was supported from 35 to 65 °C with an optimal growth temperature of 55 °C (66) . The pH range for growth was 5.0 to 8.0, with an optimum at 6.0, while the salinity range w as from 5.0 to 50 g/L, with an optimum at 19 g/L (66) . In addition, cell pellets of G. ehrlichii appear as red/pink and exhibit an absorption characteristic of c - type cytochromes (66) . G. ehrlichii strain SS015 was able to couple the oxidation of a range of electron donors, such as DL - malate, pyruvate, acetate, glutamate, propionate, butyrate, ethanol, and methanol, to the reduction of both Fe(III) and nitrate (66) . While the strain was isolated and ch aracterized to solely use insoluble Fe(III) oxides (66) , it rapidly adapted to utilize the soluble form of Fe(III), ferric citrate, when supplied ( presented within this work). The main physiological features of the organism are listed in Table 2. 1 (18, 120 124) . 24 Genome sequencing and annotation 2.5.1 Genome project history Based on a) its unique position as the only thermophilic member of the Desulfuromonadaceae available in pure cultur e; b) its ability to use acetate as an electron donor for Fe(III) reduction; and c) its ability to use sugars, starch, and several amino acids as electron donors; G. ehrlichii strain SS015 was selected for genome sequencing and annotation. This annotation thus provides information regarding the wider metabolic niche occupied by G. ehrlichii strain SS015 and the changes within the genome , when compared to the mesophilic members of the order, which likely permit growth at thermophilic temperatures. A summary of the project information is presented in Table 2. 2. 2.5.2 Growth conditions and DNA isolation G. ehrlichii strain SS015 was obtained from the Kashefi lab culture collection while it can also be obtained from the American Type Culture Collection (ATCC BAA - 635) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 15274). Media , as previously described (66) , contained 10 mM DL - malate as the electron acceptor and 50 mM soluble Fe(III) citrate as the electron acceptor. Growth on soluble Fe(III) citrate was obtained only after successive transfers in a mixture of sol uble and insoluble Fe(III) oxides. Cultures were incubated at 55 °C under a N 2 :CO 2 (80:20) atmosphere in the dark. Strict anaerobic techniques were utilized throughout (125) . Genomic DNA (gDNA) was extracted as previously described for Geoglobus ahangari (126) . In brief, G. ehrlichii cultures grown to mid - exponential phase were harvested by centrifugation, washed with an oxalate solution, an iron extraction buffer, and finally a marine wash buffer to remove any remaining chelating compounds. Cell pellets were f rozen at - 20 °C before being treated with a lytic buffer. gDNA was subsequent ly purified with the MasterPure DNA Purification Kit (E picentre Biotechnologies) according to manufacturer suggested guidelines. 25 2.5.3 Genome sequencing and assembly Two high - quality d raft genomes of G. ehrlichii strain SS015 were generated from Illumina (127) draft sequenc es obtained from Swift B ioscience s (Ann Arbor, MI). Genomic DNA was fragmented to an average size of 200 bp using a Covaris M220 ultrasonicator (Covaris, Woburn, MA). Two whole - genome libraries were made: one using the Accel - NGS 2S DNA Library kit and the other using Accel - NGS 1S DNA Library Kit (Swift Biosciences, Ann Arbor, MI). These libraries were sequenced on an Illumina MiSeq (Illumina, San Diego, CA) using MiSeq Reagent kit v2. Approximately 3 - 4 million reads were used for de novo genome assembly from each library using the assembler MIRA 4 (128) . In addition, one library was prepared, using BluePippin (Sage Science) size selected gDNA fragments , for PacBio sequencing on a single SMRT cell. 428,481 reads containing 2,052,390,107 bp af ter filtering were generated at the Weill Cornell Medical College of Cornell University and an HGAP 2.0 (129) assembly was generated by the sequencing facility for use. Table 2. 2 presents the project information. Assembly reconciliation software (130) combined with the alignment of short reads to the scaffolds before polymerase chain reactions (PCRs) were used to further clos e the genome. Minimus2 (130) was used as an assembly reconciliation program to verify, sew, and create a consensus genome from the three different assemblies. Pairwise comparisons were performed with the 1S and 2S genome s, 1S and PacBio, 2S and PacBio. F inally , these initial pairwise assemblie s were again reconciled with the third, unused, assembly. The results of the assembly reconciliation are presented in Table 2. 3. The assembly reconciled from the 2S Illumina library combined with the PacBio was determined, based on alignment of short reads to the genome , to be the optimal assembly . Attempts were then made to close any gaps and low coverage regions using primers designed to span the gaps (Table 2. 4). GoTaq Green PCR Master Mix (Promega) was used with purified gDNA from G. ehrlichii strain SS 015. All possible primer combinations were tested - - primer) . Products obtained from the closure reactions were run on an agarose gel before excision, purification with 26 Recovery Kit (Zymo Research), and submission to the Research Support and Training Facility at Michigan State University for Sanger sequencing. These reactions have yet to provide a closure based on the PCR products obtained. As such, this work is ongoing. 2.5.4 Genome annotation Gen ome annotation and ORF analyses were performed using free and publically - available software. The RAST platform (131) was used to obtain the genome annot ation, pseudogene detection was performed using the GenePRIMP pipeline (132) , pfam domains were detected using Pfam - A from EMBL_EBI (133) , signal peptides were detected using SignalP v.4.1 (134) set to Gram negative , and tran smembrane domains were detected using TMHMM (135) . Finally, CRISPR repeats and spacer region s were identified using CRISPRFinder (136) . For the 1S assembly, a total of 3,275,488 bp were assembled into 81 contigs with the largest contig containing 328,564 bp, an N 50 of 112,152 bp, 3,045 coding sequences , and 67 RNA genes. For the 2S assembly, a total of 3,279,720 bp were assembled into 84 contigs with the largest contig containing 382,770 bp, an N 50 of 110,457 bp, 3,058 coding sequences, and 53 RNA genes. For the PacBio HGAP assembly, a total of 2,450,31 3 bp were assembled into 151 contigs with the largest contig containing 84,277 bp, an N 50 of 22,144 bp, 2,377 coding sequences, and 40 RNA genes. Assembly 2S was chosen to proceed with genome annotation and analysis, during the genome - closure work describe d previously, due to the increased genome size and number of coding sequences. C - type cytochromes present within the genome, which could enable extracellular electron transfer to insoluble Fe(III) oxides, were identified as previously described for the hy perthermophilic iron - respiring archaeon G. ahangari (126) . In brief, proteins containing one or more c - type heme binding motifs (CXXCH) were selected from the genome annotation and screened for the presence of a signal peptide or an N - terminal membrane helix anchor (134) . Proteins meeting this requirement were then subjected to DELTA - BLAST analysis against the NR 27 protein database to determine h omology to known c - type cytochromes. Molecular weight estimations (137) were performed as previously desc ribed (126) on these putative cytochromes to compare them to putative c - type cytochromes identified by SDS - PAGE analysis followed by TMBZ staining (138) . Samples for the SDS - PAGE analysis we re obtained as follows. Outer - surface proteins were removed by mechanical shearing (5x in and 5x out) with a 23G needle. Prior to shearing , an aliquot was removed to represent pre - sheared cultures. Cells and cell debris were then harvested by repeated centrifugation , 2x (3,220 x g , 60 min, 25 °C) and 1x (16,873 x g , 5 min, 25 °C ) , and collected to represent the cell fraction removed of their outer - membrane associated proteins. Finally, the remaining supernatant was passed through a Pall 10 kDa centrifugal filter (1,811 x g , 25 °C ) to collect sheared proteins remaining in solution. Cell pellets were washed 1x with an oxalate solution (52) , incubated 30 min in IEB (139) to remove residual metals, and finally rinsed once with MMWB (139) . Proteins collected on the filter were washed with the same solutions and resuspended in MMWB. Samples were then run on a Tris - Tricine 4 - 20% acrylamide gel (Bio - Rad Laboratories, Inc. ) at 100V for 40 minutes and stained us ing the TMBZ protocol (138) to visuali ze heme containing bands. Genome properties The genome of G. ehrlichii strain SS015 presented within this work contains 3,279,720 bp and no plasmids. The genome size is the smallest reported within the Geobacteraceae (73) or the Desulfuromonadaceae (107) , indicating genome shrinkage; however, this may be the result of the unfinished genome. The mol percent G+C is 61.7, which closely matches the percentage (62.6 mol %) estimated b y the Identification Service of the DSMZ and within the range of both the Geobacteraceae (50.2 63.8 mol %) (73) and the Desulfuromonadaceae (46 62.3 mol %) (107) . Out of the 3,112 genes annotated within the genome, 3059 were identified as protein coding genes (98.2 %) and the remaining 53 as RNA genes (2.5 %). 1,580 of the predicted protein coding 28 genes (51.7 %) are represented by KEGG functional categories. In addition, the RAST annotation server approximates that 60 gen es may be missing from the assembled genome. The GenePrimp pipeline predicted 3 , 059 CDSs, 39 long genes, 49 short genes, 19 broken genes, 8 interrupted genes, and 163 intergenic regions with BLAST hits; totali ng 278 total anomalies. Thus, the 60 missing genes within the RAST annotation may exist in these intergenic regions. The genome characteristics and percentage representation of these genes is listed in Table 2. 5 and Table 2. 6, respectively. In addition, t he following genome characteristics have been calculated. The preferred start codon is ATG (81.4 %), then GTG (11.6 %), and finally TTG (7.0 %). There is a single rRNA gene cluster comprised of the SSU RNA (rna.42), two tRNAs (rna.43 - 44), the LSU RNA (rna. 44), and finally the 5S RNA (rna.46). The origin of replication could not be identified by Ori - Finder analysis (140) due to the draft nature of the genome. However, a gene cluster of protein s involved in chromosomal partitioning and cell division was annotated from peg.1414 - 1420, which likely indicates that this region is important for cell division and genome replication. Insights from the genome 2.7.1 Thermophilic adaptations As the only thermophilic member of the Desulfuromonadales (66) it was important to identify critical components for its ability to survive and replicate at temperatures which would be lethal to the majority of the order (73, 107) . Present in the genome are various heat shock and chaperone proteins; h owever, homologous proteins contained in similar gene clusters are present in a number of mesophilic Geobacter species (76, 77, 80, 141) . As such, pending increased expression within G. ehrlichii, these gene clusters are likely not the causative agent of the increased growth temperature of G. ehrlichii strain SS015. Outside of the af orementioned stabilizing proteins is the likelihood that G. ehrlichii proteins themselves have an increased thermostability over homologues in mesophilic Geobacter species . 29 A number of factors have been linked to increased thermal stability of proteins inc luding the use of decreased utilization of heat - labile amino acids, changes in protein packing, and the inclusion of charged amino acids (17, 142, 143) . These studies, however, have focused primarily on protein engineering (144 146) and thus lack a pan - proteome approach. This causes conflicting results as amino acid substitutions that stabilize one protein may destabilize another (142) . Thus, meta - analysis of stability data has provided insights into the subject (142) . Based on the literature it was evident that several heat - labile amino acids including cysteine, serine, methionine, asparagine, and glutamine have been implicated in reducing protein thermostability whi le others, including arginine and threonine, were implicated in increasing thermostability (17, 143) . Yet, other res ources suggest that, of these residues, only cysteine is statistically significant (142) . In addition, the amino acids glutamic acid, lysine, tyrosine, and i soleucine are used with a statistically increased rate in thermophilic and hyperthermophilic organisms (142) . To this end, the entire proteome s of all sequen ced member of the Geobacteraceae (NC_010814.1, NC_007517.1, NZ_CP009788.1, NZ_JXBL00000000.1, NC_002939.5, NC_009483.1, and NC_007498.2), with the exception of Geoalkalibacter ferrihydriticus due to the additional variable of life in an alkaline environmen t (with a pH optimum of 8.6) (147) , was used to create an amino acid bank. Comparisons of amino acid usage across these organisms (Figure 2. 3) enabled me to determine if any of these amino acids had an increased abundance over those in mesophilic species. Also included is the only psychroph ilic member of the Desulfuromonadales with a sequenced genome, Geopsychrobacter electrodiphilus (NZ_ARWE00000000.1) (116) . Several heat - labile amino acids, methionine and asparagine, showed a decreased abundance in G. ehrlichii while glutamine and the statistically - significant residue cysteine did not show a decreased abundance when compared to the mesophilic and psychrophilic strains. Ty rosine was also not significantly increased in G. ehrlichii , indicating that this amino acid may not stabilize proteins within the Desulfuromonadales . S erine, which has been linked to decreased thermostability, and arginine, which has been linked to increa sed thermostability, are utilized as expected in G. 30 ehrlichii and likely function to increase protein thermostability. Similarly, threonine and glutamic acid residues show the expected increase in abundance, suggesting their contribution to increases in th ermostability. As predicted, G. electrodiphilus shows the opposite trend as G. ehrlichii for serine, glutamic acid, valine, asparagine, glutamine, and threonine suggesting an optimization for increased protein flexibility and function within G. electrodi philus at low (4 30 °C with a T opt of 22 °C) temperatures (116) . A more telling metric for predicting the thermal stability of an orga established after the publication of these initial studies (148, 149) . T hese works state that the single most reproducible and characteri stic metric by which to assess the thermal stability of a proteome is to examine the ratio of charged to polar (non - charged) amino acids. The increased abundance of charged res idues is expected to present itself on the protein surface and stabilize the protein through ion bonds. Thus, using the same proteomes discussed above, the ratio of charged (aspartic acid, glutamic acid, lysine, and arginine) to polar (asparagine, glutamin e, serine, and threonine) amino acids was calculated (148, 149) . As above, va lues were calculated as a percentage of the proteome, and as such it is possible to determine the normalized ratio. As expected, the charged/polar ratio was correlated with growth temperature of the organisms under investigation (Figure 2.4). The psychroph ile, G. electridophilus , had the lowest ratio (1.17), followed by the mesophilic strains (1.32 +/ - 0.07), and the highest value was from G. ehrlichii (1.53). Thus, as predicted, the charged to polar ratio increased with increased optimal growth temperature of the organisms examined. This analysis thus presents a simplified and definitive reiteration of the amino acid usage presented above and confirms that the proteome has been optimized in G. ehrlichii to grow at elevated temperatures. 2.7.2 Central metabolism G . ehrlichii strain SS015 contains a near complete glycolytic pathway (Figure 2. 5 ) for the utilization of hexose sugars, even though no transporter could be found to permit glucose to enter the cell. In glycolysis, glucose is first phosphorylated to glucose 6 - phosphate by a hexokinase 31 protein. Chain s A and B of the Thermus thermophilus ROK hexokinase (3VOV), of which a crystal structure is available (150) , resulted in weak hits (7e - 11) to peg.1432 which is annotated as a polyphosphate glucokinase. DELTA - BLAST analysis of this protein shows that it is a member of the ROK family of proteins and produces strong hits to polyphosphate glucokinase proteins, which indeed take glucose to glucose - 6 - phosphate. Thus, it is likely that peg.1432 is a ROK hexokinase that could catalyze this first step in glycolysis . Glucose 6 - phosphate is next isomerized to fructose 6 - phophate, by a glucose - 6 - phosphate isomerase (peg.1967). Next, a 6 - phosphofructokinase protein (peg.1207 and peg.2741) phosphorylates the fructose 6 - phosphate to fructose 1,6 - bisphosphate in a unidirec tional reaction. Therefore, during gluconeogenesis, fructose 1,6 - bisphosphate is hydrolyzed to fructose - 6 - phosphate by fructose 1,6 - bisphosphatase proteins (peg.2234 and peg.2805). Fructose 1,6 - bisphosphate is next split into DHAP and glyceraldehyde 3 - phos phate by a class II fructose - bisphosphate aldolase (peg.2104). DHAP can next be isomerized into glyceraldehyde 3 - phosphate by a triosephosphate isomerase (peg.1641). Glyceraldehyde 3 - phosphate is next dehydrogenated and, with the addition of inorganic phos phate, produces 1,3 - bisphosphoglycerate by a glyceraldehyde - 3 - phosphate dehydrogenase. Two glyceraldehyde - 3 - phosphate dehydrogenase proteins were identified in the genome; peg.1643 is annotated as being NAD - dependent while peg.2105 is instead annotated as being NADH - dependent. Next, 1,3 - bisphosphoglycerate is phosphorylated into 3 - phosphoglycerate by a phosphoglycerate kinase (peg.1642). 3 - phosphoglycerate is next converted into 2 - phosphoglycerate by a phosphoglycerate mutase (peg.1614). Also, a 2 - 3 - bisphos phoglycerate - independent phosphoglycerate mutase can be found at peg.724 which may also catalyze this reaction. 2 - phosphoglycerate is next converted to phosphoenolpyruvate by an enolase protein (peg.2933). Finally, phosphoenolpyruvate is phosphorylated to pyruvate via a pyruvate kinase (peg.253). To proceed in the reverse direction during gluconeogenesis, a pyruvate carboxylase (peg.2794) and a phosphoenolpyruvate carboxykinase (peg.483) were identified within the genome. Therefore, with the exception of th e weak hit to the hexokinase 32 responsible for the initial step in the pathway, G. ehrlichii strain SS015 contains a complete glycolytic pathway for the processing of extracellular glucose to pyruvate to then enter the TCA cycle. Thus , G. ehrlichii strain SS 015 matches members of the Geobacteraceae in that it too contains a complete or nearly complete glycolysis pathway which likely functions primarily during gluconeogenesis (117) . Yet, the ability of G. ehrlichii to grow on starch, a trait uncommon in the Desulfuromonadales (66) , could permit the released sugars to feed into the start of glycolysis (151) . Starch is uncommon in hydrothermal vent environments while glycogen, a branched polysaccharide of glucose, can serve as a storage molecule for thermophilic archaea (reviewed in (151, 152) ). A gene cluster encoding a glycogen synthase, alpha - amylase,galactose - 1 - phosphate - uridylyltransfera se, amylopullanase (type II), and a phosphomannomutase was found in the genome (peg.2499 - 2503, respectively) in addition to a glucoamylase (peg.1002). The type II pullanase would be sufficient to degrade both the alpha - 1,6 and alpha - 1,4 glucosidic bonds pr esent within starch and glycogen (151) . In combination, these enzymes could serve to hydrolyze glycogen to glucose. Available glycogen could thus provide a source of glucose in the Bag City, and could be degraded to support the production of the EPS bags (66) found at the site . G. ehrlichii was also isolated as the first member of the Geobacteraceae able to catabolize amino acids to support growth (66) . Free amino acids are commonly pre sent in hydrothermal vent sites and provide a source of carbon, nitrogen , and energy if they are able to be degraded (59, 153, 154) . Anaerobic bacteria have been studied for decades for their ability to degrade amino acids (155) . This provides a sufficient pool of knowledge to investigate the ability of G. ehrlichii to utilize amino acids. Within the genome of G. ehrlichii are four gene clusters encoding ABC transporters for branched - chain amino acids (peg.10 13 - 1018, peg.1724 - 1729, peg.2177 - 2181, and peg.2535 - 2539). Two copies of a branched - chain amino acid aminotransferase (peg.1029 and peg.2465) and one gene cluster containing a branched - chain keto acid dehydrogenase (peg.2084 - 2086) were also identified with in the genome. In addition to these enzymes, a D - amino 33 acid dehydrogenase is required for the primary pathway for branched - chain amino acid hydrolysis. However, a D - amino acid dehydrogenase was not found in the genome. Yet, additional proteins are encoded within the genome to enable the degradation of leucine, isoleucine, and valine. Additionally, genes for glycine and serine utilization were identified by the annotation server. The combined effort of these proteins likely contribute to the ability of G. eh rlichii to couple the oxidation of these amino acids to the reduction of Fe(III) oxides. The predominance of Geobacteraceae species in anaerobic environments is partially attributed to their ability to fully oxidize acetate to CO 2 and couple this to the r eduction of metals (156) . This complete oxidation , which is conserved in the Geobacteraceae yet absent in a number of closely related ba cteria, is enabled by the complete TCA cycle in these organisms and the acquisition of a eukaryotic citrate synthase (156) . The genome a nnotation suggests that G. ehrlichii strain SS015 contains a complete TCA cycle (Figure 2. 5 ). Pyruvate can be decarboxylated to acetyl - CoA by a pyruvate dehydrogenase protein (peg.740 - 741). Next, acetyl - CoA and oxaloacetate are condensed into citrate by a citrate synthase (peg.1329) which, in G. ehrlichii strain SS015, is annotated as a Citrate Synthase (SI) and matches closely to the eukaryotic citrate synthase proteins present within Geobacteraceae genomes (156) . Several aconitate hydratase proteins, for the isomerization of citrate to D - isocitrate, were identified (peg.1005, peg.2651, and peg.2824). Blasting the acontitate hydratase 1 an d 2 (YP_006889862.1 and ADI84496.1, respectively) from G. sulfurreducens provided top hits to peg.2651 and peg.2824 respectively, and thus the functionality of the other putative aconitate hydratase (peg.1005) is unclear. Following the production of D - isoc itrate, an isocitrate dehydrogenase (peg.2812) is expected to produce alpha - ketoglutarate. Alpha - ketoglutarate is then decarboxylated to succinyl - CoA by an alpha - ketoglutarate dehydrogenase (peg.116 - 117). Succinyl - CoA is next converted into succinate by a succinyl - CoA synthetase (peg.374 - 375) and next dehydrogenated to fumarate by a succinate dehydrogenase (peg.2927 - 2929). Malate is then produced by a fumarate hydratase (peg.1890) and, subsequently, oxaloacetate by a malate 34 dehydrogenase (peg.2810). Additio nally, a NADP - dependent malic enzyme (peg.1602), a pyruvate decarboxylase (peg.2794), and a phosphoenolpyruvate carboxykinase (peg.483) were annotated in the genome. Thus, G. ehrlichii strain SS015 encodes a complete TCA cycle in addition to the eukaryotic citrate synthase, a trademark of the Geobacteraceae . An incomplete pentose phosphate pathway is present in the genome of G. ehrlichii strain SS015 . Lacking, by annotation and homology searches from related organisms, are all genes which would be used in t he oxidative phase of the pathway including a glucose - 6 - phosphate dehydrogenase, a glucolactonase, and a 6 - phosphogluconate dehydrogenase. This lack of the oxidative phase is conserved in a majority of the Geobacteraceae (77) , indicating that the a bsence of these genes is not an artifact of the assembly. Yet, a near complete non - oxidative phase is encoded within the genome. A ribulose - 5 - phosphate isomerase (peg.1302), ribulose - phosphate 3 - epimerase (peg.387), transketolase (peg.913), and a transaldo lase (peg.373) were annotated. Thus, it appears as though the non - oxidative phase of the pentose phosphate pathway is likely functional and would permit the biosynthesis of necessary amino acids. 2.7.3 Utilization of sulfur - containing compounds Unlike the majori ty of the Geobacteraceae (73) , but commo n within the Desulfuromonadales (141) , is the inability of G. ehrlichii strain SS015 to use sulfate as a terminal electron acceptor (66) . Thus, an examination of both the assimilatory and dissimilatory pathways was performed to determine why G. ehrlichii strain SS015 is unable to use sulfate. Sulfate is initially brought into the cell by two putative sulfate permease proteins (peg.3037 - 38) and subsequently activated to APS by a sulfate adenylyltransferase protein. Two copies of an annotated sulfate adenylyltransferase subunit 1/adenylylsulfate kinase were identified within the genome (peg.2438 and peg.564) and two copies of the second subunit of the adenylyltransferase were also identified (peg.736 and peg.3064). APS can then proceed to sulfite and AMP or to PAPS. APS can presumably be phosphory lated to PAPS via the previously mentioned hybrid proteins containing adenylylsulfate kinase activity (peg.564 and peg.2438). Interestingly, the 35 generation of sulfite during either pathway is likely catalyzed by a bifunctional gene at peg.2029. This gene i s annotated as a phosphoadenylyl - sulfate reductase [thioredoxin] / adenylyl - sulfate reductase [thioredoxin] and thus likely serves to take both APS and PAPS to sulfite, an enzymatic process that has been characterized in other organisms (157) . Sulfite is next reduced to sulfide by a sulfite reductase. Peg.2567 is annotated as both the alpha and beta subunits of a dissimilatory sulfite reductase while an additional, assimilatory, ferredoxin -- sulfite reductase can be found at peg.565. No str ong hits could be found to the cytochrome c nitrite and sulfite reductase of G. sulfurreducens KN400 (ADI85905.1) , yet a decaheme cytochrome (peg.568) is found near the previously mentioned ferredoxin -- sulfite reductase and peg.569 is a lipoprotein which may similarly be involved in the reduction of sulfite. Completing the assimilatory pathway are several cysteine sy nthase genes (peg.566 and peg.2657), a cystathionine gamma - synthase (peg.567), and a serine acetyltransferase (peg.1893). Yet, it appears as though the genome of G. ehrlichii encodes most, if not all, enzymes required for dissimilatory sulfate reduction, e ven though this physiology has not been observed (66) . 