Abiotic Ribose Synthesis Under Aqueous Environments with Various Chemical Conditions.

Ribose is the defining sugar in ribonucleic acid (RNA), which is often proposed to have carried the genetic information and catalyzed the biological reactions of the first life on Earth. Thus, abiological processes that yield ribose under prebiotic conditions have been studied for decades. However, aqueous environments required for the formation of ribose from materials available in quantity under geologically reasonable models, where the ribose formed is not immediately destroyed, remain unclear. This is due in large part to the challenge of analysis of carbohydrates formed under a wide range of aqueous conditions. Thus, the formation of ribose on prebiotic Earth has sometimes been questioned. We investigated the quantitative effects of pH, temperature, cation, and the concentrations of formaldehyde and glycolaldehyde on the synthesis of diverse sugars, including ribose. The results suggest a range of conditions that produce ribose and that ribose could have formed in constrained aquifers on prebiotic Earth.

Hexose phosphorylation for a non-enzymatic glycolysis and pentose phosphate pathway on early Earth.

Glycolysis and pentose phosphate pathways play essential roles in cellular processes and are assumed to be among the most ancient metabolic pathways. Non-enzymatic metabolism-like reactions might have occurred on the prebiotic Earth and been inherited by the biological reactions. Previous research has identified a part of the non-enzymatic glycolysis and the non-enzymatic pentose phosphate pathway from glucose 6-phosphate and 6-phosphogluconate, which are intermediates of these reactions. However, how these phosphorylated molecules were formed on the prebiotic Earth remains unclear. Herein, we demonstrate the synthesis of glucose and gluconate from simple aldehydes in alkaline solutions and the formation of glucose 6-phosphate and 6-phosphogluconate with borate using thermal evaporation. These results imply that the initial stages of glycolysis-like and pentose phosphate pathway-like reactions were achieved in borate-rich evaporative environments on prebiotic Earth, suggesting that non-enzymatic metabolism provided biomolecules and their precursors on prebiotic Earth.

Boron-assisted abiotic polypeptide synthesis.

The emergence of proteins and their interactions with RNAs were a key step in the origin and early evolution of life. The abiotic synthesis of peptides has been limited in short amino acid length and is favored in highly alkaline evaporitic conditions in which RNAs are unstable. This environment is also inconsistent with estimated Hadean Earth. Prebiotic environments rich in boron are reportedly ideal for abiotic RNA synthesis. However, the effects of boron on amino acid polymerization are unclear. We report that boric acid enables the polymerization of amino acids at acidic and near-neutral pH levels based on simple heating experiments of amino acid solutions containing borate/boric acid at various pH levels. Our study provides evidence for the boron-assisted synthesis of polypeptides in prebiotically plausible environments, where the same conditions would allow for the formation of RNAs and interactions of primordial proteins and RNAs that could be inherited by RNA-dependent protein synthesis during the evolution of life.

Borate-guided ribose phosphorylation for prebiotic nucleotide synthesis.

Polymers of ribonucleotides (RNAs) are considered to store genetic information and promote biocatalytic reactions for the proto life on chemical evolution. Abiotic synthesis of ribonucleotide was successful in past experiments; nucleoside synthesis occurred first, followed by phosphorylation. These abiotic syntheses are far from biotic reactions and have difficulties as a prebiotic reaction in reacting chemicals in a specific order and purifying intermediates from other molecules in multi-steps of reactions. Another reaction, ribose phosphorylation followed by nucleobase synthesis or nucleobase addition, is close to the biotic reactions of nucleotide synthesis. However, the synthesis of ribose 5'-phosphate under prebiotically plausible conditions remains unclear. Here, we report a high-yield regioselective one-pot synthesis of ribose 5'-phosphate from an aqueous solution containing ribose, phosphate, urea, and borate by simple thermal evaporation. Of note, phosphorylation of ribose before the nucleoside formation differs from the traditional prebiotic nucleotide syntheses and is also consistent with biological nucleotide synthesis. Phosphorylation occurred to the greatest extent in ribose compared to other aldopentoses, only in the presence of borate. Borate is known to improve the stability of ribose preferentially. Geological evidence suggests the presence of borate-rich settings on the early Earth. Therefore, borate-rich evaporitic environments could have facilitated preferential synthesis of ribonucleotide coupled with enhanced stability of ribose on the early Earth.