2.7.4 Nitrogen com pounds as electron acceptors Nitrate metabolism is known to take place in only a few Desulfuromonadale species including G. metallireducens, G. lovleyi, G. argilaceus , and G. ehrlichii (73, 107) . G. ehrlichii h as two nitrate/nitrite transporter genes (peg.657 - 58) for the uptake of nitrogenous compounds into the cell. Once in the cell, nitrate can proceed through an assimilatory or dissimilatory pathway. The assimilatory pathway results in t he incorporation of ni trogen in to the cell in the form of ammonium while the dissimilatory pathway is a catabolic pathway (158) where the reduction of nitrate and nitrite produce ATP (159) . Dissimilatory nitrate metabolism can be split further into two pathways: denitrification and nitrate ammonification (158) . Denitrification reduces nitrate in a stepwise fashion to nitrous oxide or dinitrogen while nitrat e ammonification is a two - step process which reduces nitrate to nitrite, and then nitrite is subsequently reduced to ammonium (159) . 36 Regardless of the pathway used, once nitrate has entered the cell it must first be reduced to nitrite. Nitrate reduction can be catalyzed by either NapAB or NarGHI nitrate reductase, which both require molybdenum cofactors to function (160) . Based on the genome annotation, G. ehrlichii encodes a NarGHI gene cluster (peg. 653 - 656) and no NapAB genes. Nitrite then proceeds down the ammo nification pathway, via NrfA, or the deni trification pathway, via NirK or NirS. Interestingly, no NrfA, NirK, or NirS genes could be found within the genome to complete the ammonification or denitrification pathway. However, two cytoch rome proteins (peg.65 and peg. 6 8) were identified near a nitrate reductase beta chain (peg. 64). Peg.65 is a member of the c 3 - cytochrome superfamily, the same superfamily as NrfA, and may serve a similar function. Since nitrite is an intermediate in the production of ammonium, most dis similatory nitrate reducing bacteria demonstrate biphasic growth when grown with nitrate as the sole electron acceptor (161) . G. ehrlichii lacks biphasic growth when nitrate is provide d as the sole electron acceptor and ammonium is accumulated in the culture vial (66) . Similar behavior has been shown in another member of the Desulfuromonadales , G. met allireducens (161) . It too pos sesses a NarGHI gene cluster, but also possesses NrfA. The lack of biphasic growth in G. metallireducens is thought to occur because nitrite is reduced immediately after production to avoid reaching toxic levels (161) . Thus, the shared physiology and genomic data suggest a similar reas oning may function in G. ehrlichii . Interestingly, the nitrate reductase of G. metallireducens is resistant to tungstate, indicating that it lacks the conserved molybdenum cofactor (161) . To provide further evidence that G. ehrlichii and G. metallireducens follow the same pathway, nitr ate reduction to ammonia in the presence of tungstate must be observed in G. ehrlichii strain SS015. 2.7.5 Motility and chemotaxis Motility has been observed in G. ehrlichii strain SS015 and this motility is presumed to be due to the presence of a bacterial fla gellum (66) . Flagellated members of the Geobacteraceae have been kn own to be chemotactic towards iron (102) . Thus, I thought it important to identify genes encoding proteins involved in both flagellar assembly, motility, and finally chemotaxis within 37 the genome of G. ehrlichii strain SS015. A single, large, and nearly uninterrupted gene cluster was identified within the genome which contains an extensive number of genes involved in flagellar biosynthesis and function. This gene cluster stretches from peg.1217 to peg.1267 with a minimal number of genes not annotated as belonging to this family of proteins. A similar organization can be seen in a number of Geobacter species (76, 77, 80, 141) . Several gene clusters involved in chemotaxis were identified within the genome. Within these gene clusters were proteins annotated as CheA, CheB, CheC, CheD, CheR, CheW, CheX, CheY, Tsr, and several Fli genes including FliG, FliM, and FliN. The presence of multiple chemotaxis gene cluster s is conserved in a number of sequenced Geobacteraceae species (162) . Interestingly, a large chemotaxis gene cluster is found almost immediat ely adjacent to the flagellar cluster (peg.1269 - 77), a gene organization which has not been reported in the Geobacteraceae (162) , but is pres ent in Pelobacter carbonolicus (141) a member of the Desulfuromonadales. The conserved alpha group of chemotaxis proteins, found only within the Geobacteraceae (162) , was not found in G. ehrlichii . P roteins within the alpha chemotaxis cluster from G. sulfurreducens (gsu0297 - 0293) did , however , match closely to proteins within one G. ehrlichii chemotaxis gene cluster (peg.1075 - 1086). Thus, the shared organization of these gene clusters, combined with the previously mentioned chemotaxis towards iron in flagellated mesophilic Geobacter species (102) may lend credence to similar strategies functioning in thermophilic environments. 2.7.6 Fe(III) as electron acceptor The Bag City hydrothermal chimney is a stable diffuse vent in the southern portion of the Axial Seamount caldera and just outside of the well - studied 199 8 eruption zone (28) . A substantial southward flow around the Axial Seamount (106, 163) combined with hy drothermal effluent exiting the caldera via a break in the southern wall (163) likely leads to the deposition of metals at the Bag City site. Phase separation at the site, due to the Axial Seamount being a shallow system, alters the redox state, temperatur e, chlorinity, and pH of the efflux water and minerals within (28) . Thus, the lowered pH and increase in H 2 S lead to changes in mineral solubility and transport 38 when compared to traditional vent systems (28) . Only after mixing with the surrounding seawater in the caldera will dissolved minerals, of which Fe(III) is a primary component, fall out and form metal - rich deposits (28) . T hese environmental conditions likely enrich for microorganisms, such as G. ehrlichii, which are able to respire these insoluble metal oxides. A direct - contact mechanism for Fe(III) respiration has been established in the Geobacteraceae and rel ies on the presence of a unique type IV pilus and c - type cytochromes (91, 164 166) . Two distinct gene clusters encoding the type IV pilus and associated assembly proteins of G. ehrlichii stra in SS015 can be found within the genome. These gene clusters are separated by a cluster of genes related to the Xap operon (discussed within 2.7.7) found in G. sulfurreducens (90) . The first of these gene cluste rs is comprised of PilSe (peg.2833), PilR (peg.2834), PilY1 (peg.2838), and then PilB, PilT, PilC, PilSe, PilR, PilA, and a hypothetical protein related to the PilA - C protein found in G. sulfurreducens (peg.2843 - 49; respectively). The PilA pilin protein al igns well to homologous pilin proteins present within the Geobacteraceae (Figure 2. 6 A and 5B) and, based on a phylogenetic tree constructed using the aligned pilin sequences (Figure 2. 6 C), is most similar to the pilin of G. metallireducens (Figure 2. 6 cont ains the references (167 169) ) . Following the previously mentioned extracellular polysaccharide gene cluster is another gene cluster of pilin genes comprised of PilD, a transcriptional regulator, PilM, PilN, PilO, PilP, and PilQ (peg.2860 - 66; resp ectively). Also found within the genome is an additional copy of PilB (peg.1883) and three additional copies of PilT (peg.951, peg.1637, and peg.1687). This organization , again, is similar to other sequenced members of the Geobacteraceae in which multiple PilT copies have been identified (76, 91) . A trademark of the Geobacteraceae is the extreme number of c - type cytochromes present within the genome of all sequen ced members of the family (73) . Several of these cytochromes, including PpcA, OmcS, and OmcZ have been implicated in G. sulfurreducens in metal respiration or in the transfer of electrons to the anode of a microbial electrochemical cell (104, 170 173) . Yet, the majority of the annotated cytochromes have not been implicated in essential processes. Some 39 suggest that the majority of these cytochromes provide a capacitor - like ability for Geobacteraceae isolates to store electrons when available electron acceptors are limiting (174) . Other possibilities exist such as custom ized cytochromes for differing electron acceptors or cytochromes poised to reduce at different voltages (175) . Regardless of which reasoning is correct, the prevalence of c - type cytochromes within the Geobacteraceae is a trademark of the family (73) . G. ehrlichii strain SS015, similarly to the remainder of the Geobacteraceae , does contain a number of c - type cytochromes. 34 putative c - type cytochromes were identified within the genome (Table 2. 7). This, while well above the number of cyto chromes present in most microorganisms sequenced to date, pales in comparison to many sequenced members of the Geobacteraceae (73) . The majority of the 34 cytochromes contain fewer than 10 heme - binding motifs while several (peg.622, peg.623, peg.810, peg.1443, peg.2155, and peg.2156) contain hig her numbers ( 13, 10, 23, 11, 19, and 57, respectively ) of heme b inding motifs. Peg.2156 containing 57 heme - binding motifs, is impressive for a c - type cytochrome yet unremarkable within the Geobacteraceae where ca. 85 % of cytochromes are multi - heme (83) . These may be too energetically taxing for G. ehrlichii to produce, as life at the Axial Seamount is likely nutrient - limited due to its location (106) , yet their existence within the genome indicates that these heme - rich proteins are widespread within the family. G. ehrlichii has an average heme/cytochrome value of 7.2. This value is similar to the values reported for G. bemidjiensis, G. metalli reducens, and G. sulfurreducens (73) . In support of this, at least 9 cytochromes can be identified, by TMBZ - stained SDS - PAGE, in ferric citrate grown cultures both before and after shearing of outer - surface cytochromes, while 5 are predicted to be outer - membrane or surface - exposed due to their d etection within the lane corresponding to the sheared fraction (Figure 2. 7 ). These cytochromes, and those identified within the genome, could serve to reduce soluble and insoluble electron acceptors, facilitate intracellular electron transfer, and enable t he capacitor - like physiology of the Geobacteraceae . 40 2.7.7 Extracellular polymeric substances G. ehrlichii strain SS015 was found to express an acidic EPS when stressed by antibiotics or when cultures were incubated outside of their optimal growth temperature (66) . This EPS likely contributes to the gelatinous bags which are th t system and, in G. ehrlichii , has been linked to growth on structural Fe(III) within phyllosilicate minerals (176) . In addition, the e xpression of an EPS matrix alongside properly anchored c - type cytochromes has, in G. sulfurreducens , been shown to be essential for growth on insolu ble oxides and in microbial fuel cells (90) . As G. ehrlichii is known to interact with insoluble Fe(III) oxides (66) , it was of interest to determine if the genes encoding the EPS of G. ehrlichii were similar to those of G. sulfurreducens . Using the XapA - K genes from G. sulfur reducens as BLAST queries against the G. ehrlichii genome I was able to identify a gene cluster (peg.2850 - 2859) which is likely involved in EPS biosynthesis. As in G. sulfurreducens (76, 90) , these genes follow a pilus - assembly gene cluster (peg.2833 - 2849) and, in addition, are followed by an additional cluster of non - redundant pilus - assembly genes (peg.2860 - 66) both of which have been discussed previously. Strong homolog ues could be found to XapA - E (peg.2853, peg.2850, peg.2851, peg.2852, and peg.2853; respectively). Two additional glycosyltransferase proteins (GT - B clan) (peg.2855 - 56), a membrane protein involved in the export of O - antigen/techoic acid/lipoteichoic acids (peg.2857), and an ADP - heptose -- lipooligosaccharide heptosyltransferase II (peg.2859) were also found within the gene cluster. Thus, this EPS cluster may serve a similar purpose to that of the Xap operon in G. sulfurreducens and permit the anchoring of es sential c - type cytochromes into an electroactive extracellular matrix to facilitate electron transfer to insoluble iron oxides (90) . Conclusions G. ehrlichii strain SS015 is the only member of the Desulfuromonad ales available in pure culture. This order contains the Geobacteraceae family, which has recently been proposed to be 41 split into the Geobacteraceae and the Desulfuromonadaceae , and the Pelobacteraceae . Genome sequences are available for a surprising number of members of these families, considering the lack of direct human impact these environmental isolates have. Yet, their use in bioelectronics, bioremediation, nanotechnology, and green energy has promoted great interest in the order. To date, members of t he Desulfuromonadales have been isolated able to grow at psychrophilic, mesophilic, and thermophilic temperatures and, with the inclusion of the Geoalkalibacter spp. available in pure culture, a range of pH values. Thus, these extremophilic members of the family environmental conditions. G. ehrlichii , for example, was isolated as the first member of the family Geobacteraceae able to grow optimally at thermophilic tem peratures. I have attributed this ability to an optimization of amino acid usage to function at elevated temperatures, a trend which is reversed in the psychrophile G. electrodiphilus . In addition, I have identified a gene cluster implicated in EPS prod uction and secretion which may be used in a similar way to the Xap operon of G. sulfurreducens. Finally, I have identified numerous c - type cytochromes, a flagellum and associated genes to enable chemotaxis, and a type IV pilin and associated assembly genes within the genome of G. ehrlichii which are undoubtedly involved in its respiratory ability to grow on soluble and insoluble Fe(III) oxides. G. ehrlichii shares a number of physiological and genetic characteristics with the Geobacteraceae ; however, its re cent classification into the Desulfuromonadaceae is upheld within this work by the absence of an alpha chemotaxis gene cluster in G. ehrlichii and its phylogenetic position within the Desulfuromonadales . While the presence of genes of interest within the g enome can be analyzed, any genes found lacking within this report may be artifactual due to the incomplete nature of the genome assembly. Efforts will continue in order to rectify this in the future to provide a high - quality closed genome for the only ther mophilic Desulfuromonadales member available in pure culture. 42 CHAPTER 3. THE COMPLETE GENOME SEQUENCE AND EMENDATION OF THE HYPERTHERMOPHILIC, OBLIGATE IRON - RED U CING ARCHAEON GEOGLOBUS AHANGARI STRAIN 234 T Abstract Geoglobus ahangari strain 234 T is an obligate Fe( III) - reducing member of the Archaeoglobales , within the archaeal phylum Euryarchaeota , isolated from the Guaymas Basin hydrothermal system. It grows optimally at 88 °C by coupling the reduction of Fe(III) oxides to the oxidation of a wide range of compound s, including long - chain fatty acids, and also grows autotrophically with hydrogen and Fe(III). It is the first archaeon reported to use a direct contact mechanism for Fe(III) oxide reduction, relying on a single archaellum for locomotion, numerous curled e xtracellular appendages for attachment, and outer - surface heme - containing proteins for electron transfer to the insoluble Fe(III) oxides. Here I describe the annotation of the genome of G. ahangari strain 234 T and identify components critical to its versat ility in electron donor utilization and obligate Fe(III) respiratory metabolism at high temperatures. The genome comprises a single, circular chromosome of 1,770 ,093 base pairs containing 2,020 protein - coding genes and 52 RNA genes. In addition, emended de scriptions of the genus Geoglobus and species G. ahangari are described. Keywords Euryarchaeota ; Archaeoglobales ; hydrothermal vent ; Guaymas Basin; Fe(III) respiration; extracellular electron transfer; autotroph. Introduction Geoglobus ahangari strain 234 T is the type strain and one of only two known members of the Geoglobus genus within the order Archaeoglobales and the family Archaeoglobaceae . It is an 43 obligate Fe(III) - reducing archaeon isolated from the Guaymas Basin hydrothermal system and grows at temp eratures ranging from 65 - 90 °C, with an optimum at about 88°C (52) . It wa s the first isolate in a novel genus within the Archaeoglobales and the first example of a dissimilatory Fe(III) - reducer able to grow autotrophically with H 2 (52) , a metabolic trait later shown to be conserved in many hyperthermophilic Fe(III) reducers (177) . G. ahangari can also couple the reduction of soluble and insoluble Fe(III) ac ceptors to the oxidation of a wide range of carbon compounds including long - chain fatty acids such as stearate and palmitate, which were previously not known to be used as electron donors by archaea (52) . It was also the first hyperthermophile reported to fully oxidize acetate to CO 2 , a metabolic function once thought to occur solely in mesophilic environments (112) . Unlike the other two genera in the order Archaeoglobales ( Archaeoglobus and Ferroglobus ), which can utilize acceptors su ch as sulfate and nitrate (52, 53, 178 183) , the two cultured members of the genus Geoglobus can only use Fe(III) as an electron acceptor (52, 178) . The obligate nature of Fe(III) respiration in Geoglobus spp. makes the genus an attractive model to gain insights into the evolutionary mechanisms that may have led to the loss and/or gain of genes involved in th e respiration of iron and other electron acceptors such as sulfur - and nitrogen - containing compounds within the Archaeoglobales . G. ahangari strain 234 T also serves as a model organism for mechanistic studies of iron reduction at high (>85 ° C) temperatures . Dissimilatory Fe(III) reduction has been extensively studied in mesophilic bacteria (reviewed in references (71, 84) ). By contrast, little is known about the mechanisms that allow (hyper)thermophilic organisms to respire Fe(III) acceptors (113, 114, 139, 184 186) . As previously observed in the thermophilic Gram - positive bacterium Carboxydothermus ferrireducens (114) , G. aha ngari also needs to directly contact the insoluble Fe(III) oxides to transfer respiratory electrons (139) . In G. ahangari , cells are motile via a single archaellum, which could help in locating the oxides, and also express numerous curled extracellular appendages, which bind the mineral particles and posit ion them close to heme - containing proteins on the outer surface of the cell to facilitate electron transfer (139) . A direct 44 contact mechanism such as this is predicted to confer on these organisms a competitive advantage over other organisms relying on soluble mediators such as metal chelators (97) and electron shuttles (187, 188) , which are energetically expensive to synthesize and are easily diluted or lost in the environment once excreted (102) . This is particularly important in hydrothermal vent systems such as the Guaymas basin chimney where G. ahangari strain 234 T was isolated, as vent fluids in these systems can flow through at ra tes as high as 2 m/s (109) . Here, I report the complete genome sequence of G. ahangari strain 234 T and summarize the physiological features that make this organism a good model system to study Fe(III) reduction in hot environments and to gain insights into the evolution of Fe(III) respiration in the family Archaeoglobales . Organism information 3.4.1 Classification and features Geoglobus ahangari strain 234 T is a euryarchaeon orig inally isolated from samples obtained from a hydrothermal chimney located within the Guaymas Basin (27° N, 111° W) at a depth of 2,000 m (52) . The sequence of the single 16S rRNA gene found in its genome was 99% identical to the previously published 16S rDNA sequence ( AF220165 ). The full length 16S rRNA gene (1,485 bp) was used to construct a phylogenetic tree in reference to 16S rRNA gene sequences from other hyperthermophilic archaea using two thermophilic bacteria ( Aquifex aeolicus and Pseudothermotoga thermarum ) as outgroups (Figure 3. 1). The closest known relative was Geo globus acetivorans (97% identical), the only other known member of the Geoglobus genus, which also is available in pure culture (178) . Closest relatives outside the genus were other hyperthermophilic archaea within the family Archaeoglobaceae such as the sulfate - reducing Archaeoglobus species A. fulgidus and A. profundus (97% and 93% identical, respectively) and Ferroglobus placidus (94% identical), which can reduce Fe(III), thiosulfate, and nitrate (112, 179) . 45 Cells of G. a hangari strain 234 T are regular to irregular cocci, 0.3 to 0.5 µm in diameter, and usually arranged as single cells or in pairs (Figure 3. 2 and Table 3. 1) (52) . Cells are motile via a single archaellum (52) , but also produce abundant extracellular curled filaments when grown with both soluble and insoluble Fe(III) (139) . Though optimum growth occurs at ca. 88 °C, growth is observed between 65 °C and 90 °C (52) . Furthermore, growth was supported at pH values between 5.0 and 7.6, with an optimum at pH 7.0, and with NaCl co ncentrations ranging from 9 to 38 g/L, with an optimum at 19 g/L (52) . A distinctive feature of the metabolism of G. ahangari strain 234 T is its obligate nature of Fe(III) respiration, with both soluble and insoluble Fe(III) species supporting growth but the insoluble electron acceptor being preferred (52) . The obligate nature of Fe(III) reduction contrasts with the wide range of electron donors tha t G. ahangari can oxidize (52) . Acetate, alongside a number of other orga nic acids (such as propionate, butyrate, and valerate), several amino acids, and both short - chain and long - chain fatty acids were completely oxidized to CO 2 to support growth during Fe(III) respiration (52, 112) . Furthermore, G. ahangari strain 234 T was also able to grow autotrophically with H 2 as the sole electron donor and Fe(III) as the electron acceptor (52) . The main physiological features of the organism are listed in Table 3. 1. Genome sequencing and annotation 3.5.1 Genome project history Based on its unique physiological characteristics (52) and use as a model system for mechanistic investigations of Fe(III) reduction by hyperthermophilic archaea (139) , G. ahangari strain 234 T was selected for sequencing. Ins ights from its genome sequence and annotation provide greater understanding of the evolution of respiratory metabolisms in the Archaeoglobales , and, in particular, about hyperthermophilic iron reduction within the Archaea . The genome project information is listed in the Genomes OnLine Database (Gp0101274) (189) and the complete 46 genome sequence has been deposited in GenBank ( CP011267 ). A su mmary of the project information is shown in Table 3. 2. Growth conditions and genomic DNA preparation G. ahangari strain 234 T was from our private culture collection and is available at the Deutsche Sammlung von Mikroorganismen und Zellkulturen ( DSM - 27542 ) , the Japan Collection of Microorganisms ( JCM 12378 ), and the American Type Culture Collection ( BAA - 425 ). The strain was grown in marine medium (52) with 10 mM pyruvate as the electron donor and 56 mM ferric citrate as the electron acceptor. Cultures were incubated under a N 2 :CO 2 (80:20 %, v/v) atmosphere at 80 °C or 85 °C in t he dark. Strict anaerobic techniques were used throughout the culturing and sampling experiments (125) . gDNA was extracted as previously reported for F. placidus (190) . Alternativel y, cells were lysed with an SDS - containing lysis buffer (5% SDS, 0.125 M EDTA, 0.5 M Tris, pH 9.4), as reported elsewhere for the preparation of whole cell extracts from G. ahangari (139) , and gDNA according to the manuf acturer suggested guidelines. Genome sequencing and assembly The finished genome of G. ahangari strain 234 T ( CP011267 ) was generated from Illumina (127) draft sequences generated independently at the Research Support and Training Facility at Michigan State University, the Deep Sequencing Core Facility at the University of Massachusetts Medical School, and the Genomic s Resource lab at the University of Massachusetts - Amherst . Table 3. 2 presents the project information. The sequencing project at the University of Massachusetts facilities used gDNA suspended in 3 ml of sonication buffer (4.95 % glycerol, 10 mM Tris - HCl (p H 8.0), 1 mM EDTA) and sonicated for 10 min (2 min on, 30 sec off) using a 550 Sonic Dismembrator (Fisher Scientific). 47 The samples were then dispensed as equal volumes into 4 tubes and mixed with 150 µl TE buffer (10 mM Tris - HCl (pH 8.0), 1 mM EDTA), 100 µ l ammonium acetate (5 M), 20 µl glycogen (5 mg/ml), and 1 ml of cold ( - 20 °C) isopropanol. Nucleic acids were precipitated at - 30 °C for 1 h, as previously described (190) and suspended in EB buffer (Qiagen) before separating the DNA fragments in the sample by agarose gel electrophoresis. DNA fragments between 300 - 500 bp were then purified with the Qi aQuick Gel Extraction Kit (Qiagen). All steps involved in end repair, reagents supplied by the TruSeq DNA Sample Prep Kit (Illumina). This resulted in the construc tion and sequencing of five independent 100 bp paired - end Illumina shotgun libraries which generated 7,970,036, 7,970,182, 7,973,896, 7,966,671 and 3,144,785 reads totaling 3.50 Gbp. The Illumina draft sequences were assembled de novo with SeqMan NGen (DNA STAR) and Velvet (191) (version 1.2.10) and optimized with VelvetOptimiser (version 2 .2.5). Reads were down - sampled to 500x coverage to increase the efficacy of the Velvet assembler while the complete depth of reads was used to verify the final genome assembly. A total of 1,780,565 bp were assembled into 25 scaffolds ranging in size from 207 bp to 510,180 bp. The scaffolds were then connected by adaptor - PCR as previously described (192) . G. ahangari gDNA was subsequently digested separately by four different restriction enzymes ( Eco RI, Bam HI, Bcl I, and Sal I). After a 1.5 - h incubation at 37 °C (or 50 °C for Sal I), the restriction digests were separated by agarose gel electrophoresis and fragments between 5 - 10 kb were overhangs generated by Eco RI, Bam HI, Bcl I, and Sal ligated with T4 DNA ligase to the fragments purified from the restriction digests of G. ahangari gDNA. The adaptor sequences used were: Eco RI adaptor AATTCCCTATAGTG Bcl I and Bam HI adaptor GATCCCCTATAGTGAGTCGTATTAAC**; and finally the Sal I adaptor TCGACCCTATAGTGAGTCGTATTAAC**. Further assembly was performed with SeqMan Pro 48 adaptor ligations were diluted 100 - fold and 1 µl of the diluted sample was used in PCR reactions with AccuTaq TM LA DNA Polymerase (50 µl total volume) according to manufacturer specifications (Sigma - Aldrich). Fifty react ions were performed with G. ahangari - specific primers designed from the various Illumina scaffolds and a non - phosphorylated primer that complemented the adaptor sequence on the gDNA (GTTAATACGACTCACTATAGGG). All PCR products were purified with the Qiagen P CR purification kit and sent for sequencing at the University of Massachusetts (Amherst) sequencing facility. This process was repeated until a single contig was obtained using SeqMan Pro assembly software. The assembly was then verified against a second, independent genome assembly generated at Michigan State University . Extracted gDNA was used to construct a single Illumina shotgun library using the Illumina DNAseq Library Kit, which was sequenced in two 150 bp paired - end runs with an Illumina MiSeq at t he Research Support and Training Facility at Michigan State University. The sequencing project generated 1,233,811 and 796,056 reads totaling 304.5 Mbp. Reads were quality trimmed using a combination of fastq - mcf (193) (ea - utils.1.1.2 - 537, using default parameters) and ConDeTri (194) (v2.2, using default parameters with th e exception of hq=33 and sc=33) to remove low - quality reads and over - represented sequences. High - quality paired and unpaired reads were assembled using Velvet (191) (v1.2.08 using default parameters N75 of 37,520 bp). The second assembly was then co mpared to the primary assembly to identify errors and low - coverage regions, which were subsequently resolved by PCR - amplifying and sequencing the regions of interest. 3.7.1 Genome annotation Initial genome annotation was performed by the RAST server (131) , the IGS Annotation Engine (195) at the University Of Maryland School Of Medicin e, and the IMG - ER platform (196) . The annotations were compared to manual annotations performed using GLIMMER (197) for 49 gene calls and DELTA - BLAST analysis to identify conserved domains and homology to known proteins. EC numbers and COG categories were determined with a combination of DELTA - BLAST analysis of each annotated gene and the IMG - ER platform. Pseudogenes were identified usi ng the GenePRIMP pipeline (132) . The data were used to create a consensus annotation before the final assembled genome was uploaded onto the IMG - ER platform. IMG - ER annotations were manually curated by comparison to the consensus annotation before submitting the final genome annotation. Potential c - type cytochromes were selected based on the presence of c - type heme binding motifs (CXXCH) within the amino acid sequen ce as previously described (89) . Predicted subcellular localization and the pr esence of signal peptides and/or an N - terminal membrane helix anchor (89) was investigated by PsortB (198) , PRED - TAT (199) , TMPred (200) , and the TMHMM Server (v. 2.0) (135) . Putative c - type cytochromes were then examined by BLAST analysis to determine homology to know n c - type cytochromes in the NCBI database. The molecular weight of putative c - type cytochromes was estimated with the ExPASy ProtParam program (137) . The weight of the signal peptide was then subtracted from the predicted weight and 685 daltons were added for each heme - binding motif to estimate the molecular weight of the mature cytochrome. The predicted mole cular weight values and subcellular localization of the mature cytochromes were compared to the masses reported for mature heme - containing proteins present in whole cells and outer - surface protein preparations of G. ahangari (139) . Genome properties The genome of G. ahangari strain 234 T comprises one circu lar chromosome with a total size of 1,770,093 bp and does not contain any plasmids. The genome size is within the range of those reported for other members of the Archaeoglobales [15, 33, 50 52, and NC_015320.1] . The mol percent G+C is 53.1 %, which is lower than the 58.7 % estimated experimentally via HPLC (52) . Out of the total 2,072 genes ann otated in the genome, 52 were i dentified as RNA 50 genes and 2,020 as protein - coding genes (Table 3. 3). There are 47 pseudogenes, comprising 2.3 % of the protein - coding genes. Furthermore, 76.5 % of the predicted genes (1,557) are represented by COG functiona l categories. Distribution of these genes and their percentage representation are listed in Figure 3. 3 and Table 3. 4. The preferred start codon is ATG (83.8 % of the genes), followed by GTG (10.4 %) and TTG (5.7 %). This distribution is similar to the sta rt codon representation of the other member of the Geoglobus genus, G. acetivorans (79.4 % ATG, 11.6 % GTG, and 9.0 % TTG) (184) and the closely related archae on F. placidus (82.5 % ATG, 10.2 % GTG, 6.1 % TTG, and 1.3 % other) (190) . There is one copy of e ach of the rRNA genes but the genes are located in two different regions of the genome: the 16S rRNA (GAH_00462) and 23S rRNA (GAH_00460) genes are in the same gene cluster and separated by a span of 139 bp encoding a single tRNA whereas the 5S rRNA (GAH_0 2069) is located 205,273 bp away in a region with genes coding for proteins with functions unrelated to ribosome function and biogenesis. Almost all origins of DNA replication identified in Archaea to date are located in close proximity to genes coding fo r a homologue of the eukaryotic Cdc6 and Orc1 proteins (204) . Interestingly, I id entified two genes encoding Orc1/Cdc6 family replication initiation proteins (GAH_00094 and GAH_00965) in the genome of G. ahangari , thus raising the possibility that the genome contains more than one functional origin of replication. Many archaeal replica tion origins consist of long intergenic sequences upstream of the cdc6 gene containing an A/T - rich duplex unwinding element flanked by several conserved repeat motifs known as ORBs (205) . A specific ORB could not be identified in the genome when compared to other archaeal origins of replication available in the DoriC database (206) . However, the 320 - bp long region upstream of GAH_00965, one of the Orc1/Cdc6 family repl ication initiation proteins, contains a long (111 bp) non - coding intergenic region with one AT - rich stretch and 8 direct repeats (3 TCGTGG, 3 CGTGGTC, and 2 GGGGATTA), which could function as a replication origin. Furthermore, the 580 - bp region directly up stream of the other Orc1/Cdc6 family replication initiation protein (GAH_00094) lacks a non - 51 coding intergenic region and/or AT - rich span but contains 8 direct repeats (2 GGTTGAGAAG, 3 TGAGAAG, and 3 AACATCCCG) and several G - o ri sites reported for haloarchaeal species (207) . Insights from the genome 3.9.1 Autotrophic growth with H 2 as electron donor G. ahangari strain 234 T was the first dissimilatory Fe(II I) - reducing hyperthermophile shown to grow autotrophically with H 2 as an electron donor (52) . In its genome, I identified genes required for the two branches of the reductive acetyl - CoA/Wood - Ljungdahl pathway (208 211) , which other members of the Euryarchaeota (212) , including most members of the Archaeoglobales (181, 203, 213 216) , use for carbon fixation. A bifunctional carbon monoxide dehydrogenase/acetyl - CoA synthase complex (encoded by GAH_01139 - 01144, and two additional copies of the beta and maturation factors encoded by GAH_00919 and GA H_00306, respectively) are present within the genome, which could initiate carbon fixation. The bifunctional nature of this enzyme also allows it to link methyl and carbonyl branches and enable acetyl - CoA biosynthesis, as reported for methanogenic archaea (217) . Complete enzymatic pathways for alternative means of carbon fixation were not identified. The g enome of G. ahangari also contains 29 genes encoding hydrogenase subunits, maturation proteins, and a cluster of genes ( hypA , hypB , hypC , hypD , and hypE ) involved in biosynthesis and assembly of Ni - Fe hydrogenases (GAH_00190 - 00195). Genes coding for the la rge, small, and b - type cytochrome subunits of a Ni - Fe hydrogenase I protein (GAH_00910 - 00912) were identified in the genome. I also found a gene cluster (GAH_00337 - 00347) encoding all subunits of a NADH - quinone oxidoreductase, which transfers electrons to the quinone membrane pool and may function as the primary generator of the proton - motive force (201) . Another large cluster of hydrogenase genes (GAH_02036 - 02044) codes for all coenzyme F 420 hydro genase subunits and proteins involved in recycling coenzyme F 420 , thus replenishing the 52 cofactor for the reductive acetyl - CoA pathway (216, 218 220 ) . The presence of multiple hydrogenases is not unusual in iron - reducing microorganisms and allows them to diversify the paths used to transfer electrons derived from the oxidation of H 2 to their acceptors (221) . Autotrophic growth in methanogens can also be suppo rted using reduced coenzyme F 420 as an electron donor to produce methane (222) . The distinctive fluorescence emission from this coenzyme has been detected in members of the Archaeoglobales (53, 181, 182, 213, 215) but not in the iron - respiring F. placidus (179) or in G. ahangari (52) . Yet, the G. ahangari genome contains genes for all coenzyme subunits of the proteins coenzyme F 420 - red ucing hydrogenase (GAH_00337 and GAH_02036 - 02038), coenzyme F 420 - dependent N 5 ,N 10 - methylene tetrahydromethanopterin reductase (GAH_01605, GAH_01835), and F O synthase (CofGH) (GAH_00662, GAH_00663) (223) . Furthermore, although G. ahangari cannot produce methane when growing autotrophically (52) , its genome codes for nearly all enzymes responsible for the reduction of CO 2 to methane (209) . Similar to A. fulgidus , F. placidus , A. sulfaticallidus , and G. acetivorans , G. ahangari has genes encoding all proteins inv olved in the formation of 5 - methyl - tetrahydromethanopterin and a gene coding for one of the 8 subunits (MtrH) of the enzyme responsible for the transfer of a methyl group to coenzyme M (GAH_01245). Yet, the genome is missing all four genes required for a f unctional coenzyme M reductase, the enzyme responsible for the final step of methane production by methanogenic archaea (2 09) . The fact that Archaeoglobale genomes have nearly all of the genes involved in methanogenesis and the high level of homology that exists between genes from the reductive acetyl - CoA pathway in both Archaeoglobales and the methanogenic archaea suggests that the Archaeoglobales may have evolved from a methanogenic archaeon that lost its ability to reduce CO 2 and produce methane over time. 3.9.2 Central metabolism Heterotrophic growth in G. ahangari is supported by a wide range of organic carbon compounds (52) , which serve as electron donor for respiration while also providing carbon for 53 assimilation in the central pathways. Similar to other hyperthermophilic archaeal species (224) , the G. ahangari genome contains a modified Embden - Meyherhof - Parnas glycolytic pathway (Figure 3. 4). The initial step of glycolysis (glucose phosphorylation to glucose 6 - phosphate) is carried out by an ATP - dependent archaeal hexokinase (GAH_00546) belongin g to the ROK family of proteins. A gene coding for the phosphoglucose isomerase enzyme, which catalyzes the next reaction in the pathway (interconversion of the aldose in glucose 6 - phosphate and the ketose in fructose 6 - phosphate) was also identified (GAH_ 01135) and was most similar to cupin - type phosphoglucose isomerases from other anaerobic Euryarchaeota , including Archaeoglobus fulgidus (224) . In A. fulgidus , fructose 6 - phosphate is phosphorylated to fructose 1,6 - bisphosphate by an ADP - dependent phosphofructokinase protein (EC:2.7.1.11) (225) . However, homologs of this enzyme were not pre sent in the genomes of G. ahangari or any other Archaeoglobale species sequenced to date. Instead, the genome of G. ahangari contained two genes (GAH_00966 and GAH_01843) coding for proteins with pfkB - like domains and ATP - binding sites, which are consisten t with the ATP - dependent phosphofructokinases (PFK - B) of other hyperthermophilic archaea such as Aeropyrum pernix and Desulfurococcus amylolyticus (226, 227) . The genome also contains two genes (GAH_00357 and GAH_01437) encoding archaeal fructose 1,6 - bisphosphatases, which catalyze the reverse reaction during gluconeogenesis but ca n also supply fructose 1,6 - bisphosphate to the glycolytic pathway from dihydroxyacetone phosphate and D - glyceraldehyde 3 - phosphate (228) . Furthermore, a triosephosphate isomerase (GAH_00576) was identified in the genome to catalyze the isomerization of dihydroxyacetone phosphate to D - glyceraldehyde 3 - phosphate. Alternatively, GAH_01502 and GAH_01751, which encode proteins homologous to archaeal type class I fructose 1,6 - bisphosphate aldolase proteins, could catalyze the conversion of fructose 1,6 - bisphosphate into D - glyceraldehyde 3 - phosphate. The next steps in the pathway involve the oxidation of D - glyceraldehyde 3 - phosphate and formation of 3 - phosphoglycerate. The G. ahangari genome contains a homolog (GAH_00413) of 54 a GAPOR, which in A. fulgidus and many other archaeal species c atalyzes the irreversible oxidation of D - glyceraldehyde - 3 - phosphate to 3 - phospho - D - glycerate bypassing the formation of the intermediate 1,3 - bisphospho - D - glycerate (224) . In addition, the genome of G. ahangari contains genes coding for an archaeal specific type II glyceraldehyde - 3 - phosphate dehydrogenase (GAH_01734) and a phosphoglycerate kinase (GAH_0157 1), which could catalyze the formation of 3 - phosphoglycerate via the 1,3 - diphosphoglycerate intermediate. These two enzymes are unidirectional and involved in formation of glyceraldehyde - 3 - phosphate from 3 - phosp h oglycerate during gluconeogenesis in most hy perthermophilic archaea (224) . As in A. fulgidus (229) , G. ahangari has 2 genes coding for cofactor - independent phosphoglycerate mutase proteins (GAH_00739 and GAH_01116), which can catalyze the interconve rsion of 3 - phospho - D - glycerate to 2 - phospho - D - glycerate. Phosphoenolpyruvate is then formed by an enolase protein (GAH_00972), which is subsequently dephosphorylated to pyruvate by pyruvate kinase. Although a gene coding for the well - characterized pyruvate kinase protein is present in the close relative F. placidus (Ferp_0744), homologs were not identified in G. ahangari or any other Archaeoglobale species. Instead, the genomes of G. ahangari (GAH_00154) and all other sequenced Archaeoglobales species conta in genes encoding PK_C superfamily proteins [15, 33, 50 52, and NC_015320.1] , which have pyruvate kinase and alpha/beta domains and are homologous to an A. fulgidus enzyme with pyruvate kinase activity in vitro (230) . Pyruvate can then be converted into acetyl - CoA via pyruvate synthase (GAH_01438 - 01441 and GAH_02021 - 02024). As in the close relative F. placidus (190) , G. ahangari lacks genes from the oxidative pentose phosphate pathway but is predicted to circumvent this limitation (231) via the use of a complete RuMP pathway (GAH_00051 and GAH_01859). The latter results in the accumulation of formaldehyde (231) , which in G. ahangari could be removed by formaldehyde - activating enzymes (GAH_00575 and GAH_00673). Ribulose 5 - phosphate formed in the RuMP pathway could then be converted into ribose - 5 - phosphate by a ribose 5 - phosphate isomerase 55 (GAH_00131), and then into PRPP by ribose - phosphate pyrophosphokinase enzymes (GAH_00743 and GAH_00557). This supplies PRPP to various anabolic pathways such as the biosynthesis of histidine and purine/pyrimidine nucleotides. S imilar to other Archaeoglobale species, a complete TCA cycle is present within the G. ahangari genome. All enzymes involved in the formation of oxaloacetate from acetyl - CoA (GAH_00258, GAH_01703, GAH_01110, GAH_02012 - 02013, GAH_00784 - 00784, GAH_00779 - 00782 , GAH_00526 - 00527, and GAH_00039), including putative aconitase proteins (GAH_00857 - 00858) (232) , were identified in the genome. Also present is a phosphoenolp yruvate carboxylase (GAH_01652), which could catalyze the reversible carboxylation of phosphoenolpyruvate to oxaloacetate, a precursor metabolite of many amino acids. 3.9.3 Fatty acids as electron donors G. ahangari strain 234 T was the first hyperthermophile rep orted to completely oxidize long - chain fatty acids anaerobically, an unsuspected capability of hyperthermophilic microorganisms prior to this discovery (52) . Long - chain fatty acids are abundant in sedimentary environments where they accumulate as byproducts of the hydrolysis of complex organic matter and the anaerobic degradati on of alkanes (233, 234) . Long - chain fatty acids are also major components of crude oil (235) , which is often present in environments inhabited by Archaeoglobale species (236) . C onsistent with the ability of Archaeoglobale members to oxidize long - chain fatty acids, the genomes of G. ahangari and other members of the family ( F. placidus , G. acetivorans , A. fulgidus , - oxidatio n pathway enzymes (184, 190, 201) . The A. fulgidus genome, for example, contains 57 genes encoding the 5 core proteins - oxidation (201) . All of these genes were used as BL AST queries against the genomes of F. placidus (190) , G. acetivorans (184) , and G. ahangari and identified 39 homologous proteins in the genomes of the F. placidus and G. acetivorans and 32 in the genome of G. ahangari . 56 Fatty acid degradation in the Archa eoglobales is thought to occur in a manner similar to bacteria and mitochondria (201) , with the initial step involving activation of a long chain fatty acid to a fatty acyl CoA by a fatty acyl CoA synthetase/ligase. I identified seven genes in G. ahangari coding for fatty acid CoA synthetase proteins (GAH_00420, GAH_00623, GAH_01111, GAH_01124, GAH_01769, GAH_01899, and GAH_02051). The next step in the pathway involves the oxidation of the fatty ac yl - CoA to a trans - 2 - enoyl - CoA by acyl - CoA dehydrogenase proteins, which in G. ahangari are putatively encoded by 11 genes (GAH_00179, GAH_00421, GAH_00484, GAH_00591, GAH_00629, GAH_00785, GAH_01331, GAH_01442, GAH_01601, GAH_01810, and GAH_02050). A water molecule is then added to trans - 2 - enoyl - CoA to form (3S) - 3 - hydroxyacyl - CoA in a reaction catalyzed by an enoyl - CoA hydratase, which in G. ahangari could be encoded by 4 genes (GAH_00487, GAH_00802, GAH_01332, and GAH_01602). Two of these genes (GAH_00487 and GAH_01602) are in fact hybrid proteins containing an enoyl - CoA hydratase domain fused to a 3 - hydroxyacyl - CoA dehydrogenase domain. Hybrid enoyl - CoA hydratase/dehydrogenase proteins such as these have been identified in other archaeal species including the Archaeoglobales species G. acetivorans , F. placidus , and A. fulgidus (184, 190, 201) . - oxidation pathway leads to the formation of 3 - oxoacyl - CoA in an oxidation reaction that generates NADH and is catalyzed by a 3 - hydroxyl - CoA dehydrog enase protein, which in G. ahangari is likely encoded by several genes (GAH_00328 , GAH_00487, GAH_01600, GAH_01602 again noting the hybrid nature of GAH_00487 and GAH_01602). Finally, acetyl - CoA is removed from the 3 - oxo - acyl - CoA molecule by an acetyl - Co A acetyltransferase and is free to enter the TCA cycle. There are 8 genes in the G. ahangari genome that could catalyze this reaction (GAH_00292, GAH_00485, GAH_00625, GAH_00626, GAH_01327, GAH_01328, GAH_01886, and GAH_02049). Additional proteins involved in fatty - acid metabolism include the alpha (GAH_01318) and beta (GAH_01319) subunits of a 3 - oxoacid CoA - - oxidation in G. ahangari and other 57 species within the Archaeoglobales provides genomic evidence s upporting the notion that long - and short - chain fatty acid oxidation is a conserved metabolic feature within the family. 3.9.4 Degradation of aromatic compounds and n - alkanes F. placidus , a member of the Archaeoglobales closely related to G. ahangari , can coupl e the complete oxidation of aromatic hydrocarbons to Fe(III) reduction (237 239) . The G. ahangari genome does not contain any benzoate degradation genes, further supporting the observation that it cannot utilize aromatic compounds as electron donors for growth (52) . Interestingly, G. acetivorans , the other member of the Geoglobus genus, has homologues of all genes coding for proteins of the benzoyl - CoA ligation pathway present in F. placidus , yet as in G. ahangari growth on aromatic hydrocarbons has not been observed in G. acetivorans (178, 184) . Another member of the Archaeoglobales , A. fulgidus , can also couple the oxidation of n - alkanes and n - alkenes with sulfur respiration (240, 241) . This archaeon uses an alkylsuccinate synthase and an activating protein (AssD/BssD; AF1449 - 1450) to oxidize saturated hydrocarbons (n - alkanes in the range of C 10 - C 21 ) (24 1) . I identified homologs of both of these proteins in the genome of G. ahangari (GAH_01645 - 01646) and G. acetivorans (Gace_0420 - 0421). A. fulgidus can also oxidize long chain n - alk - 1 - enes (C 12:1 to C 21:1 ) when thiosulfate is provided as the terminal elec tron acceptor (240) . Although enzymes inv olved in the activation of alkenes by A. fulgidus have not been characterized, the genome of A. fulgidus contains a homologue of a Mo - Fe - S containing enzyme (AF0173 - AF0176) (240) , which in Azoarcus sp. EBN1 anaerobically hydroxylates a branched alkene (242) . The Mo - Fe - S enzyme consists of 4 subunits including a chaperonin - like protein, a membrane anchor heme - b binding subunit, an Fe - S binding subunit, and a molybdopterin - binding subunit (243) . This gene cluster was identified in the genomes of G. ahangari (GAH_01285 - 01288) and F. placidus (Ferp_0121 - 0123), but not in the other member of the Geoglobus genus, G. acetivorans . 58 3.9.5 Nitrogen compounds as electron acceptors Except for F. placidus (179, 215) , all of the Archaeoglobales , including G. ahangari (52) , are unable to use nitrate or nitrite as electron acceptors for respiration (53, 178, 180 183) (Table 3. 5). Yet, surprisingly, the genome of G. ahangari contains several 4Fe - 4S domain - containing nitr ate and sulfite reductase proteins (GAH_01242 and GAH_02063) as well as all four subunits (NarGHIJ) of a nitrate reductase (GAH_01285 - 01288). A nitrate/nitrite transporter is also annotated in the genome (GAH_00501), though it does not cluster with genes i nvolved in nitrate/nitrite respiration and thus may function in the transport of alternative compounds. In addition, I identified a gene in this region of the genome (GAH_01290) coding for an uncharacterized channel protein, which could potentially functio n as a nitrate transport protein. The presence of genes encoding both nitrate reductase proteins (NarGHIJ and NirA) combined with the inability of G. ahangari to use nitrate for respiration (52) suggests a role for these proteins in assimilatory, rather than dissimilatory, nitrate reduction (244) . Similar to F. placidus , the G. ahangari genome does not contain any nir or nrf genes (for the NADH - and formate - dependent nitrite reductase proteins, respectively), with th e exception of several homologues of NirA (GAH_00501, GAH_00506, GAH_01242, and GAH_02063), a nitrite reductase protein that catalyzes the reduction of nitrite to ammonia and is involved in assimilatory nitrate reduction in other organisms (244) . Also missing are genes coding for nitric and nitrous oxide reductase proteins, which the genome of F. placidus contains (190) , again supporting the observation that G. ahangari is not capable of dissimilatory nitrate reduction (52) . The lack of these enzymes helps explain the physiological separation of G. ahangari from its close phylogenetic relative F. placidus , which is capable of dissimilatory nitrate reduction to N 2 O (215) . Furthermore, it is unlikely that the reduction of nitrogen - containing compounds exerts any significant selective pressure on hydrothermal vent microorganisms, as concentrations of these compounds are often low in vent systems (245) . 59 N 2 gas, on the other hand, is the largest reservoir of nitrogen in the ocean (245, 246) and nitrogen fixation supplies hydrothermal vent systems with nitrogen sources for assimilatory growth (245) . Ammonium is particularly abundant in the heavily sedimented Guaymas Basin hydrothermal system (51) , from which G. ahangari was isolated (52) , and this could select for organisms with assimilatory rather than dissimilatory nitrogen metabolisms and inhibit nitrogen fixation. Not surprisingly, the annotated genome of G. ahangari and homology searches for the primary enzymes from the nitrogen fix ation pathway ( nifH , nifD , and nifK ) provided no significant hits, as previously reported for other members of the Archaeoglobales . The genome does contain genes coding for a glutamine synthetase (GAH_01658), a glutamate synthase (GAH_01667 - 01669), and a glutamate dehydrogenase (GAH_00573 and GAH_01931). The enzymes glutamine synthetase - glutamate synthase comprise the GS - GOGAT pathway, and together with the GDH pathway, function as the two major paths for ammonium assimilation in archaea (244) . While the GDH pathway does not use ATP as an energy source, as the GS - GOGAT pathway does, it has a lower affinity for ammonium (244) . The presence of these enzymes and two ammonium transporter proteins (GAH_00438 and GAH_01767) for the formation of 2 - oxoglutarate and glutamate from ammonium, is c onsistent with the notion that G. ahangari is under pressure to assimilate ammonium for anabolic processes. 3.9.6 Sulfur compounds as electron acceptors Most members of the Archaeoglobales are dissimilatory sulfate - reducing organisms and able to use several sulf ur - containing compounds as electron acceptors to fuel their metabolism (53, 179 183) (Table 3. 5). By contrast, G. ahangari cannot couple the oxidation of electron donors that supported Fe(III) reduction to the respiration of commonly considered sulfur - containing electron acceptors such as sulfate, thiosulfate, sulfite, or S 0 (52) . Interestingly, the genome of G. ahangari contains two genes (GAH_02067 and GAH_01481) coding for sulfate adenylyltransferase, which can initiate the first step in both the dissimilatory and assimilatory sulfate reduction pathways by catalyzing the formation of APS from ATP and inorganic sulfate. 60 The enzyme is also present in the genome of F. placidus which, like G. ahangari , is unable to respire sulfate (179) (Table 3. 