Mineralogical and Genomic Constraints on the Origin of Microbial Mn Oxide Formation in Complexed Microbial Community at the Terrestrial Hot Spring.

Manganese (Mn) oxides are widespread on the surface environments of the modern Earth. The role of microbial activities in the formation of Mn oxides has been discussed for several decades. However, the mechanisms of microbial Mn oxidation, and its role in complex microbial communities in natural environments, remain uncertain. Here, we report the geochemical, mineralogical, and metagenomic evidence for biogenic Mn oxides, found in Japanese hot spring sinters. The low crystallinity of Mn oxides, and their spatial associations with organic matter, support the biogenic origin of Mn oxides. Specific multicopper oxidases (MCOs), which are considered Mn-oxidizing enzymes, were identified using metagenomic analyses. Nanoscale nuggets of copper sulfides were, also, discovered in the organic matter in Mn-rich sinters. A part of these copper sulfides most likely represents traces of MCOs, and this is the first report of traces of Mn-oxidizing enzyme in geological samples. Metagenomic analyses, surprisingly, indicated a close association of Mn oxides, not only in aerobic but also in anaerobic microbial communities. These new findings offer the unique and unified positions of Mn oxides, with roles that have not been ignored, to sustain anaerobic microbial communities in hot spring environments.

Complex History of Aerobic Respiration and Phototrophy in the Chloroflexota Class Anaerolineae Revealed by High-Quality Draft Genome of Ca. Roseilinea mizusawaensis AA3_104.

Roseilinea is a novel lineage of Chloroflexota known only from incomplete metagenome-assembled genomes (MAGs) and preliminary enrichments. Roseilinea is notable for appearing capable of anoxygenic photoheterotrophy despite being only distantly related to well-known phototrophs in the Chloroflexia class such as Chloroflexus and Roseiflexus. Here, we present a high-quality MAG of a member of Roseilinea, improving our understanding of the metabolic capacity and phylogeny of this genus, and resolving the multiple instances of horizontal gene transfer that have led to its metabolic potential. These data allow us to propose a candidate family for these organisms, Roseilineaceae, within the Anaerolineae class.

Experimental Investigation of the Formation of Formaldehyde by Hadean and Noachian Impacts.

Formaldehyde (FA) is an important precursor in the abiotic synthesis of major biomolecules including amino acids, sugars, and nucleobases. Thus, spontaneous formation of prebiotic FA must have been crucial for the chemical origin of life. The frequent impacts of meteorites and asteroids on Hadean Earth have been considered one of the abiotic synthetic processes of organic compounds. However, the impact-induced formation of FA from CO2 as the major atmospheric constituent has not been confirmed yet. This study investigated the formation of FA in impact-induced reactions among meteoritic minerals, bicarbonate, gaseous nitrogen, and water to simulate the abiotic process experimentally. Products were analyzed with ultra-high-performance liquid chromatography/tandem mass spectrometry and powder X-ray diffraction techniques. The results show the formation of FA and oxidation of metallic iron to siderite in the impact shock experiments. This indicates that this important prebiotic molecule was also synthesized by impacts of iron-bearing meteorites/asteroids on the Hadean oceans. The impact events might have generated spatially and temporally FA-enriched localized environments. Moreover, the impact-induced synthesis of FA may have also occurred on Noachian Mars given the presence of liquid water and a CO2-N2-rich atmosphere on the planet.

Vestiges of Adaptation to the Mesophilic Environment in the Genome of Tepiditoga spiralis gen. nov., sp. nov.