5). APS can then be used as substrate in the assimilatory (247 250) or dissimil atory (251, 252) pathway, depending on the needs and capabilities of the microorganism (249) . The assimilatory pa thway converts APS to the intermediate PAPS in a reaction catalyzed by an adenylsulfate kinase, which in G. ahangari is encoded by GAH_01478. The genome of G. ahangari contains genes coding for both the alpha and beta subunits of adenylsulfate reductase (G AH_02065 - 02066), an FAD dependent oxidoreductase protein that reduces APS to sulfite in the dissimilatory pathway. However, the genome lacks genes coding for a dissimilatory sulfite reductase ( dsrAB ), which catalyzes the reduction of sulfite to hydrogen su lfide in the final step of the dissimilatory sulfate reduction pathway (253) . Strong matches could not be found even when the alpha ( AAB17213 .1) and beta ( AEY99618 .1) subunits of the sulfite reductase from A. fulgidus were used as queries in manual searches. It is interesting to note that, despite the absence of dsrAB genes in the genome, G. ahangari does have a nitrite and sulfite reductase 4 Fe - 4S domain - containing protein (GAH_02063) located in a cluster of genes involved in sulfur metabolism (GAH_02063 - 02067). Whether these genes code for functional proteins of the dissimilatory pathway, perhaps with electron donor/acceptor pairs not tested yet, remains to be elucidated. F. placidus , for example, has homologs of all of these genes, except for dsrAB , and it grows with thiosulfate as the sole electron acceptor when hydrogen is provided as an electron donor (179) . This ca pability may be due to the presence of several molybdopterin oxidoreductase proteins within the genome of F. placidus that show high similarity to a predicted thiosulfate reductase (NP_719592.1) from Shewanella oneidensis . However, strong homologs of this protein were not present in the genome of G. ahangari . 3.9.7 Fe(III) as the sole electron acceptor for respiration The most distinctive physiological feature of G. ahangari strain 234 T is its dependence on Fe(III) as an electron acceptor for respiration (52) . Both insoluble Fe(III) oxides and soluble 61 species of Fe(III), such as Fe(I II) citrate, support growth, though the original isolate did not grow readily with the soluble electron acceptor and required prolonged adaptation under laboratory conditions to grow in its presence (52) . Key to the ability of G. ahangari to respire the insoluble Fe(III) oxides is the ability of the cells to locate the oxides, attach to them, and position electron carriers of the outer surface close enough to favor the transfer of electrons (139) . Hence, I examined the genome of G. ahangari for genes that code for cellular components that could be involved in motility and attachment and extracellular electron transfer. Motility in this organism is enabled by a single flagellum (52) , which in archaea is designated as an archaellum to reflect its distinct evolutionary origin (254) . Arch aeal flagellar genes can be organized into one of two very well conserved clusters ( fla 1 and fla 2) based on the type and order of genes in the cluster: flaBC(D / E)FGHIJ in fla 1 and flaBGFHIJ in fla 2 (255) . The fla 1 clusters are exclusively found in Euryarchaea while fla 2 clusters are generally associated with the Crenarchaea , which includes the Desulfurococcales and Sulfolobales orders, and are also present within the Eur yarchaeal order Archaeoglobales (255) . Interestingly, the Archaeoglobales have members with both types. I identified, for example, a fla 1 gene cluster in the genome of G. ahangari (GAH_01994 - 02001), as in F. placidus (Ferp_1456 - 1463) (190) , while the flagellar gene s of Archaeoglobus spp. [15, 33, 50 52, and NC_015320.1] and G. acetivorans (184) were of the fla 2 type. It has been suggested that a horizontal gene transfer (HGT) event occurr ed in the Ferroglobus lineage after divergence from the Archaeoglobus and Geoglobus lineages (184) . Yet, the presence of a fla 1 gene cluster in the flagellated and motile G. ahangari (52) , when compared to the fla 2 gene cluster foun d in the non - motile and non - flagellated G. acetivorans (178) , would lend credence to a possible second HGT event within the family. The genome of G. ahangari also encodes several glycosyltransferase genes (GAH_00218 , GAH_00870, and GAH_01279) and an oligosaccharyltransferase (GAH_01455), which could glycosy late the growing archaellum (256) and post - translationally modify surface pr oteins, as is commonly observed in the Archaea (257) . However, chemotaxis proteins, which 62 are present in nearly all sequenced members of the Archaeoglobales [15, 33, 50, 51, and NC_015320.1] , with the exception of A. sulfaticallidus (203) , and are typically found i mmediately upstream or downstream of the fla gene cluster, were absent in G. ahangari . The lack of chemotaxis genes in G. ahangari contrasts with their presence in most Archaeoglobales genomes, including G. acetivorans (184) , the other member of the genus. Both Geoglobus species were isolated from hydrothermal vent chimneys: G. acetivorans from the Ashadze field on the Mid - Atlantic Ridge at a depth of 4 , 100 m (178) and G. ahangari from a Guaymas Basin chimney at a depth of 2 , 000 m (52) . The hydrothermal fluids spewed from chimneys with in the Guaymas Basin system are likely enriched in nutrients after passage through the 300 - 400 m thick, organic - rich sediments underneath (110) . Furthermore, hydrothermal circulation at this site is high (109) , which would rapidly replenish nutrients, both electron donors and fresh Fe(III) oxides, and thus organisms living in this environment may not need to utilize chemotactic mechanisms to seek out these nutrients. By contrast, hydrothermal fluids from offshore spreading systems, such as the Ashadze field, flow through thin sediment layers before reaching the chimney (110) . This likely increases the selectiv e pressure on resident microbes to evolve chemotactic mechanisms to locate nutrients. The genome of G. ahangari also encodes proteins potentially involved in the assembly of extracellular protein appendages such as pili. I identified, for example, a prepi lin peptidase (GAH_00760), numerous type II secretion system proteins (GAH_01195 - 01196, GAH_00173, GAH_00290, GAH_01412 - 01413), and a putative twitching motility pilus retraction ATPase (GAH_00960). Homologous genes are also present in the genomes of G. ac etivorans (184) , F. placidus (190) , and A. fulgidus (201) . In addition, G. ahangari has two genes encoding proteins with DUF1628 or DUF1628 - like domains (GAH_01202, GAH_01671), which are associate d with previously described archaeal pilin proteins (258) and present in all sequenced members of the Archaeoglobales [15, 33, 50 52, and NC_015320.1] . Any of these proteins could be involved in the assembly of the curled ext racellular appendages that G. ahangari produces to attach to Fe(III) 63 oxides and facilitate the transfer of electrons from electron carriers located on the outer surface to the insoluble electron acceptor (139) . G. ahangari uses heme - containing proteins to transport electrons across the cell envelope and to the insoluble Fe(III) oxides (139) . The most commo n heme - containing proteins used by mesophilic Fe(III) reducers for extracellular electron transport are c - type cytochromes (259) . Archaea are known to have a variant form of the cytochrome c maturation (Ccm) system, whereby the CcmE protein has a CXXXY - type motif, rather than the HXXXY motif found in eukaryotic and most bacterial c - cytochromes, and CcmH is absent (89) . Similar to other sequenced Archaeoglobales , G. ahangari has an archaeal - type CcmE protein (GAH_01977), a CcmC protein (GAH_00620) with a tryptophan - rich motif (WG[S,T][F ,Y]WNWDPRET), a CcmF protein (GAH_01976 and GAH_01093) with the motif WGGXWFWDPVEN, and a gene coding for a CcmB homolog (GAH_00449) lacking the conserved FXXDXXDGSL motif. Although previously reported archaeal cytochrome maturation pathways do not contain CcmH (89) , I identified two putative CcmH proteins in the genomes of not only G. ahangari (GAH_01092 and GAH_01094), but also in G. acetivorans (GACE_2070 and GACE_2068) and F. placidus (Ferp_1362 and Ferp_1364). All of these proteins contain cysteine - rich motifs consisting of LX[S,N]C[E,D,H]C but lack the LRCXXC motif characterist ic of most CcmH proteins. However, they all flank a duplicate CcmF - encoding gene found only in G. ahangari (GAH_01093), G. acetivorans (184) , and F. placidus (190) . In addition to having a distinct cytochrome c biogenesis pathway, the iron - reducing Archaeo globales , Geoglobus and Ferroglobus species, also have more c - type cytochromes than any other archaeon, and many of these c - type cytochromes have multiple heme groups (113, 18 4, 190) . The genome of G. ahangari contains 21 genes (Table 3. 6) encoding putative c - type cytochromes, 7 of which have more than 5 heme groups; F. placidus has 30 c - type cytochromes (12 with more than 5 heme groups); and G. acetivorans has 16 c - type cytoc hromes (8 with more than 5 heme groups). By contrast, Archaeoglobus species, which do not use Fe(III) electron 64 acceptors (Table 3. 5), have significantly fewer c - type cytochromes. Within this genus, the greatest number of c - type cytochrome encoding genes wa s found in the genome of A. veneficus , which has 16 c - type cytochromes (3 with more than 5 hemes). Other species such as A. profundus and A. sulfaticallidus have only 1 monoheme c - type cytochrome and A. fulgidus has 3 c - type cytochromes (none of which have more than 5 heme groups). The subcellular localization of the putative c - type cytochromes of G. ahangari was also investigated. The ExPASy TMPred program (137) revealed that a majority (62%) of the c - type cytochromes have at least 1 transmembrane helix, consistent with their association to the cytoplasmic membrane. One of these c - type cytochrome proteins (G AH_00504) was predicted to be extracellular. I also identified several c - type cytochromes (GAH_01306, GAH_00286, GAH_01534, and GAH_01253) with predicted sizes once in mature form (46.3, 39.1, 18.5, and 16.9 kDa, respectively) matching those reported for o uter - surface heme - containing proteins required for the reduction of insoluble Fe(III) oxides, but not soluble Fe(III) citrate, by G. ahangari (139) (Table 3. 6). Hence, these 4 c - type cytochromes likely function as the terminal electron carriers between the cells and the oxides. In addition to c - type cytoc hromes, I identified other potential electron carriers such as quinones, flavoproteins, and various Fe - S proteins ( i.e. ferredoxins). I identified a number of uniquinone/menaquinone biosynthesis proteins in the genome of G. ahangari ( Table 3. 7 ), which coul d create a quinone pool in the membrane to promote electron transfer. The genome also contains a great number of Fe - S binding domain proteins and ferredoxins, which could participate in electron transfer pathways ( Table 3. 8 ). Fe - S proteins and ferredoxins were also abundant in the genome of G. acetivorans and F. placidus , which, like G. ahangari , also utilize Fe(III) respiration as their primary metabolism. Fe - S proteins and ferredoxins are regarded as some of the most ancient of electron transfer carriers (260) and also have high thermostability (261) , which is critical to ensure maximum rates of electron transfer in the hot hydrothermal vent systems. Th us, the abundance of electron carrier proteins, some known to have increased thermostability, 65 and c - type cytochromes, some of them localized to the outer surface, is consistent with a mechanism evolved for efficient extracellular electron transfer in hot e nvironments. Conclusions G. ahangari strain 234 T is only one of three members of the Archaeoglobales capable of dissimilatory Fe(III) respiration. Furthermore, it is an obligate Fe(III) reducer that grows better with insoluble than soluble Fe(III) species. Consistent with this, the genome contains a large number of c - type cytochromes within and on the cell surface, as well as other redox - active proteins such as thermostable ferredoxin and Fe - S proteins. The paucity of c - type cytochromes within non - Fe(III) r espiring members of the Archaeoglobales ( Archaeoglobus species) is consistent with the physiological separation between these archaea and F. placidus , G. acetivorans , and G. ahangari , which can gain energy for growth from the reduction of Fe(III) electron acceptors. Additionally, some genes required for both dissimilatory sulfate and nitrate metabolism s are absent in G. ahangari and G. acetivorans . This supports the physiological separation of Geoglobus spp. from F. placidus , which is capable of Fe(III) - , t hiosulfate - , and nitrate respiration, and from Archaeoglobus species which are primarily sulfur - respiring organ isms. Genomic data also support the reported physiological similarities between G. ahangari and other Archaeoglobales such as autotrophic growth with H 2 via the reductive acetyl - CoA/Wood - Ljungdahl pathway and the use of similar electron donors, including short - and long - chain fatty acids. Noteworthy is the fact that genomic evidence supports the synthesis of the methanogenic coenzyme - F 420 in G. aha ngari which is responsible for the characteristic fluorescence detected in all Archaeoglobus spp. except for G. ahangari or F. placidus . Hence, the genome sequence of G. ahangari provides valuable insights into its physiology and ecology as well as into th e evolution of respiration within the Archaeoglobales . 66 Taxonomic note The initial publication (52) of the Geoglobus genus and Geoglobus ahangari species was accepted for publication with extenuating circumstances at several culture - collection agencies. Thus, upon the original publication G. ahangari strain 234 T was accepted on ly at a single agency. In addition, the G+C mol% determined from the complete genome sequence (53.1 mol%) differs from that originally published (58.7 mol%), representing a discrepancy of over 5 mol%. This publication thus warrants an emended description o f the genus Geoglobus and the type species, Geoglobus ahangari . Emended description of Geoglobus Kashefi et al. 2002 The description of the genus Geoglobus is the one provided by Kashefi et al. 2002 (52) , with the following modifications. In addition to the single monopolar flagellum, numerous curled filaments can be seen per cell (139) . The G+C content of the genomic DNA of t he type species is 53.1 mol%. Emended description of Geoglobus ahangari Kashefi et al. 2002 The description of the species Geoglobus ahangari is the one provided by Kashefi et al. 2002, with the following modifications. The type strain is strain 234 T and has been deposited at three culture collection agencies which include the Deutsche Sammlung von Mikroorganismen und Zellkulturen ( DSM - 27542 ), the Japan Collection of Microorganisms ( JCM 12378 ), and the American Type Culture Collection ( BAA - 425 ). Thereby, G eoglobus ahangari is now a validly published species name. 67 CHAPTER 4. EXTRACELLULAR ELECTRON TRANSFER TO FE(III) OXIDES BY THE HYPERTHERMOPHILIC ARCHAEON GEOGLOBUS AHANGARI VIA A DIRECT CONTACT MECHANISM Copyright © American Society for Microbiology, Appl Environ Microbiol. 2013 Aug;79(15):4694 - 700. doi: 10.1128/AEM.01566 - 13. Epub 2013 May 31. Abstract The microbial reduction of Fe(III) plays an important role in the geochemistry of hydrothermal systems yet it is poorly understood at the mechanistic level. Here I show that the obligate Fe(III) - reducing archaeon Geoglobus ahangari uses a direct contact mechanism for the reduction of Fe(III) oxides to magnetite at 85 ° C. Alleviating the need to directly contact the mineral with the addition of a chelator or the ele ctron shuttle anthraquinone - 2,6 - disulfonate (AQDS) stimulated Fe(III) reduction . By contrast , entrap ment of the oxides within alginate beads to prevent cell contact with the electron acceptor prevented Fe(III) reduction and cell growth unless AQDS was prov ided . Furthermore, f iltered culture supernatant fluids had no effect on Fe(III) reduction, ruling out the secretion of an endogenous mediator too large to permeate through the alginate beads. C onsistent with a direct contact mechanism, electron micrographs revealed cells in intimate association with the Fe(III) mineral particles, which once dissolved revealed abundant curled appendages. The cells also produced several h eme - containing bands . Some of them were uter surface and were required for the reduction of insoluble Fe(III) oxides but not for the reduction of the soluble electron acceptor Fe(III) citrate. The results thus support a mechanism in which the cells directly attach and transfer electrons to the F e(III) oxides using redox - active p roteins exposed on the cell surface . This strategy confers on G. ahangari a competitive advantage for accessing and reducing Fe(III) oxides under the extreme physical and chemical conditions of hot ecosystems . 68 I ntroductio n The accumulation of Fe(III) in hot sediments surrounding marine hydrothermal vents and the availability of electron donors for microbial growth, such as H 2 and acetate (31) , provide ideal conditions to support the growth and activit y of dissimilatory Fe(III) - reducing microorganisms at elevated (>80 ° C) temperatures (262) . Fe(III) reduction is, in fact, a conserved metabolic capability of deeply - branching hyperthermophilic microorganisms (32) and t he advent of isolation methods us ing Fe(III) oxides as the sole electron acceptor s have enabled the recovery , in pure culture , of many novel hyperthermophilic Fe(III) reducers with previously unsuspected metabolic capabilities (reviewed in (177) ). Evidence to date also indicates that the reduced Fe(III) minerals produced by dissimilatory Fe(III) reducers have distinctive signatures (263 266) which could serve as geological markers for Fe(III) biomineralization in hot ecosystems. Despite the critical role that Fe(III) biomineralization has in t he geoch emistry of hydrothermal systems , its underlying mechanism s remain largely unknown . By contrast, Fe(III) biomineralization has been extensively studied i n moderate temperature environments . In general, mesophilic, dissimilatory metal - reducing bacter ia, both Gram - negative and Gram - positive, rely on c - type cytochromes to transfer metabolic electrons to the outer surface (185, 259, 267) . Some transfer the electrons directly from the cell surface electron carriers to the Fe(III) oxides or indirectly v ia the secretion of soluble redox mediators, such as electron shuttles and/or metal chelators (reviewed in (84) ) . Recent studies in the thermophilic Gram - positive bacterium Carboxydothermus ferrireducens have also provided evidence for a di rect contact mechanism for the reduction of Fe(III) oxides at moderately elevated temperatures ( 65 ° C ) (114) . Mesophilic Fe(III) reducers can also transfer electrons to exogenous elec tron shuttles such as dissolved humic substances (187) and s olid phase humics (188) generated during the degradation of organic matter . Both humic forms can be recycled through cycles of microbial reduction and abiotic reoxidation by the Fe(III) oxides, thus stimulat ing the rates of Fe(III) reduction in sediments (96, 188, 268) . A 69 has also been propose d in species of Geobacter and Shewanella , which use a direct and an indirect strategy for Fe(III) reduction, respectively (91, 94, 269) . The finding that Fe(III) reducers with a direct contact mechanism predominate over those producing endogenous shuttles and chelators in a wide variety of subsurface environments (270 273) has led to the proposal that a direct strategy may confer on microorganisms a competitive advantage for reducing insoluble Fe(III) oxides (102) . This is because these microorganisms do not divert energy towards the synthesis of endogenous soluble mediators, which often need to be replenished due to their loss to diffusion and advection in the bulk fluid and their adsorption onto redox - inactive solid phases (102) . Such losses could be more pronounced in areas of high fluid circulation such as in hydrothermal environments, where seawat er seeps down through fissures of the volcanic bed and is pushed back up again through the volcanic rock once heated by the underlying magma (31) . However, mechanistic understanding of microbial Fe(III) reduction in hydrothermal environments is lacking. Hence , I investigated the mechanism used by the model hyperthermophilic archaeon Geoglobu s ahangari to reduce Fe(III) oxides. G. ahangari is an obligate Fe(III) - reducing archaeon isolated from the Guaymas Basin hydrothermal system at a depth of 2,000 m that grows at temperatures between 65 and 90 o C, with an optimum at ca. 85 - 88 ° C (52) . It was the first isolate in a novel genus within the Archaeoglobales and the fi rst example of a dissimilatory Fe(III) - reducer growing autotrophically on H 2 (52) , a metabolic trait later found to be conserved in many hyperthermophilic Fe(III) reducers (177) . In addition to H 2 , it oxidizes a wide range of organic acids, amino acids and long - chain fatty acids for the reduction of Fe(III). Here I show that G. ahangar i reduces Fe(III) oxides with a direct contact mechanism and I identify cellular components necessary for the cell to establish electronic contact with the oxide minerals . To the best of our knowledge , this is the first mechanistic study of Fe(III) reducti on by a hyperthermophilic archaeon and provides novel insights into the microbial adaptive responses that contribute to Fe(III) mineralization in hydrothermal systems . 70 Materials and Methods 4.3.1 Bacterial strains, culture conditions and mineral characterization G. ahangari strain 234 T (JCM 12378 T , ATCC BAA - 426 T ) was routinely grown anaerobically in modified marine (MM) medium (52) with pyruvate (10 mM) as electron donor and free poorly - crystalline Fe(III) oxides (100 mM for routine transfers or 50 mM for experiments with free Fe(III) oxides ) or soluble Fe(III) citrate (50 mM) as elec tron acceptor . Incubations were in the dark and at 85°C . These nominal concentrations of electron donor and acceptor were selected so the electron donor was provided in excess supply of electrons (one molecule of pyruvate, 10 electrons) to yield the full r eduction of the electron acceptor (1/3 of the Fe(III) in Fe(III) oxides and all the Fe(III) in Fe(III) citrate). The cultures were transferred (5% (v/v)) into fresh medium when ca. 1/3 (Fe(III) oxides) or 2/3 (Fe(III) citrate) of the Fe(III) had been reduc ed (early stationary phase and late - exponential phase, respectively) . The extent of Fe(III) oxide reduction in the cultures was periodically monitored by extracting the Fe(II) from the mineral phase with 0.5 N HCl (274) (HCl - extractable Fe(II)) and measuring it with the ferrozine method (275) . When indicated, soluble Fe(II) was also measured in filtered (0.22 m) culture supernatant fluids with the ferr ozine method. The supernatant fluids were also treated with hydroxylamine hydrochloride under acidic conditions to reduce soluble Fe(III) to Fe(II) (276) and the total soluble Fe content was measured with the ferrozine method. The difference between the total Fe content and the Fe(II) fraction was used to estimate the amount of soluble Fe( III). When indicated, t he reduced Fe(III) oxide mineral was characterized by harvesting the mineral particles from early stationary phase cultures by centrifugation, dr ying the sample with N 2 gas, and examin ing it by electron diffraction using a JEOL 2000 FX MARK II, 200kV transmission electron microscope. 4.3.2 Assays for endogenous mediators The secretion of low - molecular - weight mediators by G. ahangari was investigated using Fe(III) oxides ent rapped in alginate beads (12 - kDa pore size, 3 - mm diameter), prepared as previously described (27 7, 278) and modified for Fe(III) oxides (98) . The bead - entrapped Fe(III) 71 oxides were subjected to 30 cycles of pressurization with N 2 gas followed by aspiration to vac uum to remove any residual oxygen trapped in the beads and were then dispensed at 50 mM concentrations in tubes with 10 ml of MM medium with 10 mM pyruvate. Tubes were periodically sacrificed to measure the extent of Fe(III) reduction in the beads as HCl - e xtractable Fe(II). The beads were first washed with autoclaved, anaerobic marine wash buffer (NaCl, 19.0 g/L; MgCl 2 . 6H 2 O, 9.0 g/L; CaCl 2 . 2H 2 O, 0.3 g/L; KCl, 0.5 g/L; pH to 7.0) and the Fe(II) in the beads was then extracted with 0.5 N HCl for 12 h and measured with the ferrozine method (275) . Cell growth was also monitored by counting the number of acridine orange - st ained cells in the culture supernatant fluids by fluorescence microscopy, as described previously (279) . The secretion of endogenous mediators larger than the bead pore size was investigated by measuring the effect of culture supernatant fluids o n the reduction of free Fe(III) oxides (10 mM) by washed cell suspensions, using a protocol adapted from a previously published procedure (98) . The supernatant fluids were harvested from 200 - ml cultures grow n with 10 mM pyruvate and 50 mM Fe(III) oxide until ca. 1/3 of the Fe(III) was reduced. After being filtered and oxidized as previously described (98) , the supernatant fluids were concentrated 100 - fold in an Ultracel ® YM - 10 centrifugal filter unit (Millipore, 10 kDa nominal molecular weight limit), made anaerobic by gassing with N 2 :CO 2 (80:20) for 20 min and diluted 100 - fold in MM medium supplemented with 10 mM pyruvate and, when indicated, 10 µ M AQDS. Approximately 9.7 ml of these supernatant fluids were dispensed anaerobically into culture tubes. Tubes with 9.7 ml of MM medium were also used as controls. These tubes were inoculated with cells harvested by centrifugation (8,000 x g , 10 min, 20 ° C) from 100 ml Fe(III) citrate (50 mM) cultures grown to mid - exponential phase (ca. 25 mM Fe(III) reduced). After washing them twice with autoclaved, anaerobic marine wash buffer, the cells were suspended in 5 ml of MM medium and f lushed with N 2 :CO 2 (80:20) gas for 20 min. Approximately 0.1 ml of this cell suspension (~ 0.6 mg of total cell protein) was inoculated into each tube containing 9.7 ml of concentrated supernatant fluids or fresh medium. 72 4.3.3 Microscopy Acridine orange - stained cells were routinely examined and counted using a Zeiss Axioskop 20 phase - contrast microscope using an oil - immersion objective x 100/1.25, equipped with an UV lamp, an excitation filter (LP 420) and a red - attenuation filter (BG 38). Cells from stationary - p hase cultures grown with free Fe(III) oxides or Fe(III) citrate were also fixed with 2.5% (w/w) glutaraldehyde , adsorbed onto Formvar - coated nickel grids (200 - mesh, Electron Microscopy Sciences) for 15 min , washe d with ddH 2 O, and negatively stained with 1% uranyl acetate for 15 min . N egatively stained cells were then examined by Transmission Electron Microscopy (TEM) using a JEOL100 CXII TEM operated at 75 mV . When indicated, the culture samples were first treated with an oxalate solution for 15 min at room temperature to dissolve any cell - associated iron deposits before fixing and staining them for TEM analyses . The biofilms associated with the bead - entrapped Fe(III) oxides were examined by Confocal Laser Scanning Microscopy (CLSM) using an inverted Olympu s FluoView 1000 LSM equipped with a UPlanFLN 40x objective. Prior to CLSM, the beads were washed with PBS buffer and stained with the BacLight TM viability kit (Invitrogen) for 5 min , recommendations. After another wash with PBS, th e beads were transferred to a glass bottom 35 - mm microwell dish (MatTek Co.) and imaged. The fluorescence from the SYTO9 dye (green, live cells) was detected with a 488 nm argon line using a 505 - 525 band - pass filter, whereas the fluorescence from the propi dium iodide dye was detected with a 560 long - pass filter. Images were collected at 1.0 - µ m increments. 4.3.4 Denaturing Polyacrylamide Gel Electrophoresis (SDS - PAGE) and heme staining Cultures with 10 mM pyruvate and 50 mM Fe(III) citrate were grown to late - expon ential phase at 85 ° C and treated with oxalate (52) for 15 min to remove Fe precipitates that formed around the cells. The oxalate - treated cells were harvested by centrifugation (8,000 x g , 30 min, 25°C), washed once with marine wash buffer, and incubated at 37 °C for 30 min in iron extraction buffer, prepared as previously des cribed (280) but containing 100 mM EDTA. The cells were 73 harvested by centrifugation and stored as a pellet at - 20 °C until use. Loosely - bound proteins were mechanical ly sheared off the cell surface by repeated (20 x) passages through a 23G needle (91) . The cells were pelleted by centrifugation (453 x g , 60 min, 25 °C) and the supernatant containing the solub le, sheared protein fraction was removed and concentrated using an Amicon Ultra - 15 centrifugal filter unit equipped with a 100 - kDa vertical membrane (Millipore). The protein sample was then washed repeatedly with ddH 2 O, re - suspended in 10 mM Tris buffer (p H 8.0), and stored at - 20 °C until use. All protein samples were mixed with an equal volume of a 2X Laemmli Sample Buffer (Bio - R ad Laboratories, Inc. ) and subjected to SDS - PAGE . Reducing agents were omitted from the SDS - sample buffer and the samples were l oaded onto the gel without boiling to prevent the loss of heme groups (138) . The protein samples were separated by SDS - PAGE in a 4 - 20 % Tris - Glycine Mini - PROTEAN gel ( Bio - R ad Laboratories, Inc. ) at 250 V for 30 min. Heme staining after SDS - PAGE was performed as previously described (138) . The heme - stained gels were then de - stained (138) and stained with Coomassie Bril Organics) to visualize all the proteins in the samples. 4.3.5 Effect of mechanical shearing of outer surface proteins on Fe(III) reduction The role of exposed outer surface redox proteins in electron transfer to poorly crystalline Fe(III) ox ides was examined by testing the ability of cells subjected to mechanical shearing (as described above) or untreated cells to resume Fe(III) oxide reduction. All culture manipulations were performed inside a glove bag (Coy laboratories) containing a H 2 :CO 2 : N 2 (7:10:83) atmosphere and all the cultures were prepared in MM medium (pH 6.8 ) lacking s odium bicarbonate to minimize pH drops during exposure to the glove bag atmosphere. Untreated or sheared cells grown with 10 mM pyruvate and 50 mM Fe(III) citrate we re harvested by centrifugation (8000 x g , 10 min, 25 °C) when approximately 2/3 of the Fe(III) had been reduced. After being washed anaerobically with the same medium, the cell pellets were gently resuspended with a Pasteur pipette into tube s containing 10 ml of MM medium supplemented with 10 mM 74 pyruvate as the electron donor and 100 mM Fe(III) oxides or 5 0 mM Fe(III) citrate as the electron acceptor. Incubation was at 85 ° C in the dark. Results and discussion 4.4.1 Stimulation of Fe(III) reduction by exogenous m ediators As shown in Figure 4. 1A, G. ahangari coupled the oxidation of pyruvate to the reduc tion of poorly crystalline Fe(III) oxide at 85 ° C with d oubling times for three independent experiments, each containing at least triplicate samples, rang ing from 4 .3 to 5.2 h, which is within the ranges previously reported for the growth of the original isolate at optimum temperatures (52) . The reduced mineral harvested from stationary phase cultures was black and magnetic and contained 20 (± 2) mM of HCl - extractable Fe(II) . This contrasts with the less than 4 mM Fe(II) produced in contr ols lacking the electron donor (Figure 4. 1A), which accounts for any extractable Fe(II) produced abiotically by reducing agents in the medium such as cysteine and FeCl 2 or carried over in the inoculum. Thus, 16 ( ± 2) mM of the Fe(II) extracted from the red uced mineral was generated during the biological reduction of the Fe(III) oxides. This Fe(II) concentration is approximately 1/3 of the nominal concentration of poorly crystalline Fe(III) oxides (50 mM) provided as electron acceptor, consistent with the fo rmation of a mixed valence mineral such as magnetite, which has 1/3 Fe(II) and 2/3 Fe(III) . To further confirm this, I analyzed the electron diffraction pattern of the reduced magnetic mineral generated by G. ahangari . As shown in the inset in Figure 4. 1A, the electron diffraction analyses showed the characteristic diffuse ring electron diffraction pattern of magnetite (281) . I al so gained insights into the mechanism of Fe(III) reduction by G. ahangari in experiments in which an exogenous electron shuttle was added to partially alleviate any need of the cells to directly contact the Fe(III) oxides. Addition of low (50 µM) concentra tions of the humics analog anthraquinone - 2,6 - disulfonate (AQDS) stimulated the reduction of Fe(III) oxides over that observed in controls without AQDS, as evidenced by the increased rate of accumulation of HCl - 75 ext ractable Fe(II) over time (Figure 4. 1A). E x tracellular quinones such as AQDS function as e lectron shuttle s between the cell and the Fe(III) oxides (282) . The cells reduce AQDS to anthrahydroquinone - 2,6 - disulfonate (AHQDS), which then abiotically reduces Fe(III) to Fe(II) , thus regenerating the quinone and making it available for a new cycle of biotic reduction and abiotic oxidation . This partially alleviat es the need for the cell to directly contact the insoluble electron acceptor and stimulates the rates of Fe(III) reduction (282) . The stimulatory effect of AQDS on Fe(III) reduction was not dose - dependent. As shown in Figure 4. 1B, concentrations of AQDS up to 100 µM stimulated growth, as indicated by the decreases in doubling times, whereas 200 µM concentrations did not significantly affect the doubling times. By contrast, doubling times were ca. two - fold higher in the presence of 500 µM concentrations of AQDS. This suggests that AQDS is toxic to G. ahangari above relatively low threshold concent respire AQDS when provided as sole electron acceptor for growth, which requires mM concentrations of the humic analog in the medium (52) . High (10 mM) concentrations of AQDS also inhibited the growth of the mesophilic Fe(III) - reducing bacterium Sh ewanella oneidensis (283) . However, S. oneidensis reduced and re spired AQDS at 1 mM concentrations (283) . The higher threshold o f AQDS toxicity in S. oneidensis has been linked to the activity of the outer membrane protein TolC, which functions as a pump to extrude the AQDS (283) . The increased sensitivity to AQDS noted in G. ahangari could be due to the absence of an outer membrane in its cell envelope, which has the characteristic archaeal st ructure composed of a cytoplasmic membrane and an S layer (52) . Consisten t with this, the rate of Fe(III) reduction at 100 ° C by resting cell suspensions of another hyperthermophilic archaeon Pyrobaculum islandicum was highest at 200 µM concentrations of AQDS, but decreased at 500 µM concentrations (284) . As with AQDS, t he addition of the synthetic metal chelator nitrilotriacetic acid ( NTA , 4 mM) also stimulated the reduction of Fe(III) oxide s by G. ahangari (Fig ure 4. 1A). NTA solubili zes Fe(III) from the Fe(III) oxides an d provides a soluble, chelated form of Fe(III) as an electron acceptor for 76 its microbial reduction (96, 285) . As a result, NTA partially alleviates any need of the cell to directly contact the Fe(III) oxides and stimulates Fe(III) reduction (96, 285) . Consistent with this , Fe(III) oxide cultures supplemented with NTA solubilized 3 - times more Fe than cultures with no additions and most of the soluble Fe spec ies were in the reduced form (Fe(II) ) (Figure 4. 1C). 4.4.2 G. ahangari does not secrete endogenous mediators for Fe(III) reduction . I further investigated the mechanism of Fe(III) reduction in G. ahangari by growing the cells with Fe(III) oxides that had been e ntrapped in microporous alginate beads. As with the experiments with free Fe(III) oxides, the nominal concentration of entrapped Fe(III) oxides per tube was 50 mM and there was little variability between replicates (49.8 ± 1.8 mM of entrapped Fe(III) per t ube). As shown in Figure 4. 2A, G. ahangari produced 3.9 ( ± 0.9) mM Fe(II) over the course of 72 h in cultures with the entrapped Fe(III) oxide. This amount is approximately 7.8 ( ± 1.8) % of the total Fe(III) entrapped in the alginate beads , which correspon ds well with the amount of Fe(III) (10 %) that is exposed on the beads surface (98) . This suggests that only the Fe(III) oxide exposed on the surface of the beads was a ccessible for microbial reduction. As a result, cell growth was limited in these cultures (Figure 4. 2A). By contrast , addition of 50 µM AQDS stimulated Fe(III) reduction and cell growth (Figure 4. 2A). AQDS is small enough to pass through the pores of the a lginate beads and can, therefore, shuttle electrons between the cell and the entrapped oxides (98) . Approximately one third ( 17 ± 1 .0 mM ) of the Fe(III) entrapped in t he beads was reduced to Fe(II) in the cultures supplemented with AQDS (Figure 4. 2A) , consistent with the complete reduction of the entrapped Fe(III) oxide to the mixed - valence (Fe[II]:Fe[III], 1:2) mineral magnetite . This yield of HCl - extractable Fe(II) is 13 mM higher than in the cultures without AQDS, which were about 4 mM. The discrepancy between the amount of Fe(III) reduced in the presence of AQDS (13 mM) and the electron - accepting capacity of the AQDS provided (50 µM) indicates that the AQDS molecules underwent several (ca. 260) cycles of microbial reduction and abiotic oxidation until all the entrapped Fe(III) oxides had been reduced to magnetite. By stimulating the 77 reduction of the entrapped Fe(III), AQDS also supported the concomitant growth of a pl anktonic population (Figure 4. 2A ). Thus, AQDS alleviated, at least partially, the need of the cells to directly contact the insoluble Fe(III) oxides . Biofilms also grew on the bead surface in cultur es supplemented with AQDS (Figure 4. 2 B ). This contrasted w ith the cultures lacking AQDS, which only contained a few live and dead cells at tached to the bead surface (Figure 4. 2 B , inset). This is because the ability of AQDS to serve as an electron shuttle also facilitates long - range electron transfer across multil ayered biofilms (286 ) . By contrast, the synthetic metal chelator NTA did not stimulate the reduction of the entrapped Fe(III) oxides (1.6 ± 0.3 mM Fe(II) produced over 72 h ). This is because although NTA is small enough to permeate through the bead pores, it solubilize s Fe(I II) at rates too slow to support cell growth (98) . The alginate beads experiments ruled out the secretion by G. ahangari of electron - shuttling compounds with molecular weights smaller than the pore size of the microporous beads. However, they cannot rule out the presence of larger mediator molecules or molecules. To bypass this limitation, I also tested the ability of cell - free culture supernatants obtained from station ary phase cultures grown with free Fe(III) oxides to stimulate Fe(III) reduction by washed cell suspensions of G. ahangari . Concentrated filtered supernatant fluids did not stimulate Fe(III) reduction compared to cell suspensions in fresh medium unless sup plemented with AQDS (Figure 4. 2C). These results further suggest that G. ahangari does not produce endogenous mediators to reduce Fe(III) oxides. 4.4.3 Cellular components involved in Fe(III) reduction in G. ahangari In the absence of mediators, cells need to d i rect ly contact the Fe(III) oxides in order to reduce them. Consistent with this, TEM micrographs of G. ahangari cells from cultures with free Fe(III) oxide revealed cells densely coated by Fe(III) oxide particles (Figure 4. 3A) . Treatment of the cells with oxalate to dissolve most of the Fe(III) oxide particles revealed abundant filaments ( Figure 4. 3B). Similar appendages were also present in cells grown with Fe(III) citrate and were associated with mineral particles, presumably Fe precipitates as they were eas ily dissolved with 78 oxalate (Figure 4. 3C) . T (52) was easily distinguishable from abundant curled , thin appendages (Figure 4. 3C) . The production of a flagellum and the tumbling motility exhibited by this microorganism in cultures with Fe(III) oxides (52) provide a mechanism for locat ing the Fe(III) oxides , similar to what has been observed in mesophilic Fe(III) redu cers (102) . On the other hand, the production of the curled appendages associated with the Fe(III) oxides suggest s a mechanism for binding the mineral particles close to the cell. Close associat ion between the cells and the insoluble Fe(III) oxides is necessary to Most of the redox - active proteins catalyzing this last step of electron transfer in mesophilic Fe(III) reducers are heme - containing, c - cytochromes (267) . These proteins are especially suited for electron transfer at high temperatures because t he covalent attachment of heme groups to proteins stabilizes their secondary structure and increases th eir thermal stability (287) . H owever, dithionite - reduced versu s air - oxidized spectral analyse s of resting cell suspensions of G. ahangari failed to identify the spectral signatures characteristic of c - type cytochromes (52) . Similarly, cytochromes were not detected by redox - difference spectroscopy in mesophilic metal - reducing bacteria from the genus Pelobacter (288 290) . Yet , when whole - cell protein s from cells of Pelobacter carbonolicus were concentrated and subjected to electrophoresis on an SDS - PAGE gel and heme - stained , three bands were detected and identified as cytochromes by peptide mass fingerprinting (291) . F ollow ing a similar approach, I detected several heme - stained bands in whole cell extracts of G. ahangari (Figure 4. 4A and B) . At least 4 of these bands (with relative molecular masses of 46, 38, 19 and 16 kDa) were also present in protein fractions that had been mechanically sheared from the cell surface (Figure 4. 4 A and B). The presence of he me - containing bands in the sheared protein fraction is consistent with redox - active proteins having the insoluble Fe(III) oxides (166) . To further test this, I investigated the ability of cells missing these outer surface proteins after mechanical shearing to resume growth and reduce Fe(I II) 79 oxides. Shearing the outer surface proteins prevented the cells from reducin g insoluble Fe(III) oxides (Figure 4. 4C), although the cells still produ ced the curled appendages (Figure 4. 4D). Furthermore, shearing did not compromise cell viability, as ind icated by the fact that these cells did not have a growth defect when the soluble electron acceptor Fe(III) citrate was provided as the el ectron acceptor for growth (Figure 4. 4E). This is analogous to the mesophilic Fe(III) reducer Geobacter sulfurreducens , which requires c - cytochromes exposed on the outer surface to reduce insoluble metal oxides but not Fe(III) citrate (166) , and is consistent with their role in the direct transfer of electrons to the mineral. This is because their exposure minimizes the distance with the Fe(III) oxides and facilitates the direct tunneling of electrons between the heme groups a nd the mineral surface (292) . Conclusions The results presented above demonstrate that G. ahangari uses a direct contact mechanism to reduce Fe(III) oxides, strongly associating with the mineral particles to prom ote electron transfer between redox - active proteins exposed on the outer surface and the mineral. TEM micrographs of other hydrothermal vent isolates consistently show cells in intimate association with the Fe(III) oxide particles (65, 293, 294) , suggesting that this may be a widespread mechanism for Fe(III) reduction in these environments. A direct contact mechanism is likely to confer on the se microorganisms a competitive advantage over microorganisms relying on mediators. Fluid circulation is especially high in hydrothermal systems (31) , which limits the bioavailability of soluble electron shuttles and favors the growth of microorganisms that can directly contact the oxides. Colonization of the mineral surface can also lead to the formation of biofilms, as shown for G. ahangari , thus providing a microenvironment for the absorption and concentration of nutrients, including soluble electron shuttles. Solid phases of humic acids are also abundantly produced during the degradation o f organic matter and can serve as an electron acceptor to support the growth of dissimilatory Fe(III) - reducing microorganisms (188) . The 80 insoluble nature of the humic phases and their co - association with Fe(III) oxides in sediments is likely to also select for direct contact mechanisms. Like G. ahangari , most hyperthermophilic Fe(III) reducers isolated to date are flagellated and motile (177) . Flagellar motility is especially advantageous to these organisms as it provides a mechanism to access Fe(III) oxides freshly deposited in the hot sediments. The heated waters that exit the vents dissolve minerals from the vo lcanic rock and deposit them in the nearby sediments. As a result, reactive ferric minerals are continuously being deposited as a dispersed layer on top of the hot sediments around hydrothermal vents and could be accessible to motile microorganisms. Energy expenditure for flagellar synthesis and operation is relatively modest (about 2 % and 0.1 %, respectively, of the total energy expenditure of the cell during normal growth (295) ), yet yields high returns, as it enables microorganisms to rapidly access nutrients and increases their chances of survival (296) . Hence, motilit y and direct contact mechanisms for the reduction of Fe(III) oxides are likely to have coevolved to maximize the fitness of microorganisms growing under the extreme physical and chemical conditions of hydrothermal systems. 81 CHAPTER 5. MICROBIAL NANOWIRES: A CONSERV ED MECHANISM OF EXTRACELLULAR ELECTRON TRANSFER Abstract Microbial nanowires have been identified in only two genera of mesop hilic iron reducers, Geobacter and Shewanella . Here I demonstrate the presence of conductive filaments isolated from both the bact erium Geothermobacter ehrlichii and the archaeon Geoglobus ahangari which are predicted to be essential for iron respiration at (hyper)thermophilic temperatures. CP - AFM analysis of the motility structures (flagella and putative archaella , respectively ) and the putative nanowires ( t ype IV pili and pilus - like filaments , respectively ) obtained by mechanical shearing demonstrated that the motility structures were insulating while the pil i or pilus - like filaments were conduct ive under the same conditions. These data were reproduced using exhaustively - purified filaments , again demonstrating their conductivity . Since these organisms are both able to grow at extreme temperatures, it was of interest t o determine if these novel nanowires exhibited increased thermal st ability over those of mesophilic Geobacter species . A S ypro Orange thermal shift assay was carried out and w ho le - cell extracts from these organisms present ed the appropriate t rends. Unfortunately, sufficient data could not be generated for the highly - purif ied filaments . However, the available data support a mechanism whereby microbial nanowires function in thermophilic and hyperthermophilic organisms to avoid the use of soluble compounds which could be lost to the surrounding environment, and to increase th e surface area available for extracellular electron transfer. Introduction Iron is the fourth , and as such it represents a significant electron sink for surface a nd sub surface microorganisms (84) . Yet, iron presents a challenge as, unlike the electron acceptors us ed by the majority of organisms on the surface ( i.e. oxygen), it is unable to enter the cell in its most common form (84) . At circumneutral 82 pH , iron is insoluble and exists as an oxidized (Fe(III)) or reduced (Fe(II) ) solid - phase mineral (84) . Still , numerous microorganisms are able to utilize these insoluble oxides (84) . T he most comm only studied dissimilatory iron - respiring microorgan isms are Geobacter sulfurreducens (74) and Shewanella oneidensis (297) . W hile these bacteria can solve the problem of reducing an insoluble electron acceptor , they accomplish this goal with different mechanisms. S. oneidensis has the ability to utilize a range of extracellular electron transfer mechanisms to reduce insoluble Fe(III) oxides (94, 298, 299) . Chelation of iron i nto a soluble form is known to assist in the reduction of Fe(III) oxides (84, 10 0) . A weak chelator is produced by S. oneidensis , while the rate of solubilization has been shown to be too low to permit the demonstrated growth rates (300) . The predicted metal chelator, an en dogenously - produced flavin, instead serve s as an electron shuttle (100) . This flavin, i n addition to any exogenously supplied shuttles , facilitates growth on Fe(III) oxides and within microbial electrochemical cell s (MEC s ) (300) . If p roduced in a MEC, cells remain planktonic and electrons are passed to the electrode surface via the excreted flavin (286) . I f no electron shuttle is present , a biofilm is formed on the electrode surface (286) . In addition, a unique microbial nanowire can be produced by S. oneidensis (94) . T extension of the cell membrane, forcing any cytochromes present in the periplasm or on the membrane outward (95) . Studies on G. sulfurreducens have resulted in a range of proposed mechanisms for iron respiration (see (103) for review) , and even after 20 years (74) a consensus has not been reached. However , after extensive study, reproducible ass ays were developed to permit mechanistic studies on this organism and others (97, 113, 114, 139) . It is now firmly cemented that G. sulfurreducens , the model of the Geobacteraceae , is an obligate direct - contact reducer of insoluble Fe(III) oxides (91, 164 166) . If an electron shuttle or a chelator is available , the cells will utilize this more readily - available electron acceptor (96, 187) , but they are unable to produce shuttles themselves (164, 301, 302) . 83 These two model systems thus demonstrate that multiple pathways exist to enable direct - contact respiration (see (84) for review). The simplest of these mechanisms is the expression of a terminal reducta se on the cell surface (84, 1 14) . In dissimilatory iron - reducing microorganisms t hese terminal reductases are commonly c - type cytochromes (72) . Via their expression on the outer surface of the cell, they are placed into close p roximity with the mineral surface and enable electron transfer from the cell interior to the extracellular electron acceptor (84, 8 6, 166, 303, 304) . Facilitating this interaction could be the expression of an extracellular polysaccharide (EPS) matrix to maintain a biofilm on the mineral surface (90) , production of a motility apparatus to locate or maintain proximity to the electron acceptor (102) , and/or the production of filaments to attach and bind to the mineral surface (91 ) . Yet, the inadequacy of these mechanism s is that the cell surface has a limited area, and as such the amount of contact with the mineral is restricted (91) . The extension of the surface area for red uction can be accomplished by the secretion or loss of outer - surface cytochromes in to an EPS matrix (90) or by the production of conductive filaments (91, 95) . Deletion of an operon (Xap) encoding the EPS of G. sulfurreducens generated strains that were un able to reduce Fe(III) oxides . This phenotype was attributed to the loss of EPS - associated cytochromes from the biofilm matrix (90) . Cytochrome s, however, are not the sole reductase of iron in G. sulfurreducens. The first proposed mechanism for the extension of the electroactive cell surface was based on the discovery that the type IV pili of G. sulfurreducens were conductive and able to transfer respiratory electrons to the cell surface and beyond (91) . resemble typical t ype IV pili with one important distinction. These g eopili ns lack the conserved globular head domain and have therefore a lower subunit molecular weight (91) . The loss of this domain alters the function of the pilus away from typical t ype IV pili functions, such as motility and attachment, and towards being a conduit for metabolic electrons to exit the cell (91, 118) . Dissimilatory iron respiration is also found outside of the mesophiles. In f act, the majority of ther mophiles and hyperthermophiles are able to respire Fe(III) oxides as a terminal electron 84 acceptor when ex amined by cell - suspension assays (32) . Yet, little attention has been spent on the diversity of t hermophilic iron reducers until recently (113, 114, 126, 139, 184) . Genome evidence demonstrating the presence of c - type cytochromes, flagella, and other filament morphologies is available for several (hyper)thermophilic bacteria and archaea (190, 201, 305) . The first thermophile to have a n eluci dated iron - respiration mechanism was the bacterium Carboxydothermus ferrireducens , with a T opt of 65 °C (306) . This bacterium was capable of using exogenous electron shuttles or chelators, but produced none of its own (114) . Thus, a mechanism based on c - type cytochromes, flagella for motility, and pili for attachment was proposed (114) . Another thermophilic bacterium, Thermincola potens, has been studied and c - type cytochromes expressed on the cell surface are predicted to be the termin al reductase (186) . Similar studies have also been performed within t he Archaea. T he hyperthermophilic archaeon Geoglobus ahangari (52) , prese nted within this work (Chapter 4), belongs to the archaeal family Archaeoglobaceae and similarly lacks endogenously produced mediators a nd contains an archaellum, pilus - like filaments, and numerous c - type cytochromes (139) . This physiology is conserved within the Archaeoglobaceae , as both Ferroglobus placi dus (179) and Geoglobus acetivorans (178) have both been shown to rely on the same strategy for iron respiration (113, 184, 190) . Thus, all of the thermophilic and hyperthermophilic organisms, regardless of their kingdom (18) , appear to forego the use of excreted compounds in favor of direct contact with the mineral surface. Due to the presence of both pilus - like filaments and c - type cytochromes (113, 114, 126, 139, 184, 1 90) , however, it is difficult to speculate which is (or are) the terminal reductase(s) of iron in these organisms. T herefore, t he next step is to determine if the filaments are conductive under physiologically - relevant conditions (307) . I f the fi laments produced are conductive , it would indicate that microbial nanowires are a wide - spread mechanism for dissimilatory iron reduction. To this end, G. ahangari and the only thermophilic relative of the Geobacteraceae , 85 Geothermobacter ehrlichii , were selected for biochemical and electrochemical characterization of their extracellular filaments. Materials and Methods 5.3.1 Bacterial strains and culture conditions G. ahangari strain 234 T (JCM 12378 T , ATCC BAA - 426 T ) was grown an aerobically in a m odified marine (MM) medium with pyruvate (10 mM) as electron donor and soluble Fe(III) citrate (50 mM) as electron acceptor. Incubations were in the dark and at 85 °C ( 18 ) . G. ehrlichii strain SS015 (ATCC BAA - 635, DSM 15274) was grown anaerobically in MM medium with malate (10mM) as the electron donor and soluble Fe(III) citrate (50mM) as the electron acceptor (Chapter 2). Incubations were in the dark and at 55 °C (66) . Cultures were transferred (10% (v/v)) into fresh medium when 2 / 3 of the Fe(III) had been reduced (late - exponential phase). The extent of Fe(III) oxide reduction by growing cultures was monitored by extracting the Fe(II) from the mineral phase with 0.5 N HCl ( 23 ) and measuring the extracted Fe(II) by the ferrozine method ( 46 ) . 5.3.2 Isolation and purification of extracellular filaments Both a rapid and exhaustive method were developed for the purification of the extracellular filaments of G. ehrlichii and G. ahangari. The rapid preparat ion was developed to produce filament preparations containing both the pilus - like filaments and archealla of G. ahangari, and both the pili and flagella of G. ehrlichii . This protocol involves repeated mechanical shearing , via a 23G needle ( 10 passages ), o f the filaments from the cell surface followed by centrifugation (20 min, 3,220 x g , RT) to remove whole cells and cell debris. Culture supernatant, containing the sheared filaments, was concentrated via a 30 kDa Centriprep centrifugal filter (Millipore) a nd washed with an oxalate solution (52) , an iron extraction buffer (139) , and a final wash with MMWB (139) . By utilizing the preparatory cell gel electrophoresis (prep - cell) p rotocol developed in our lab (92) , it was possible to use the SDS - insolubility of the pili of G. ehrlichii and the pilus - like filament s of G. ahangari to purify these filaments to homogeneity. 86 5.3.3 Thermostability measurements by t hermal shift assay Thermal shift assay protocols are derived from those in (308 310) . G. sulfurreducens strain PCA , G. ehrlichii strain SS015, and G. ahangari strain 234 T were used to represent mesop hilic (74) , thermophilic (66) , and hyperthermophilic (52) dissimilatory iron reducers with direct - contact mechanisms involving microbial nanowires ( (91, 139) and Chapter 2 ) . Whole - cell preparations , which were washed as per the culture supernatant above to remove residual iron, were lysed by repeated (3 x ) sonica tion (duty cycle = 50, time = 1 min, output = 1 , on ice ) of cell pellets from late - exponential stage ferric citrate cultures. Cell debris was removed by centrifugation (20 min, 3,220 x g , RT) and the soluble protein fraction was collected. Sypro Orange (Si gma - Aldrich ) was diluted to a 10 x stock (from 5 , 000 x ) and 5 µ l were added to 40 µ l of the desired pH buffer. The buffers used were 50 mM s odium acetate (pH 4.0), 50 mM HEPES (pH 7.0 ), and 50 mM CHES (pH 9.4) . Finally, 5 µ l of the desired sample was added to the well and mixed. T o create the appropriate blank for each condition , 5 µl of 10x Sypro Orange was placed in 45 µ l of the appropriate buffer. Samples, including the soluble fraction of whole - cell lysate and purified filaments, were placed into an iQ 96 - Well PCR Plate (Bio - Rad Laboratories, Inc.) and covered with iCycler iQ optical tape (Bio - Rad Laboratories, Inc.) before being placed in the i Q5 multicolor real - time PCR detection system ( Bio - Rad Laboratories, Inc.). The thermal cycler was ramped from 25 to 99 ° C in 0.5 ° C increments with a ho ld time of 30 seconds per step. Fluorescence measurements were performed using an excitation wavelength of 545 nm while the emission of the dye was measured at 585 nm . Due to limited preparations, protein quantific ation was foregone. T wo biological replicates and t hree technical replicates were performed for each organism and the average and standard deviation of each T m was calculated from these replicates. T m was calculated as the median temperature obtained betwe en the trough and the peak fluorescence. However, calculations could not be performed on the prep - cell purified pilus or pilus - like filaments under investigation as these samples failed to produce sufficient fluorescence data using the experimental paramet ers discussed above. 87 5.3.4 Conductive probe atomic force microscopy Mechanically - sheared samples obtained from both G. ehrlichii or G. ahangari were immobilized onto independent highly oriented pyrolytic graphite (HOPG) surfaces, th oroughly dried under a stre am of N 2 gas , and imaged by a Cypher atomic force m icroscope (Asylum Research). Topographic scans by atomic force microscopy (AFM) were performed at a 4.88 Hz scan rate or a 2.44 Hz scan rate , for scans below 2 µ m , using an AC240TS tip (Asylum Research). U pon location of an appropriate area for conductivity measurements, one containing a number of diffuse fibers, the tapping tip was exchan ged for a conductive tip ( ASYELEC01 ) (Asylum Research) and the instrument was reengaged for conductive probe atomic forc e m icroscopy (CP - AFM) . When possible, m ultiple points (2 - 4) were selected across 1 - 2 filaments per frame, including points on both thick and thin areas, representing bundled and what I believe to be less aggregated filaments. Analysis of current/voltage ( I /V ) output was performed within the Igor software suite (WaveMetrics) . Averages were taken for measurements at each bias voltage and these values were smoothed using a Savitzky - Golay algorithm (311) with the end points an d averaged. Finally, resistance was calculated as 1 x slope - 1 and compared to the resistance obtained from th e other filaments under investigation. 5.3.5 Elemental analysis of purified filament preparations Quantitative elemental analysis of p urified filament preparations are awaiting additional sample preparations, as the prep - cell protocol yield is low, yet will proc eed as previously described (92) by inductively coupled p lasma atomic emission s pectrometry (ICP - AES) . Analyses will be performed at the Chemical Analysis Laboratory at the University of Georgia (Athens , GA ) using a Thermo Jarrell - Ash Enviro 36 Inductively Coupled Argon Plasm. In brief, samples obtained from each microorganism under investigation will be resuspended in 10 mM CHES buffe r (pH 9.5, 1 mM EDTA) and this same buffer will be used as a reference during the experimental procedure. In order to obtain a value for the number of elemental atoms per filament 88 subunit it will be important to quantify the samples before submission. As p reviously stated, the quantification of filament samples from these organisms is difficult, but will be performed on three prep - cell samples per organism to create a n average per preparatory gel. Thus, I can use this concentration and the data from the ICP - AES protocol to calculate the number of elemental atoms per filament subunit . For G. ahangari , where the molecular weight of the nanowire subunit is unknown, it will be possible to calculate the number of elemental atoms per microgram of protein while the predicted molecular weight of pilin of G. ehrlichii (peg.2848) is estimated (137) to be 6.95 k Da , or 1.1 5 x 10 - 11 µg . 5.3.6 Examination of the curli - like nature of G. ahangari nanowires M ethods for the identification of amyloid - fib ril f orming filaments inclu de microscopic analysis of fibril formation after incubation at 4 ° C in a pH neutral buffer , Congo Red bindi ng, and Thioflavin T binding (312) . Preparations of sheared pilus - like filaments from G. ahangari were incubated (4 °C, pH 7.0) for 72 h before examination by transmission electron microscopy on a JEOL CXII transmission microscope (operated at 75 mV) to qualitati vely determine if the number of individual filaments decreased and if there was an increase in the amount of fibrils formed. Samples before and a fter incubation were placed on F ormvar - coated 300 - mesh copper grids (Electron Microscopy Supplies), washed twic e with ddH 2 O and stained with 1% ammonium molybdate . Additionally, while not yet performed, Thioflavin T (ThT) binding assays will be performed on sheared samples from G. ahangari . These will be incubated for 24 h in a range of buffers ( pH 4.0, 7.0 , and 9 .4 ) and mixed with ThT and examined for the increased fluorescence of the bound dye (313) . ThT binding was chosen over that of Congo Red due to the specificity of ThT for amyloid fibrils (312, 313) . S heared samples will be of a lesser purity than those purified via the prep - cell , yet have the advantage in that they exhibit decreased amounts of bundling and aggregation ( see Figure 5. 1) when compared to the exhaustive 3 - day protocol performed on the prep - cell samples. This aggregation will likely block ThT binding and lessen the efficacy of the 89 a ssay (31 2) . ThT binding will be performed on the pilus - like filaments of G. ahangari ( as per (312) ) using the QuantaMaster spectrometer (Photon Technology International) available at Michigan State University. In tandem, the nanowires of G. ehrlichii , which are presumed to not be curli - like in nature, will be used as a negative control to assess the accuracy of the protocol within (hyper)thermophil ic filaments from dissimilatory iron - reducing microorganisms. In addition to the se biochemical studies, DELTA - BLAST analysis was conducted using curli - associated proteins from Escherichia coli against the genome of G. ahangari . CsgA ( CDU39169.1 ), CsgB ( ACB59306.1 ), CsgC ( ACB59308.1 ), CsgD ( ACB59305.1 ), CsgE ( ACB59297.1 ), CsgF ( ACB59303.1 ), and CsgH ( ACB59302.1 ) from E. coli wer e used as queries. Results and discussion 5.4.1 Isolation and purification of filaments Rapidly - purified samples from G. ehrlichii and G. ahangari , prio r to the iron - cleanup steps of o xalate and IEB treatment, appear visually to contain a significant amount of i nsoluble iron oxide particles, which are presumed to be iron clays (139) . In most cases , the sheared filaments at the end of the rapid protocol, even following the iron - cleanup steps, appear from faint orange to black, depending on the extent of iron remaining in the preparation that was resilient to the i ron - cleanup steps. This is unlike the majority of samples acquired from the prep - cell protocol. In most cases these samples, from either organism, contain small black filaments which are visible to the naked eye and occasionally a fine white precipitate. B oth of these remain following the vigorous cleanup steps after the preparatory electrophoresis setup runs. The black filaments are presumed, as in G. sulfurreducens , to be bundles of highly - purified filaments while the fine white precipitate is presumed to be acrylamide particles released from the surface of the gel during collection of the protein filaments. TEM micrographs of filaments purified from G. ehrlichii by the rapid method clearl y show both t he bacterial flagella and the t ype IV pili (Figure 5. 2A and B ). The bacterial flagella produced 90 by G. ehrlichii appear morphologically similar , i.e. relatively s traight with an average diameter of 30 nm , to those reported in other members of the Desulfuromonadaceae (107, 108, 116, 147) and flagellated members of the Geobacteraceae (73, 102, 314, 315) . While reportedly rare within the extremophilic members of the Desulfuromonadaceae (108, 116, 147) , type IV pili ( ca. 2.5 nm in diameter ) were purified by both purification protocols (Figure 5. 2B - D ). Thereby , their SDS - insolubility, like the pili of G. sulfurreducens (92) , was confirmed . These filaments appear morphol ogically similar by TE M to the g eopili present in Geobacteraceae species and, based on previous alignments of the pilin sequence to other members of the Geobacteraceae, are predicted to be most similar to the pili of G. metallireducens (Chapter 2). TEM micrographs of filaments purified , by the rapid method, from G. ahangari clearly show both t he archaellum and numerous pilus - like filaments (Figure 5. 1A ). The archaella of G. ahangari exhibited the same structure as seen by TEM (139) , i.e. relatively straight with an average diameter of 4 6 nm . The pilus - like filaments (139) again appear smaller than the archaella ( ca. 2.5 nm in diameter) and exhibit a curled structure resembling the amyloid - fibril forming curli of sev eral mesophilic organisms (316, 317) . These filaments are also seen in the iron - respiring members of the Archaeoglobales , F. placidus (113) and G. acetivorans (184) . The pilus - like filaments remained intact throughout both purification procedures (Figure 5. 1A and B ), indicating their SDS - in solubility and resistance to depolymerization, while the archaella were only present within the rapidly - purified sample preparations. These pilus - like filaments are thus morphologically and physiologically similar to the curli of several mesophilic isolate s in that they appear curled or kinked and are resistant to stressors that would denature or depolymerize the majority of extracellular appendages (316, 317) . However, without additional studies this behavior may be proven to be circumstantial and/or related to the predicted thermal and chemical stability of these filaments. 91 5.4.2 G. ehrlichii expresses multiple filaments whi le only the Geopili are conductive As expected, when examined by AFM, the filaments of G. ehrlichii exhibited the same structure as seen by TEM (Figure 5. 3 A ). In p reliminary CP - AFM experiments, performed with Dr. Sanela Lampa - Pastirk (Reguera lab), sheared pili from G. ehrlichii , while shown to be conductive, were covered in an insulating layer of what was presumed to be EPS (Figure 5.3A) . This EPS layer made measurements difficult, yet an average resistance was calculated for the sheared pili and was deter mined to be ca. 37,458 ( ± 9,381 ) MOhms (Figure 5. 3 B and when compared to graphite (Figure 5. 3 B , insert ) ). The flagella, visualized within the same preparation, were found to be insulating ( Dr. Lampa - Pastirk, personal communication ). These results were expe cted , as the flagella are used primarily as motility structures (52, 66) and the relatedness of G. ehrlichii to members of the Geobacteraceae (66, 73, 107, 108) suggests their pili may function similarly to those of G. sulfurreducens inasmuch as they are produced to increase the electroactive surface area of the cell and permit the reduction of metal oxides at a distance (91) . Conductivity measurements by CP - AFM were repeated on an additional biological replicate of purified pili from G. ehrlichii purifi ed using the prep - cell protocol. T he resistance was found to be dependent on the location of measurement (Figure 5.4A) and the bias vo ltage sweep that was performed. At a 1 V sweep (from - 1.0 to 1.0), two spots were analyzed. The first, Po sition 4, w as located within a bundled, or globular , region o f the micrograph while the second, Position 3, was located on a thinner, more pilus - like filament section . The calculated resistance at Position 4 was 3,775 ( ± 2,088) MOhms while the resistance at Position 3 was calculated as 454 ( ± 64) MOhms . These values can be compared to the resistance of the HOPG surface (Position 1) analyzed within the same micrograph (45.8 ± 21.6 MOhms) (Figure 5. 4A ). This trend, of Position 4 being more insulating than any of the les s - bundled positions, was replicated at 0.6 V, 0.3 V, and 0.1 V sweeps (Figure 5. 4C ). The resistance o f Position 4 at 0.6 V was 5,905 ( ± 2,205 ) M O hms while Positions 3, 6, and 7 averaged a resistance of 127 ( ± 20) MO hms. Yet, the resistance of graphite rema ine d approximately the same at 51 MO hms (n=1 within this micrograph). Th is trend 92 further continued at lowe r bias voltage sweeps, with Position 4 being more insulating than Position 3 . However, sample resistances begin to approach those of graphite at these lower bias voltage sweeps, and as such will not be reported. The pili of G. ehrlichii thus rep resent the first nanowires identified within a thermophile and demonstrate that microbial nanowires are likely a conserved structure within the Geobacteraceae. 5.4.3 G . ahangari expresses multiple filaments while only the smaller, pilus - like filaments are conductive Both filament morphologies in G. ahangari exhibited the same structure as seen by TEM (139) (Figure 5. 5A and B ). Preliminary experiments on the filaments of G. ahangari , with Dr. Sanela Lampa - Pastirk, were p erformed on sheared preparations and prep - cell samples (Figure 5.5C and D) . The sheared samples, possibly containing residual metal ions, had a resistance of 266 ± 147 MOhms. The archaella, in comparison, were found to be insulating ; an average resistance was calculate d and determined to be 465,825 ± 84,953 MO hms. The conductivity of purified pili, by the prep - cell protocol, was also assessed at the time and the resistance (993 ± 405 MOhms) was determined to be higher than those from the sheared preparation s, yet still far lower than the insulating archaella (Figure 5. 5C and D ). Conductivity measurements were again performed on the pilus - like filaments of G. ahangari , purified using the prep - cell protocol, and confirmed the conductive nature of the pilus - lik e filaments (Figure 5. 6B and C ). Initial measurements, performed at a range of forces (8 - 15 nN) and voltage sweeps (0.3, 0.6, and 1 V) on two different positions (Figure 5. 6A ) showed that the pilus - like filaments were less conductive than previously foun d (18,338 ± 9,799 MOhms) while the large standard deviation indicates substantial variability (Figure 5. 6C ). However , after repeated measurements and dithering of the CP - AFM tip on the graphite surface, to remove any insulating material on the tip, it was possible to break through this insulating layer and, I believe, examine the true conductivity of the pilus - like filaments. Averaged resistance values were calculated from 2 independent positions (Position 3 and Position 4) (Figure 5. 6A ) and over the full 93 a ssessed range of bias voltages. As opposed to the nanowires of G. ehrlichii , as bias voltages narrowed (1.0, 0.6, and 0.3 V) an increase in resistance was seen (292, 497, and 731 MOhms, respectively), although this trend was not as drastic as in G. ehrlich ii . The average resistivity after the removal of the insulating layer, over a total of 13 measurements, was 540 ± 246 MOhms (Figure 5. 6C ). A s the archaella are structurally and genetically similar to type IV pili (318) , it was predicted that these filaments would be conductive. Y et , the abundance of the curled filaments on the cell surface (139) makes them phy siologically more similar to bacterial nanowires . However, the identification of protein nanowires in this organism is the first report of such nanowires in the Archaea and in a hyperthermophile. Based on the shared metabolic capabilities and phylogenetic relatedness between G. ahangari, G. ac etivorans, and F. placidus , the conductivity of the pilus - like filaments from these species should be assessed to determine if they too could function as microbial nanowires. Identification of additional microbial nanowires among other hyperthermophiles, d ue at least in part to their proposed relatedness to the last common ancestor (32) , would permit speculation regarding the antiquity of this strategy for iron respiration. 5.4.4 T hermostability of soluble cell extracts and ther mophilic nanowires Soluble fractions of whole - cell extracts from G. sulfurreducens, G. ehrlichii, and G. ahangari (representing a mesophile, a thermophile, and a hyperthermophile; respectively) were examin ed via the Sypro Orange thermal shift assay. As exp ected, the T m of the soluble c ell fraction increased from 57.2 ( ± 2.9 ) , to 64.6 ( ± 1.5 ) , to 81.6 ( ± 2.3 ) °C as the growth temperature of the organism increased (Figure 5. 7). These temperatures match the approximate upper limit of growth temperatures for ea ch organism (52, 66, 74) , with the exception of G. ahangari . Thus, cessation of cell growth at their upper temperature limit , in most cases, is due to an overall instability of protein and thereby reduced function at increased temperatures. In addition, increasing (9.4) or decreasing (4.0) the pH away from a neutral (7.0) buffer negatively affected the T m , as expected for proteins derived from neutrophilic micro organisms (52, 66, 74) (Figur e 94 5. 7A - C ) . Thus, the presence of stabilizing proteins and solutes in the cell interior, especially in the case of G. ahangari , likely functions to increase the in vivo stability of these proteins, which is lost upon their release from the cell. Thus, whil e the soluble protein fractions demonstrate an increased thermal stability, the thermal stability of the nanowires themselves must be assessed to evaluate their use in nanodevices or biomimetic interfaces (307, 319) in which this stability is desired or required . The majority of proposed uses for these filaments, including electroactive nanobrushes for contaminant removal (307, 319) , requ ire the filaments to remain functional under conditions that would destabilize mesophilic proteins (17) . Thermal stability has been linked to increased overall stability (17) , and as such these thermoph ilic nanowires will likely exhibit increased resistance to a number of stressors. A s the stability of the nanowires of mesophilic Geobacter species has never been assessed, it was also important to identify conditions and temperatures which would result in the denaturation of these, in addition to the (hyper)thermophilic nanowires. While the pili of G. sulfurreducens are being developed into nanodevices at Michigan State University (Chemical Engineering and Materials Science department), the p ili of G. meta llireducens were chosen to compare to those of G. ahangari and especially G. ehrlichii due to the phylogenetic relatedness of the pili of G. metallireducens to those of G. ehrlichii (Chapter 2). Sypro Orange binding to the highly - purified filaments, i.e. t he T m , was expected to be dependent on the pH of the incubating buffer. However, after repeated attempts to obtain thermostability data for these filaments I was unable to obtain any useable results (Figure 5. 8). Initial samples were introduced in low quan tities and produced no increase in fluorescence, indicating no Sypro Orange binding to exposed hydrophobic domains. Subsequent attempts were made with increased relative protein concentrations, including an entire prep - cell sample being loaded, and the exp eriment was again unsuccessful. Of the samples which produced a fluorescence peak, each was present at the same temperature, regardless of organism or buffer pH ( see Figure 5. 8, G. ahangari for example). This is unlike sheared samples which were 95 introduced into the Sypro Orange assay. These samples, like the whole cells, reflected changes in the growth temperature of the organism and the incubating buffer. However, these matched the results obtained for whole - cell extracts and thus, the results were deemed to be acquired from contaminant, non - filament proteins. One additional possibility for the failure of prep - cell samples to provide sufficient fluorescence could be the presence of residual acrylamide from the purification protocol. Acrylamide is a well - kno wn fluorescence quenching compound (320 322) , and as such may interfere wi th the Sypro Orange fluorescence. Further attempts will be made to assess the chemical and thermal stability of these nanowires in comparison to those of mesophilic strains, primarily G. sulfurreducens . This may include further Sypro Orange assays on filam ents not purified using acrylamide , or circular dichroism after filament incubation in a range of chemical and pH stressors. 5.4.5 Pilus - like filaments from G. ahangari demonstrate curli - like behavior Amyloid fibrils produced by the curli of mesophilic bacteria , primarily within the Enterobacteriaceae (see (316, 317) for review) , raised the exciting possibility of studying am yloid - fibril formation in a non - e ukaryotic system . If an archaeal system, which would potentially be phylogenetically more related to systems in eukaryotes (18) , was identified it could lead to major breakthroughs in the st udy of amyloid - based diseases such a (317) . The smaller, pilus - like filaments from G. ahangari are superficially similar to the curli of several mesophilic bacteria by both transmission electron microscopy and atomic force microscopy , as previously described (Figure 5. 9A and B ). Upon shearing of the pilus - like filaments f rom the cell and incubation at 4 °C, the filaments did function to enable the formation of amyloid - like bun dles (Figure 5. 9C and D ). However , t his alone is insufficient to permit speculations regarding the curli - like nature of these filaments. A definitive test is the incubation of sheared cell preparations with the amyloid - specific (323, 324) dye Thioflavin T. This dye, upon the switch from alpha helix to beta - pleated sheet, binds to the amyloid - like protein and alters its fluorescence. ThT fluorescence incr ease was seen in biofilms of the archaeon Haloferax volcanii (325) , 96 indicating that functional amyloids may be present within archaeal biofilms, as they are in bacterial. T he next step in the identification of the first reported amyloid - forming filaments in the Archaea would be to determine if incubations of filament preparations from G. ahangari strain 234 T bind Thioflavin T. Sheared filament preparations using the rapid protocol from G. ahangari and G. ehrlichii , which will serve as the negative control, will be incubated at pH 4.0, 7.0, and 9.4, and in the presence of denaturing agents. A negative result, indicating that amyloid fibrils are not formed, would indicate that the morphological similarity of the pilus - like filaments from G. ahangari to bacterial curli is coincidental and that the aggregates forme d after incubation at 4 °C are artifactual or non - amyloid aggregates. This finding would be supported by genome evidence. H omology searches conducted using the curli - associated Csg genes of E. coli against the genome of G. ahangari resulted in no homologou s proteins ; with the exception of the master regulator, CsgD, which provided weak hits to CheY and other response regu lators within the genome. T he probability that such proteins would be homologous across kingdoms is low, so this alone is not sufficient e vidence to disprove the presence of curli - like filaments in G. ahangari . If I do see an increase in ThT fluorescence, indicating the presence of amyloid - like bundles, it would be th e first report of amyloid fibrils forming in an archaeon outside of a biofi lm environment. Conclusions Previous result s demonstrate that both G. ehrlichii and G. ahangari use direct contact mechanisms to reduce insoluble Fe(III) oxides in the laboratory. T he numerous c - type cytochromes identified within both genome s , and via SDS - PAGE , were speculated to be the terminal reductases of Fe(III) oxides. This indicated that the flagellar structures from both organisms were primarily used for motility and that the pili or pilus - like filaments were for attachment to the mineral surface ( Chapters 2 and 3). A similar strategy is propose d for the thermophilic bacteria C. ferrireducens (114) and T. potens (186) a nd the hyperthermophilic 97 archaea F. placidus (113) and G. acetiv orans (184) based on mechanistic and genomic evidence. Thus, based on available evidence, this was proposed to be a conserved mechanism for iron respiration at thermophilic and hyperthermophilic temperatures. However, with the inclusion of the evidence presented within this chapter , it may be possible that these proposed mechanisms were missing one important consideration. The conductivity measurements presente d within this chapter for G. ehrlichii and G. ahangari indicate that both of the hyper(thermophilic) nanowires under investigation are conductive ( 127 and 540 MOhm , respectively). While possibly fortuitous, the identification of nanowires in two physiologi cally - and geographically - distinct iron reducers suggests that microbial nanowires are more widespread than initially though t . TEM micrographs of other hydrothermal vent isolates able to grow on iron oxides show cells in direct contact with the mineral sur face ( 15 - 17 ) , and this may be linked to the presence of extracellular filaments . A thorough investigation of the prevalence of microbial nanowires has yet to be performed in mesophilic iron reducers, and as such this represents the first manuscript to provide data from a multi - species approach to examine the diversity of microbial nanowires. In addition, these represent the first microbial nanowires functioning ou tside of the m esophilic bacteria and the first report of microbial nanowires within the Archaea . The development of a genetic system for either G. ehrlichii or G. ahangari would facilitate additional studies of these nanowires and their involvement in iro n reduction. The pilus - like filament s from G. ahangari are predicted to be produced by GAH_01202 and/or GAH_01671 , yet this is solely based on the presence of a DUF1628 domain, which has been found in pilin proteins throughout the archaea (258) . The development of a genetic system for this model organism would permit mutagenesis assays and gene knockouts to help identify the gene(s) responsible for the production of these pilus - like filaments. However, g iven the sequence similarity between the pilin subunit sequences of G. ehrlichii and the Geobacteraceae (Chapter 2), it is likely that the mechanism present within the Geobacteraceae extends to the closely related 98 Desulfuromonadales . This simil arity presents a unique opportunity for the G. ehrlichii pilin sequence to be heterologously expressed within apiliated strains of G. sulfurreducens (91) or G. metallireducens (82) and examined for their ability t o transfer electrons to iron oxides or the anode of a microbial electrochemical cell. Thus, the use of the nanowires of G. ehrlichii and G. ahangari may serve as a better developmental system for the future of microbial nanowires if I can determine their thermal and chemical stability . The Sypro Orange thermal shift assay presented here is ineffective due to the reasons discussed above, yet other assays may be performed. Determination of the stability of these proteins under a range of stressors would allo w for the customization of nanodevices, employing one of the known microbial n anowires . Environmental sites where the use of nanowire - based nanodevices has been proposed have a range of stressors including pH, surfactants, and temperature (326) . Sim ilarly , the differences in resistance of these nanowires could be utilized for further customization. An additional point of interest regarding these thermophilic and hyperthermophilic nanowires is the fact that they are operational at temperatures 30 6 0 °C below the temperature optimum of the organism s from which they were purified (52, 66) . Enzymatic studies demonstrate increased reaction rates at increasing temperatures (17) . If this trend holds true for the nanowires under investigation, c onductivity measurements performed at thermophilic or hyperthermophilic temperatures on the nanowires of G. sulfurreducens , G. ehrlichii , and G. ahangari ma y provide further insight into their pathway for electron transfer and its stability at elevated temperatures. G. sulfurreducens nanowires, being mesophilic, may show an increase in resistance while those of the (hyper)thermophiles, being thereby closer to their optimal temperature, may increase in conductivity. T he finding that microbial nanowires are a conserved mechanism by which both the Bacteria and the Archaea can interact with and reduce insoluble Fe(III) oxides is of great interest to the field. Th is suggests that these conductive filaments are an ancestral mechanism , rather 99 than a recent acquisition by the Geobacteraceae. Evolution of extracellular appendages from attachment structures to conductive filaments would have been an efficient way for (h yper)thermophiles living in a biofilm environment (29) to ex tend their electroactive surface area and ensure the availability of electron acceptors to support growth . 100 CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS Review of project Dissimilatory iron reduction has been intensively studied in mesophilic microo rganisms, primarily those residing within the bacterial genera Shewanella and Geobacter . Other models for iron reduction are available, yet until recently ( (113, 114, 184 186) and (139) presented within this work) there has been no study of th ermophilic and hyperthermophilic bacteria and archaea. The relatedness of these organisms to the last common ancestor has been established in the literature (32) . Thus, the study of these microorganisms represents a unique opportunity to study the origins of one of the earliest forms of respiration (32) and of life on Earth (5, 20) . These organisms, most often found surrounding geothermally - heated sites, can be a challenge to samp le and culture due to their stringent growth requirements and low growth yields (34) . Yet, much can be learned from the study of hyperthermophiles. The fields of genetics, microbiology, and genomics owe much to a single species Thermus aquaticus . This bacterium was isolated in 1969 from Yellowstone National Park (Wyoming) and g rows at temperatures between 40 °C and 79 °C, with an optimum of 70 °C (327) . Studies on T. aquaticus led to the identification and development of a number of enzymes which are still at the forefront of research in many fi elds (17, 328) ; foremost of which is the DNA polymerase from T. aquaticus which is . Since its discovery (329) , this enzyme has revolution ized the field of genetics and a number of other fields (17, 330) . T. aquaticus demonstrates the power of ther mophilic studies, and their ability to shape our world (17, 328, 331, 332) . If I apply this same formula to other systems it may be possible to reach new heights in almost any field . The general formula which applies, regardless of what enzyme or property is bein g utilized, proc eeds as follows: 1) identify, characterize, and sequence the product of interest within a (hyper)thermophilic organism, 2) heterologously express the 101 thermostable product in a mesophilic organism which is easy to culture and reaches high density ( i.e. E. c oli ), and 3) verify that the properties sought for are still present for the heterologously - expressed product (330) . A pplying this same technique to almost any gene product could enable the expression and mass production o f thermostable gene products. Within this project I have proceeded through the initial stages of this workflow. Selection of potential model organisms was a key initial step. Following the selection of G. ehrlichii and G. ahangari as potential models for thermophilic and hyperthermophilic iron reduction it was possible to examine their mechanisms of iron reduction by both physiological and genomic studies. Finally, key proteins were identified which can, in the future, be applied to bioelectronic devices i n development at Michigan State University to replace or augment the use of homologous, mesophilic, proteins. 6.1.1 Review of Geothermobacter ehrlichii genome studies The genome of G. ehrlichii strain SS015 was targeted for s equencing based on its phylogenetic position (66) , within the Desulfuromonadaceae yet close to the wel l - studied Geobacteraceae family (108) , and due to its phylogenetically - unique ability to grow at thermophilic temperatures (66) . A high - quality draft genome was constructed from a single Illumina library and contains 3.27 Mbp across 84 contigs . Present within these contigs are 3,059 protein - coding genes, 53 RNA genes, and a predicted 60 missing genes. This genome , while not complete, enabled the identification of key components which differentiate G. ehrlichii from the rest of the Desulfuromonadales and others which cement its inclusion within the order. A phylogenetic tree was created using the complete small sub unit r RNA from the genome and populated with the increased diversity within the order, provided key insights into the diversity of the Geothermobacter genus and its position within the Desulfuromonadales . The thermophilic nature of G. ehrlichii was attribu ted to the increased usage of thermostable amino acids and the decreased usage of thermolabile resid u es. These trends were in opposition to those for the psychrophile G. electrodiphilus . 102 Central m etabolic pathways within the genome were identified and a near - complete glycolytic and a complete gluconeogenic pathway were encoded . In addition, a complete TCA cycle was identified within the genome and contained the conserved eukaryotic citrate synthase (156) . A near complete non - oxidative phase of the pentose phosphate pathway was also identified within the genome while the oxidative phase was absent. G. ehrlichii is unable to respire sulfur - containing compounds to support growth (66) . However , a nea r comple te dissimilatory pathway was discovered within the genome in addition to the identified assimilatory pathway. Nitrate reduction has been identified in G. ehrlichii and, based on the work presented here, may proceed in a manner similar to G. metallireducens (77, 3 33) where nitrite is produced by the identified NarGHI gene cluster and immediately consumed, although the enzyme for this could not be definitively determined. Mechanistic studies of dissimilatory iron reduction have been ongoing for years within the Ge obacteraceae (103) . Thus, it was essential to this project to identify, within the G. ehrlichii genome, proteins involved in the respiration of insoluble Fe(III) oxides. A bacterial flagellum was identified and this, comb ined with the presence of numerous chemotaxis gene clusters, may be involved in chemotaxis to iron (102) . In addition, 34 putative c - type cytochromes were identified in the genome which likely f unction as in Geobacter spp . to enable the capacitor - like physiology of the order (174) . Finally, a type IV pilus and associated genes were identified within the genome. This Geopilus is most similar, by sequence similarity, to that of G. metallireducens and is predicted to be involved in binding or electron transfer to insoluble metal oxides. Finally, an EPS gene cluster was discovered between the type IV pilus gene clusters which may function as in G. sulfurreduc ens to facilitate the inclusion of c - type cytochromes into an EPS matrix to facilitate extracellular electron transfer (90) . 6.1.2 Review of Geoglobus ahangari genome studies G. ahangari was chosen for genomic sequen cing due to its obligate iron - reducing nature and its phylogenetic position within a family, the Archaeoglobaceae (52) , which predominantly 103 respire sulfur - containing compounds (334) . G. ahangari (52) , G. acetivorans (178) , and F. placidus (179) are the only members of the Archaeoglobales able to respire inso luble Fe(III) oxides. A complete and finished genome was constructed from Illumina sequences, in collaboration with researchers at Western New England University. This genome, available at GenBank (CP011267), contains 1.77 Mbp, 2,072 genes, 52 RNA coding g enes, and a predicted 47 pseudogenes. Numerous hydrogenases were identified within the genome and these, combined with a near - complete methanogenesis pathway for carbon fixation, provides genomic evidence for the ability of G. ahangari to grow autotrophic ally with hydrogen as an electron donor (52) . A modified Embden - Meyerhof - Parnas glycolytic pathway was identified in the genome and, based on the available sequence data, gluconeogenesis is expected to function. As in F. placidus , the incomplete nature of the oxidative pentose phosphate pathway is predicted to be overcome by th e use of the RuMP pathway (231) . In addition, a complete TCA cycle is present within the genome of G. ahangari , as is conserved among the Archaeoglobales (335) . G. ahangari was the first anaerobic hyperthermophile reported to utilize long - chain fatty acids and completely oxidize them to CO 2 (52) . 39 proteins were identified within the G. ahangari genome that enc ode for - oxidation pathway proteins and likely enable this metabolism. Proteins likely to be involved in extracellular electron transfer, with a focus on c - type cytochromes and cellular components involved in motility and attachment to iron oxides, were a lso identified in the genome of G. ahangari . An archaeal fla1 gene cluster (255) was identified within the genome of G. ahangari to enable the production of an archaell um. This is surprising, as the majority of the family contains fla2 gene clusters (255) , including the only other member of the Geoglobus genus available in pure cultur e (184) . Two putative pilus proteins, GAH_01202 and GAH_01671, were identified within the genome by the presence of a DUF1628 domain (258) . These pilus proteins likely work in conjunction with the 21 genes encoding putative c - type cytochromes to enable extracellular electron transfer by G. ahangari . 104 6.1.3 Review of Geoglobus ahangari iron - respiration mechanism Within t his work I was able to demonstrate that Geoglobus ahangari employs a direct - contact mechanism for the reduction of insoluble Fe(III) oxides . This reduction results in the production of fine - grain magnetite, as shown by electron diffraction data. Addition o f metal chelators or electron shuttles to the culture media stimulated the reduction of the Fe(III) oxides. No electron shuttles or chelators were produced by the cells. Thus, growth was not supported using alginate - entrapped Fe(III) oxides as the sole ele ctron acceptor unless an exogenous el ectron shuttle was added. T he addition of AQDS, the electron shuttle, enabled growth on these entrapped oxides and resulted in the accumulation of a biofilm on the bead surface in addition to supporting a planktonic pop ulation. The presence of a large electron shuttle, which would be unable to enter the pores in the alginate beads, was disproven by spent culture supernatant having no spurring effect on freshly inoculated G. ahangari cultures. In addition, no significant chelation of the Fe(III) oxides was shown in culture tubes unless an exogenous chelator was provided. Thus, I proposed that a direct - contact mechanism was employed by G. ahangari to enable the respiration of insoluble metal oxides. Consistent with the prop osed direct contact mechanism is the presence of numerous proteins which could facilitate extracellular electron transfer. Transmission electron microscopy analysis of cultures of G. ahangari show cells in direct contact, and encased in many cases, with Fe (III) oxides. Dissolution of the metal oxides revealed two different morphologies of extracellular filaments. One, the archaellum identified in the genome (Chapter 3), is predicted to function solely for motility. In contrast, numerous curled pilus - like fi laments were found on soluble and insoluble Fe(III) oxide grown cultures . G. ahangari cells also, identified by a heme - stained SDS - PAGE gel, produced a number of h eme - containing bands , several of which were able to be sheared from the outer surface of the cell. Mechanical shearing of outer - surface proteins, including the predicted outer - surface c - type cytochromes, resulted in the inability of cells to grow on insoluble, 105 but not soluble, Fe(III) oxides. These data thus supported the proposed mechanism that t he archaellum provides a motility apparatus to locate iron oxides in the environment, the pilus - like filaments facilitate attachment and electron transfer, and the cytochromes act as the terminal reductase. 6.1.4 Review of (hyper)thermophilic microbial nanowires Previous data suggested that a direct - contact mechanism permitted the respiration of Fe(III) oxides in both G. ehrlichii and G. ahangari. Due to the presence of extracellular filaments in these organisms their biochemical properties and contributions to extracellular electron transfer were assessed. Mechanical shearing of both species produced crude purifications of extracellular filaments. Both the motility apparatus and the putative nanowires were purified in this matter, concentrated, and visualized by both TEM and AFM. The pili of G. ehrlichii were numerous and ca.2 - 2.5 nm in diameter while the flagella were much larger, ca. 30 nm. The pilus - like filaments from G. ahangari, which were again the more numerous of the filament morphologies, were ca. 2.5 nm in diameter and present a curli - like morphology as seen by TEM and AFM. Incubation of these curli - like filaments at 4 °C produced, after 72 h, protein aggregates which are hypothesized to be am yloid in nature. The archaella produced by G. ahangari are r elatively straight and have a diameter of ca. 4 nm. The use of a preparatory gel (92) to purify these filaments to homogeneity resulte d in highly - purified samples containing only the pilus and pilus - like filaments, demonstrating their SDS insolubility. These purification techniques provided sufficient sample to determine if, like G. sulfurreducens , these filaments function ed as microbi al nanowires in G. ehrlichii and G. ahangari. From the mechanical shearing protocol it was determined that the motility structures from both organisms were insulating. This provided an internal control to ensure that contaminant metals were not contributin g significantly to the conductivity of the putative nanowires. When the more numerous cellular filaments were assayed by CP - AFM both of these filaments were shown to be conductive ( 37,458 and 266 MOhms for G. ehrlichii and G. ahangari , respectively ) . Purif ied 106 samples obtained from the prep - cell protocol retained their conductivity ( 127 and 540 MOhms for G. ehrlichii and G. ahangari , respectively ) . This indicates that contaminant metal ions and proteins were not the cause of the detected conductivity, and, i n the case of G. ehrlichii, contaminants caused an insulating effect. These filaments are predicted to increase the electroactive surface area available to the cell (91) , an ability thought to be evolut ionarily beneficial in biofilm and subsurface environments where soluble compounds could easily be lost (102, 139) . Discovery of additional microbial nanowires expands our knowledge regarding the diversity of this mechanism of extracellular electron transfer. Additionally, du e to the increased growth temperatures of both G. ehrlichii (66) an d G. ahangari (52) , the thermal stability of cell proteins and the filame nts was assessed by the Sypro Orange thermal shift as say. Unfortunately, the thermal and chemical stability of the nanowires under investigation, in addition to the pili of G. metallireducens , could not be assessed due to limited fluorescent output attribu ted to fluorescence quenching, protein aggregation, or low protein concentrations. However, soluble cell extracts were produced from the mesophile G. sulfurreducens in addition to the two (hyper)thermophilic species under investigation and provided reprodu cible results. As expected, the thermal stability of these cell extracts approximately matched the upper limit of growth temperatures for these organisms, with the exception of G. ahangari which is expected to stabilize its proteins in vivo . Future direct ions 6.2.1 Geoglobus ahangari As discussed in Chapter 3, the protein - coding gene (s) for the pilus - like filaments of G. ahangari has been hypothesized but has not been confirmed by biochemical methods. Pending the development of a genetic system for this archaeon , a method to depolymerize the nanowires into their individual subunits must be elucidated. If taken from prep - cell samples, which are assumed to be comprised solely of the nanowires, a single band may be obtained by SDS - PAGE 107 analysis and stained using a h igh - sensitivity protein stain such as the Pierce Silver Stain Kit (Life Technologies). Yet, the predicted thermal stability of these filaments, which must also be successfully assessed, combined with their proposed curli - like nature will likely cause diff iculties with their depolymerization (17, 316) . I f this can be overcome , it may be possible to obtain the protein sequence of the subunit and, due to the availability of the sequenced genome, match peptide fragments obtained by m atrix - assisted lase r desorption/ionization (MALDI - TOF) of trypsin - digested subunits to proteins encoded within the genome. The production of protein aggregates after extended incubation at 4 °C by the pilus - like filaments of G. ahangari is a promising preliminary result to i dentifying the first amyloid - forming protein in an archaeon. However, th e available data, which include their curled appearance by TEM and AFM, do not provide sufficient evidence to definitively make this claim. Within this work, the use of Congo Red and T hioflavin T as amyloid dyes has been proposed. These dyes, when bound to amyloid - like beta sheets, exhibit altered spectra (317) . Incubation of the pilus - like filaments from G. ahangari at 4 °C for 72 h, as previously demonstrated to be sufficient for aggregates to form, in the presence of these dyes would provide sufficient evidence to cement the cur li - like nature of these filaments. 6.2.2 Geothermobacter ehrlichii Due to the phylogenetic and physiological similarities between G. ehrlichii and the intensely studied members of the Geobacteraceae (66) , there exist a staggering number of possibilities for future studies in G. ehrlichii . Heterologous expression of the pilin ge ne in G. sulfurreducens , for example, would enable comparison of current production in microbial electrochemical cells, where the contribution of the pilus is expected to be important for electron transfer. Changes in current production would indicate that the pili of G. ehrlichii are more or less conductive than those of G. sulfurreducens when expressed heterologously. In addition, a similar strategy could be employed to determine if the EPS operon identified in the G. ehrlichii genome could comple ment the lack of the Xap operon of G. sulfurreducens when grown on insoluble 108 Fe(III) oxides. In additio n to these studies, the thermal and chemical stability of the pili must be assessed. While the genome of G. ehrlichii presented within this work has been suffici ent to identify complete metabolic pathways and components critical to iron respiration, it is not closed or finished. Genome reconciliation attempts by Minimus2 (130) have been successful, but must continue. In addition, additional sequencing must be attempted by next - generation sequencing te chnologies or using the primer - based approach that is currently underway. The closure of the genome, while not essential, would strengthen the use of G. ehrlichii as the model thermophile within the Desulfuromonadales . Looking forward While the data prese nted within this work have shed light on a possible mechanism for the origins and evolution of ancient (hyper)thermophiles able to grow on and respire insoluble Fe(III) oxides, this has been discussed in depth previously. Looking forward, instead of back i n time to early Earth, these nanowires may be critical components in the next generation of biomimetic nanodevices. Following successful heterologous expression in a mesophilic strain which is able to produce the nanowires in high numbers, as is in process for the pilus nanowires of G. sulfurreducens , it will be possible to examine their biochemical and electrochemical capabilities with greater ease. Self - assembly of the nanowires into films or as an assembly would permit their use in all systems being deve loped for G. sulfurreducens . While not expressly proven within this work, these thermophilic filaments are exp ected to have increased thermal and chemical stabilities over those currently in use. Thus, if they can be introduced into the current nanodevice framework, these nanowires will likely have a broader range of applicability in bioremediation sites where conditions are often far from pH 7.0 and 25 °C. Such applications are beyond the scope of this work, but researchers are only now beginning to scratc h the surface in terms of applications 109 for these novel nanowires. The future is bright, and may someday be powered by (hyper)thermophiles and microbial nanowires. 110 APPENDICES 111 APPENDIX A TABLES 112 Table 2.1 . Class ification and general features of G. ehrlichii MIGS ID Property Term Evidence code a Current classification Domain Bacteria TAS (18) Phylum Proteobacteria TAS (121) Class Deltaproteobacteria TAS (122, 123) Order Desulfuromonadales TAS (123) Family Desulfuromonadaceae TAS (108) Genus Geothermobacter TAS [1] Species Geothermobacter ehrlichii TAS [1] Type strain SS015 TAS [1] Cell shape Rod TAS [1] Motility Mo tile TAS [1] Sporulation Non - sporulating NAS Temperature range 35 - 65 °C (optimum 55 °C) TAS [1] Salinity range 5.0 - 8.0 (optimum 6.0) TAS [1] Salinity 5.0 - 50 g/L (optimum 19 g/L) TAS [1] MIGS - 22 Oxygen requirement Anaerobe TAS [1] Carbon so urce Organic acids, CO 2 TAS [1] Energy metabolism chemoorganotrophic TAS [1] MIGS - 6 Habitat Marine geothermally heated areas TAS [1] MIGS - 15 Biotopic relationship Free - living TAS [1] MIGS - 14 Pathogenicity None NAS Biosafety level 1 NAS Isolatio n TAS [1] 113 MIGS - 4 Geographic location Axial Seamount - Juan de Fuca Ridge TAS [1] MIGS - 5 Isolation time Unknown MIGS - 4.1 Latitude 46° N TAS [1] MIGS - 4.2 Longitude 130° W TAS [1] MIGS - 4.3 Depth 1400 m TAS [1] MIGS - 4.4 Altitude Not applicable a) Evidence codes IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non - traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project (124 ) . Classification and general features are a ccording to the MIGS recommendations (120) . 114 Table 2. 2 . Genome sequencing project information for the genome of G. ehrlichii MIGS ID Property Term MIGS - 31 Finishing quality High - quality draft MIGS - 28 Libraries used 2 independent 100 bp paired - end Illumina shotgun libraries, and 1 Pacific Biosciences library run on a single SMRT cell MIGS - 29 Sequen cing platforms Illumina MiSeq, PacBio MIGS - 31.2 Sequencing coverage 300 × coverage (Illumina libraries), 600 × coverage (Pacific Biosciences library) MIGS - 30 Assemblers Velvet, Minimus2 MIGS - 32 Gene calling method RAST INSDC ID N/A Genbank Date of Release N/A GOLD ID N/A NCBI project ID N/A MIGS - 13 Source material identifier ATCC BAA - 635 , DSMZ DSM - 15274 Project relevance Phylogenetic diversity, biotechnology, metal respiration in thermophiles, and microbial nanowires 115 Table 2. 3 . As sembly reconciliation statistics for the G. ehrlichii genome 1S + 2S 2S + PacBio 1S + PacBio (1S + 2S) + PacBio # contigs (>= 0 bp) 38 23 21 19 # contigs (>= 1000 bp) 37 23 21 19 Total length (>= 0 bp) 2,997,257 3,307,005 3,254,057 3,070,722 Total le ngth (>= 1000 bp) 2,996,328 3,307,005 3,254,057 3,070,722 # contigs 38 23 21 19 Largest contig 386,328 676,668 721,357 782,787 Total length 2,997,257 3,307,005 3,254,057 3,070,722 GC (%) 61.9 61.75 61.8 61.87 N50 121,299 228,051 227,920 234,024 N75 9 2,840 128,803 128,724 126,606 L50 7 5 5 4 L75 13 9 9 8 # N's per 100 kbp 2.64 1.15 2.09 2.77 116 Table 2. 4 . Primers used for gap closure of the G. ehrlichii genome Primer - Amplification Geh_A+H_01_S CTG CCA TCA CCA AGG TTC TT Off of the 5 Geh_A+H_02_S TGA TCC CCA TGT ATC TGC TG Geh_A+H_03_S GTT TCG GTC ACC TTC TGC TG Geh_A+H_04_S CGG ACG GTA ACC TTT GAA GA Geh_A+H_05_S ACC GAC ACC AAC AGC AAA GT Geh_A+H_06_S GCA GTT GTT GGC GTA GAG AG Geh_A+H_07_S GGC AAC ATC CTC TGC TAC ATC Geh_A+H_08_S GTG TTG AAA GCA CGA CCA AA g 8 Geh_A+H_09_S GTC ACC GGC GTC TAC TAT CC Geh_A+H_10_S AGG GTG ACG AAC AAC AGC TT Geh_A+H_11_S ATC AGG CTC CAG GTC TCC TT Geh_A+H_12_S ATG ACC TTC AGC TCG GTC AG Geh_A+H_13_S AGA AAA TTT GGC AGC CTT CA Geh_A+H_14_S CCT GCG GTG TAA GAT GTG TG Geh_A+H_15_S GGC CAA CAA GTT CAA AGA GC Geh_ A+H_16_S CGC CTT ACT CTT TGG TGC TC Geh_A+H_17_S ACA ACA GCG TCA GGA GGA CT Geh_A+H_18_S AGA CCG TAC TTG GCC TTG TG Geh_A+H_19_S CAC CGC AAC GAT TTC ATC AT Off Geh_A+H_20_S GAA GAG ATC CGG GAG GAA GT Geh_A+H_21_S CAG ATC GCT CTC TTC GAT CC Geh_A+H_22_S CCT CTA GTC ATC CCT GGC TTT 117 Table 2.4 Geh_A+H_23_S GGC CTC GTC CAC TAT CTT GA Geh_A+H_01_E TCA TCG AGG GGA TCA AGA AG Geh_A+H_02_E AAA ACC AAC GCC TGA AAA TG Geh_A+H_03_E AGG TCG CAA GCA AAA TCA AA Geh_A+H_04_E GTT CAA GGC CTT TGA CCA GA Geh_A+H_05_E CCT GGC TTC ATC GAA CAC TT Geh_A+H_06_E AAG CTC GAA GCT GAA CCA AA G eh_A+H_07_E GTT CAT CAC CCC CGG ATA CT Geh_A+H_08_E GGG GAT GAA CCG ATT TCT TT Geh_A+H_09_E GTT GAT CGC CAG GAT GAA GT Geh_A+H_10_E TTT GAA GAA CGG GGT GTG AC Off Geh_A+H_11_E AGG TCG CAA ACC ATC AAA AG Geh_A+H_12_E AAA GTC GCT GTA TGC CAG GT Geh_A+H_13_E GAT CGA AGA GAG CGA TCT GG Geh_A+H_14_E GCC ATC GAC GGC AAT ATC Geh_A+H_15_E CGA GAA CCA GGA GAA TGA GC Geh_A+H_16_E CGG TTT TGC TTT TGA TGG TT Geh_A+H_17_E GGG GCT TCT GGT AGT GTT GA nd of contig 17 Geh_A+H_18_E CGA GTG CAT CAG CAC CAC Geh_A+H_19_E CTG GCT GTT GGT GTG CAG Geh_A+H_20_E CAG ACC TTC GTG GCA GAA AT Geh_A+H_21_E CAA GGT GGT TGT CGA GGT CT Geh_A+H_22_E TTG GGC ACT GTC TCA ACA AG Geh_A+H_23_E GAT CAA CAC CGA CCC TGA AG 118 Table 2. 5 . Nucleotide content and gene count levels of the G. ehrlich ii genome Attribute Value % of total a Size (bp) 3,276,179 100.0% Coding region (bp) 2,845,273 86.8% G+C content (bp) 2,022,946 61.8% Number of replicons 1 Extrachromosomal elements 0 Total genes 3 , 112 100.0% RNA genes 53 1.7% rRNA operons 1 Protein - coding genes 3 , 059 100.0% Pseudogenes 115 3.7% Genes with function prediction 2 , 289 74.8% Genes assigned to KEG pathways 1 , 580 51.7% Genes assigned Pfam domains 2 , 496 81.6% Genes with signal peptides 206 6.7% Genes with transmembrane helic es 709 23.2% CRISPR repeats 2 a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome. 119 Table 2. 6 . Number of genes within G. ehrlichii associated with the 27 subsystem categories in RAST Value %age a Description 296 9.7% Amino acids and d erivatives 265 8.7% Protein m etabolism 259 8.5% Cofactors, vitamins, prosthetic groups, and p igments 219 7.2% Carbohydrates 177 5.8% Respiration 156 5.1% RNA m eta bolism 118 3.9% Membrane t ransport 108 3.5% Motility and c hemotaxis 105 3.4% Fatty acids, lipids, and i soprenoids 102 3.3% DNA m etabolism 94 3.1% Stress r esponse 86 2.8% Virulence, disease, and d efense 79 2.6% Cell w a ll and c apsule 72 2.4% Nucleosides and n ucleotides 33 1.1% Cell division and cell c ycle 33 1.1% Phosphorus m etabolism 28 0.9% Nitrogen m etabolism 27 0.9% Miscellaneous 21 0.7% Sulfur m etabolism 19 0.6% Potassium m etabolism 19 0.6% R egulation and cell s ignaling 120 4 0.1% Secondary m etabolism 4 0.1% Metabolism of aromatic c ompounds 3 0.1% Dormancy and s porulation 1 0.0% Iron acquisition and m etabolism 0 0.0% Photosynthesis 0 0.0% Phages, prophages, transposable e lemen ts, and p lasmids 731 23.9% Not in s ubsystems 121 Table 2. 7 . Putative c - type cytochromes identified in the genome of G. ehrlichii Feature id Best hit by BLAST H eme motifs Final MW (kDa) peg.33 Cytochrom_c 3 superfamily protein 5 21.6 peg.301 Cytochrom_c 7 superfamily protein 9 72.9 peg.357 Hypothetical protein 2 16.9 peg.481 Cytochrome c oxidase 1 14.2 peg.491 Cytochrome c family protein 1 8.9 peg.499 DUF3365 family protein 1 19.1 peg.569 Decaheme c - type cytochrome / Lipoprotein cytochrome c 1 77 peg.622 Hydroxylamine reductase / Cytochrome c family protein 13 104 peg.623 Hydroxylamine reductase / Cytochrome c family protein 10 35 peg.652 Cytochrome c fami ly protein 1 15.7 peg.707 Cytochrome c family protein 3 13.1 peg.810 Cytochrome c family protein 23 83.4 peg.812 Cytochrome c family protein 8 38.8 peg.814 Cytochrome c family protein 4 23.6 peg.874 Hydroxylamine oxidoreductase 8 53.2 peg.1066 Cytoch rome c family protein 4 22.4 peg.1403 Cytochrome c family protein 4 30.4 peg.1404 Cytochrome c family protein 7 43.1 peg.1443 Cytochrome c family protein 11 57.5 122 peg.1444 Cytochrome c family protein 5 18.3 peg.1570 Hydroxylamine reductase 9 51.6 peg.1636 Cytochrome c peroxidase 1 10.4 peg.1714 Cytochrome b 1 9.6 peg.2110 Cytochrome c family protein 9 32 peg.2155 Cytochrome c family protein 19 244.2 peg.2156 Cytochrome c family protein 57 310.5 peg.2157 Cytochrome c family pr otein 4 29.6 peg.2276 Cytochrome cbb 3 1 15.3 peg.2316 Hypothetical protein 1 16.3 peg.2495 Octaheme tetrathionate reductase 8 54.5 peg.2497 Cytochrome c family protein 1 10.4 peg.2725 Hypothetical protein / DnaJ 4 11.6 peg.2734 Cytochrome cbb 3 2 29.8 peg.2766 Cystathione beta synthase 7 31.6 123 Table 3.1 . Classification and general features of G. ahangari MIGS ID Property Term Evidence code a Current classification Domain Archaea TAS (18) Phylum Euryarchaeot a TAS (18, 336) Class Archaeoglobi TAS (337) Order Archaeoglobales TAS (335) Family Archaeoglobaceae TAS (334) Genus Geoglobus TAS (52) Species Geoglobus ahangari TAS (52) Type strain 234 T TAS (52) Gram stain Variable NAS Cell shape Irregular coccus TAS (52) Motility Motile TAS (52) Sporulation Non - sporulating NAS Temperature range 65 - 90 °C TAS (52) Optimal temperature 88 °C TAS (52) pH range; Optimum 5.0 - 7.6 ( optimum 7.0) TAS (52) Carbon source CO 2 TAS (52) Energy metabolism Chemolithoautotrophic, chemolithotrophic, chemoorganotrophic TAS (52) MIGS - 6 Habitat Marine geothermally heated areas TAS (52) MIGS - 6.3 Salinity 9.0 - 38 g/L NaCl TAS (52) MIGS - 22 Oxygen requirement Anaerobe TAS (52) 124 MIGS - 15 Biotopic relationship Free - living TAS (52) MIGS - 14 Pathogenicity Non - pathogen NAS Isolation Hydrothermal vent chimney TAS (52) MIGS - 4 Geographic location Guaymas Basin hydrothermal system TAS (52) MIGS - 5 Sample collection time Unknown NAS MIGS - 4.1 Latitude 27° N TAS (52) MIGS - 4.2 Longitude 111° W TAS (52) MIGS - 4.3 Depth 2000 m TAS (52) MIGS - 4.4 Altitude Not applicable a) Evidence codes IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non - traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project (124) . Classification and general features are based on the MIGS recommendations (120) . 125 Table 3.2 . Genome sequenc ing project information of G. ahangari MIGS ID Property Term MIGS - 31 Finishing quality Finished MIGS - 28 Libraries used 5 independent 100 bp paired - end Illumina shotgun libraries, 150 bp paired - end Illumina shotgun library MIGS - 29 Sequencing platforms Illumina MiSeq MIGS - 31.2 Sequencing coverage 1,977 × coverage (100 bp libraries) 100 × (150 bp library) MIGS - 30 Assemblers SeqMan NGen, Velvet, SeqMan Pro MIGS - 32 Gene calling method JGI - ER, GLIMMER INSDC ID CP011267 Genb ank Date of Release May 11, 2015 GOLD ID Gp0101274 NCBI project ID 258102 MIGS - 13 Source material identifier ATCC BAA - 425 , DSMZ DSM - 27542 , JCM JCM 12378 Project relevance Phylogenetic diversity, biotechnology, evolution of metal respi ration in hyperthermophiles, and anaerobic degradation of hydrocarbons 126 Table 3.3 . Nucleotide content and gene count levels of the G. ahangari genome Attribute Value % of total a Size (bp) 1,770,093 100.0 % Coding region (bp) 1,662,832 93.9% G+C c ontent (bp) 940,071 53.1% Number of replicons 1 Extrachromosomal elements 0 Total genes 2072 100.0% RNA genes 52 2.5% rRNA operons 2 Protein - coding genes 2020 100.0% Pseudogenes 47 2.3% Genes with function prediction 1677 83.0 % Genes in para log clusters 1406 69.6 % Genes assigned to COGs 1470 72.8 % Genes assigned Pfam domains 1667 82.5 % Genes with signal peptides 55 2.7% Genes with transmembrane helices 409 20.2 % CRISPR repeats 7 a) The total is based on either the size of the genome i n base pairs or the total number of protein coding genes in the annotated genome. 127 Table 3.4 . Number of genes in G. ahangari associated with the 25 general COG functional categories Code Value %age a Description J 155 7.6% Translation, riboso mal structure and biogenesis A 2 0.1% RNA processing and modification K 68 3.3% Transcription L 58 2.9% Replication, recombination and repair B 6 0.3% Chromatin structure and dynamics D 20 1.0% Cell cycle control, Cell division, chromosom e partitioning Y 0 0.0% Nuclear structure V 7 0.3% Defense mechanisms T 24 1.2% Signal transduction mechanisms M 35 1.7% Cell wall/membrane biogenesis N 15 0.7% Cell motility Z 0 0.0% Cytoskeleton W 0 0.0% Extracellular structur es U 23 1.1% Intracellular trafficking and secretion O 57 2.8% Posttranslational modification, protein turnover, chaperones C 160 7.9% Energy production and conversion G 37 1.8% Carbohydrate transport and metabolism E 139 6.8% Amino aci d transport and metabolism F 49 2.4% Nucleotide transport and metabolism H 103 5.1% Coenzyme transport and metabolism I 55 2.7% Lipid transport and metabolism 128 P 86 4.2% Inorganic ion transport and metabolism Q 16 0.8% Secondary metabolites biosynthesis, transport and catabolism R 244 12.0% General function prediction only S 198 9.7% Function unknown - 477 23.5% Not in COGs a) The total is based on the total number of protein coding genes in the genome 129 Table 3.5 . Terminal electron acceptors in the Archaeoglobales Electron acceptors Organism Sulfate Sulfite Thiosulfate Nitrate Fe(III) Geoglobus spp. - - - - + Ferroglobus placidus - - + + + Archaeoglobus spp. +/ - + + - - 130 Table 3.6 . Putative c - type cytochromes in G. ahangari Gene ID: Annotation: # of heme binding motifs: Calculated molecular weight: TM domains: GAH_00015 Hypothetical protein 4 58.4 0 GAH _00283 Cytochrome c7 4 21.2 a 1 GAH_00286 Nitrate/TMAO reductases, membrane - bound tetraheme cytochrome c subunit 12 39.1 0 GAH_00301 Putative redox - active protein (C_GCAxxG_C_C) 2 31.5 3 GAH_00504 Hypothetical protein 10 54.5 1 GAH_00505 Hypothetical pr otein 4 26.8 2 GAH_00506 Cytochrome c3 9 48.6 0 GAH_00507 Cytochrome c7 4 27.4 a 1 GAH_00508 Hypothetical protein 5 28.5 1 GAH_00510 Hypothetical protein 4 27.3 1 GAH_00817 Seven times multi - haem cytochrome CxxCH 8 53.7 1 GAH_01091 Hypothetical protei n 1 11.7 1 GAH_01235 Hypothetical protein 5 21.5 0 GAH_01236 Hypothetical protein 5 22.3 b 0 GAH_01253 Hypothetical protein 4 16.9 0 GAH_01256 NapC/NirT cytochrome c family, N - terminal region 10 43.6 a 1 131 GAH_01296 Cytochrome c fam ily protein 4 17.2 1 GAH_01297 Seven times multi - haem cytochrome CxxCH 8 61.0 1 GAH_01306 Class III cytochrome C family 8 46.3 0 GAH_01534 Hypothetical protein 1 18.5 1 GAH_01700 Hypothetical protein 3 9.9 0 a) No signal peptide detected b) Signal peptide d etected by PRED - SIGNAL 132 Table 3.7 . Uniquinone and menaquinone biosynthesis proteins in G. ahangari Protein name Abbreviation Homologs in G. ahangari 1,4 - dihydroxy - 2 - naphthoate octaprenyltransferase M enA GAH_01919 N aphthoate synthase M enB GAH_01602, GAH _00487, GAH_01332 C horismate dehydratase M qnA GAH_00872 C yclic dehypoxanthinyl futalosine synthase M qnC GAH_00873, GAH_00871, GAH_00663 1,4 - dihydroxy - 6 - naphthoate synthase M qnD GAH_02003 A minodeoxyfutalosine synthase M qnE GAH_00871, GAH_00873, GAH_0066 3 4 - hydroxybenzoate polyprenyltransferase M biA GAH_00304, GAH_00157 P henylphosphate carboxylase subunit beta U biD GAH_01625, GAH_00517 4 - hydroxy - 3 - polyprenylbenzoate decarboxylase U biD GAH_01570 UbiD family decarboxylase U biD GAH_01625 Demethylmenaqui none M ethyltransferase UbiE/M enG GAH_00100, GAH_01336, GAH_00796 133 Table 3.8 . Fe - S binding domain proteins and ferredoxins within the genome of G. ahangari Locus Tag Gene Product Name GAH_00040 Ferredoxin - like domain protein GAH_00110 Uncharacterized Fe - S oxidoreductase GAH_00113 Fe - S oxidoreductase GAH_00114 Predicted Fe - S oxidoreductases GAH_00137 Fe - S oxidoreductase GAH_00138 Fe - S oxidoreductase GAH_00140 Iron - sulfur cluster - binding protein GAH_00158 Ferredoxin - like domain protein GAH_00165 P redicted Fe - S oxidoreductases GAH_00413 Aldehyde:ferredoxin oxidoreductase GAH_00488 Electron transfer flavoprotein beta GAH_00537 Heterodisulfide reductase, subunit A and polyferredoxins GAH_00538 Iron - sulfur cluster - binding oxidoreductase GAH_00570 Pyruvate ferredoxin oxidoreductase GAH_00615 Uncharacterized Fe - S protein PflX GAH_00622 Radical SAM domain iron - sulfur cluster - binding oxidoreductase with cobamide - binding - like domain GAH_00678 Predicted Fe - S - cluster oxidoreductase GAH_00684 Ferredox in GAH_00822 Fe - S oxidoreductase GAH_00845 Ferredoxin GAH_00886 Fe - S oxidoreductase 134 GAH_00924 Ferredoxin GAH_01011 Indolepyruvate ferredoxin oxidoreductase GAH_01136 4Fe - 4S binding domain/Putative Fe - S cluster GAH_01180 Iron - su lfur cluster - binding oxidoreductase GAH_01225 Ferredoxin GAH_01254 Fe - S - cluster - containing hydrogenase GAH_01276 Fe - S oxidoreductase GAH_01286 Fe - S - cluster - containing hydrogenase GAH_01295 Fe - S - cluster - containing hydrogenase GAH_01344 Predicted F e - S oxidoreductase GAH_01351 Uncharacterized Fe - S center protein GAH_01440 Ferredoxin GAH_01646 Ferredoxin GAH_01669 Ferredoxin GAH_01685 Rubredoxin GAH_01686 Uncharacterized flavoproteins GAH_01728 Aldehyde:ferredoxin oxidoreductase GAH_01737 P redicted Fe - S oxidoreductases GAH_01738 Aldehyde:ferredoxin oxidoreductase GAH_01822 Aldehyde:ferredoxin oxidoreductase GAH_01856 Aldehyde:ferredoxin oxidoreductase GAH_01866 Ferredoxin - thioredoxin reductase 135 GAH_01870 Fe - S oxidored uctase GAH_01921 Dehydrogenases (flavoproteins) GAH_01948 Ferredoxin GAH_01960 Ferredoxin GAH_01981 Fe - S oxidoreductase GAH_02012 Pyruvate ferredoxin oxidoreductase GAH_02033 Heterodisulfide reductase, subunit A 136 APPENDIX B FIGURES 132 Figure 1 . 1 . Geologic timescale of the Precambrian Supereon Taken from the Geological Society of America ® and can be found at the following web address: http://www.geosociety.org/science/timescale/timescl.pdf 133 Figure 1 . 2 . Location of the Axial Seamount and the layout of the Axial Seamount caldera Location of the Axial Seamount in relation to the continental United States (A) and the layout of the Axial Seamount caldera (B). Image A is taken from the Monterey Bay Aquarium Research Institute and can be found at the web address http://www.mbari.org/news/homepage/2012/axial - mapping/axial - locationmap - 350.jpg . Image B is taken from the Interactive Oceans website and can be found at the web address http://w ww.interactiveoceans.washington.edu/files/axial.seamount.webcopy_med.jpg and has been altered to include the approximate location of the Bag City site. Bag City S ite 134 Figure 1 . 3 . Location of the Guaymas Basin Location of the Axial Seamo unt in relation to the continental United States, the Baja Peninsula, and Mexico. Image taken from the University of California Museum of Paleontology and can be found at the web address http://www.ucmp.berkeley.edu/images/science/profiles/guaymas.jpg 135 Figu re 2 . 1 . Phylogenetic tree of G. ehrlichii Tree c onstructed with the maximum likelihood algorithm comparing the 16S rRNA gene sequence from G. ehrlichii strain SS015 to other members of the Des ulfuromonales. Bootstrap values were determined from 100 replicates and Escherichia coli was used as an outgroup. 136 Figure 2 . 2 . Transmission electron micrograph of G. ehrlichii Cultures were g rown on soluble Fe(III) oxides. Bar, 500 nm. 137 Fig ure 2 . 3 . Amino acid usage of G. ehrlichii and G. electrodiphilus in comparison to mesophilic members of the Geobacteraceae Amino acid usage of the psychrophile G. electrodiphilus ( white ) and the thermophile G. ehrlichii ( black ) compared to the averaged a mino acid usage of examined mesophilic Geobacter spp (gray) . Protein sequences were obtained from NCBI. Solid bar at bottom indicates amino acids which are implicated in increasing thermal stability while those indicated by the dashed line indicate amino a cids implicated in reducing thermal stability. 138 Figure 2 . 4 . Ratio of charged to polar amino acids from G. ehrlichii, G. electrodiphilus, and mesophilic members of the Geobacteraceae Charged to polar amino acid ratios of the psychrophile G. electr odiphilus ( white ) and the thermophile G. ehrlichii ( black ) compared to the averaged ratio of mesophilic Geobacter spp (gray) . Protein sequences were obtained from NCBI. 139 Figure 2 . 5 . Central metabolism in G. ehrlichii 140 Figure 2 . 6 . Sequence rel atedness of the pilin subunit protein to members of the Desulfuromonadales 141 (A) Alignment of the pilin subunit of G. ehrlichii strain SS015 to members of the Geobacteraceae and Desulfuromonadaceae generated using the Muscle alignmen t tool (167) within MEGA 6.0 (168) . (B) Graphical representation (169) of the multiple sequence alignment highlighting the conserved N - terminus region. (C) Phylogenetic tree deri ved from the multiple sequence alignment created using the Maximum Likelihood algorithm within MEGA 6.0 (168) and bootstrap values represent 100 replicates. 142 Figure 2 . 7 . SDS - PAGE gel of sheared proteins stained with the TMBZ protocol H eme - stained S DS - PAGE of outer - surface proteins sheared from the cell surface (lane 2 ) , sheared cells (lane 3), and whole - cell extracts from G. ehrlichii (lane 4). Lane 1 shows prot ein markers and numbers at left show their corresponding molecular weights in kDa. The Novex molecular weight marker used is shown (left). Black boxes indicate id entified cytochromes 143 Figure 3 . 1 . Phylogenetic tree of G. ahangari The phylogenetic tree was constructed with the maximum likelihood algorithm comparing the 16S rRNA gene sequence from G. ahangari to other hyperthermophilic archaea. Bootstrap values were Aquifex aeolicus Pseudothermotoga thermarum were used as outgroups. 144 Figure 3.2 . Scanning electron micrograph of cells of G. ahangari Cells were grown on insoluble Fe(III) oxides. Bar, 100 nm. 145 Figure 3.3 . Graph ical circular map of the G. ahangari genome From outside to the center: Genes on forward strand (colored by COG categories), genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, and GC ske w. 146 Figure 3.4 . Central metabolism in G. ahangar i 147 Figure 4.1 . Effect of electron shuttles or metal chelators on the reduction of Fe(III) oxides by G. ahangari (A) Stimulation of Fe(III) oxide reduction (calculated as HCl - extractable Fe(II)) with 50 µM of the electron shuttle AQDS (squares) and 4 mM of the metal chelator NTA (triangles) in reference to cultures with no additions (circles). Controls lacking electron donor (pyruvate, 10 mM) are also shown (open symbols). Inset shows electron diffraction pattern of the reduced mineral, which is consistent with magnetite. (B) Doubling times (calculated from the rates of HCl - extractable Fe(II) production) as a function of the AQDS concentration present in the medium. Horizontal dashed line marks the doublin g time in cultures without AQDS. (C) Soluble Fe(II) and Fe(III) in filtered supernatant fluids from stationary phase (ca. 17 mM of HCl - extractable Fe(II)) cultures with no additions or supplemented with NTA). All data points show averages and standard devi ations of five replicates. 148 Figure 4.2 . G. ahangari lacks the ability to produce endogenous electron shuttles (A) Growth (dashed lines) and Fe(III) reduction (solid lines) of G. ahangari with entrapped Fe(III) oxides in the presence (squares) or absence (circles) of 50 uM AQDS. Controls lacking electron donor (pyruvate) are also shown (open symbols). The data points represent averages and standard deviations of triplicate cultures. (B) CLSM micrographs of biofilms formed on the Fe(III) oxi des exposed on t cells in red. Bar, 50 µm. (C) Reduction of free Fe(III) oxides (10 mM) by washed cells resuspended in filter - sterilized supernatant flu ids with (squares) or without (circles) AQDS. 149 Figure 4.3. TEM micrographs of negatively - stained cells of G. ahangari C ultures were grown with free Fe(III) oxides (A) or Fe(III) citrate (B and C). Partial solubilization of cell - associated iron precipit ates with oxalate reveals abundant filaments, some still associated with the mineral particles (B). At higher magnification, the c archaeal flagellum (arrow labeled Fl ) and curled filaments (arrow labeled Cu ) are revealed (C). 150 Figure 4.4 . Surface - exposed c - type cytochromes are essential for the reduction of insoluble Fe(III) oxides (A - B) Coomassie Blue - stained (A) and heme - stained (B) SDS - PAGE of whole - cell (lanes 1 - 4, decreasing concentrations of protein) and sheared outer surface (lane 6) prot ein extracted from G. ahangari . Lane 5 shows protein markers and numbers at right (top) show their corresponding molecular weights in kDa. Arrows in B point at 4 discreet heme - containing proteins sheared from the outer surface. (C - E) Effect of shearing on the reductio n of Fe(III) oxides (10 mM) (C) , production of extracellular appendages, which are visible after dissolving the cell - associated Fe(III) oxides with oxalate (D), and growth with Fe(III) citrate (E); Y and X axes are as in panel C. Closed circles indicate sheared cultures while open circles indicate unsheared cultures. Scale bar in panel D is 100 µ m. 151 Figure 5.1 . Transmission electron micrographs of G. ahangari filaments Transmission electr on micrographs of the sheared (A ) a nd prep - cell ( B ) prep arations of G. ahangari filaments. Archaellar structures (black arrow) and pilus - like filaments (white arrow) are indicated as noted . Scale bars are at 100 nm. 152 Figure 5.2 . Transmission electron micrographs of G. ehrlichii filaments TEM micrographs of the sheared flagella ( A) and pili (B) and prep - cell preparations (C and D) of G. ehrlichii filaments. Scale bars are as labelled. 153 Figure 5.3 . Preliminary AFM data on G. ehrlichii AFM micrograph (A ) of G. ehrlichii strain SS015 filaments purified from th e shearing protocol containing the flagellum (green) and the pili (red) compa red to graphite (blue). Panel B contains representative I/V curves for the pili (red) and the insert shows the conductivity of the pili compared to the HOPG surface (blue). 154 Figur e 5.4 . Atomic force microscopy on G. ehrlichii purified pili (a) AFM micrograph of G. ehrlichii strain SS015 pili purified by the prep - cell proto col containing pure pili which appea r as bundles of wires. Panel B contains representative I/V curves for gra phite ( gray ) and the pilus filaments (Positions 3, 6, and 7 represented as dashed black and Position 4 as solid black ) across a 0 .6 V bias voltage sweep. Panel C contains averaged resistivity for the pilus filaments across both sites and graphite as a cont rol (col ors as in B). Scale bar in A is 200 nm. 155 Figure 5.5. Preliminary AFM data on G. ahangari AFM micrographs of G. ahangari strain 234 T filaments purified from the shearing protocol contai ning the pilus - like filaments (A) and the archaella (B ). Panel C contains representative I/V curves for sheared pilus - like filaments ( dashed black ), prep - cell purified pilus - like filaments ( solid black ) an d the archaella (dashed gray). Panel D contains averaged resistivity for the pilus - like filaments at a bias vol tage of 0.6 V (colors are as in (C ) ) . 156 Figure 5.6 . Atomic force microscopy on G. ahangari purified pilus - like filaments AFM micrograph of G. ahangari strain 234 T filaments purified from the p r ep - cell protocol containing only the pilus - like filaments ( A ). Panel B contains representative I/V curves for graphite ( gray ) and both the less conductive ( solid black ) and more conductive ( dashed black ) measu rements. Panel C contains averaged resistivity for the pilus - like filaments at a bias voltage of 0.6 V bot h before and after removal of the in sulating layer (colors as in C). Scale bar in (A ) is 200 nm. 157 Figure 5.7 . Sypro Orange thermal shift assa ys on soluble protein extracts Effect of pH on the T m of G. sulfurreducens, G. ehrlichii, and G. ahangari (A - C , respectively). Representati ve thermal shift assay output (D ) of G. sulfurreducens (gray), G. ehrlichii (dashed black), and G. ahangari (black ) soluble proteins at pH 7.0. (E ) Averaged thermal shift data represented as a column chart ( labels are as above) . 158 Figure 5.8 . Failure of the Sypro Orange thermal shift assay to characterize the T m of prep - cell purified filaments 159 Figure 5.9 . 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