A novel anaerobic heterotrophic strain, designated strain sy52T, was isolated from a hydrothermal chimney at Suiyo Seamount in the Pacific Ocean. A 16S rRNA gene analysis revealed that the strain belonged to the family Petrotogaceae in the phylum Thermotogae. The strain was mesophilic with optimum growth at 48°C and the phylum primarily comprised hyperthermophiles and thermophiles. Strain sy52T possessed unique genomic characteristics, such as an extremely low G+C content and 6 copies of rRNA operons. Genomic analyses of strain sy52T revealed that amino acid usage in the predicted proteins resulted from adjustments to mesophilic environments. Genomic features also indicated independent adaptions to the mesophilic environment of strain sy52T and Mesotoga species, which belong to the mesophilic lineage in the phylum Thermotogae. Based on phenotypic and phylogenetic evidence, strain sy52T is considered to represent a novel genus and species in the family Petrotogaceae with the proposed name Tepiditoga spiralis gen. nov., sp. nov.

Impact-induced amino acid formation on Hadean Earth and Noachian Mars.

Abiotic synthesis of biomolecules is an essential step for the chemical origin of life. Many attempts have succeeded in synthesizing biomolecules, including amino acids and nucleobases (e.g., via spark discharge, impact shock, and hydrothermal heating), from reduced compounds that may have been limited in their availabilities on Hadean Earth and Noachian Mars. On the other hand, formation of amino-acids and nucleobases from CO2 and N2 (i.e., the most abundant C and N sources on Earth during the Hadean) has been limited via spark discharge. Here, we demonstrate the synthesis of amino acids by laboratory impact-induced reactions among simple inorganic mixtures: Fe, Ni, Mg2SiO4, H2O, CO2, and N2, by coupling the reduction of CO2, N2, and H2O with the oxidation of metallic Fe and Ni. These chemical processes simulated the possible reactions at impacts of Fe-bearing meteorites/asteroids on oceans with a CO2 and N2 atmosphere. The results indicate that hypervelocity impact was a source of amino acids on the Earth during the Hadean and potentially on Mars during the Noachian. Amino acids formed during such events could more readily polymerize in the next step of the chemical evolution, as impact events locally form amino acids at the impact sites.

Racemization of Valine by Impact-Induced Heating.

Homochirality plays an important role in all living organisms but its origin remains unclear. It also remains unclear whether such chiral molecules survived terrestrial heavy impact events. Impacts of extraterrestrial objects on early oceans were frequent and could have affected the chirality of oceanic amino acids when such amino acids accumulated during impacts. This study investigated the effects of shock-induced heating on enantiomeric change of valine with minerals such as olivine ([Mg0.9, Fe0.1]2SiO4), hematite (Fe2O3), and calcite (CaCO3). With a shock wave generated by an impact at ~0.8 km/s, both D- and L-enriched valine were significantly decomposed and partially racemized under all experimental conditions. Different minerals had different shock impedances; therefore, they provided different P-T conditions for identical impacts. Furthermore, the high pH of calcite promoted the racemization of valine. The results indicate that in natural hypervelocity impacts, amino acids in shocked oceanic water would have decomposed completely, since impact velocity and the duration of shock compression and heating are typically greater in hypervelocity impact events than those in experiments. Even with the shock wave by the impact of small and decelerated projectiles in which amino acids survive, the shock heating may generate sufficient heat for significant racemization in shocked oceanic water. However, the duration of shock induced heating by small projectiles is limited and the population of such decelerated projectiles would be limited. Therefore, even though impacts of asteroids and meteorites were frequent on the prebiotic Earth, impact events would not have significantly changed the ee of proteinogenic amino acids accumulated in the entire ocean.

Effects of Glycine, Water, Ammonia, and Ammonium Bicarbonate on the Oligomerization of Methionine.

The abiotic oligomerization of amino acids may have created primordial, protein-like biological catalysts on the early Earth. Previous studies have proposed and evaluated the potential of diagenesis for the amino acid oligomerization, simulating the formation of peptides that include glycine, alanine, and valine, separately. However, whether such conditions can promote the formation of peptides composed of multiple amino acids remains unclear. Furthermore, the chemistry of pore water in sediments should affect the oligomerization and degradation of amino acids and oligomers, but these effects have not been studied extensively. In this study, we investigated the effects of water, ammonia, ammonium bicarbonate, pH, and glycine on the oligomerization and degradation of methionine under high pressure (150 MPa) and high temperature conditions (175 °C) for 96 h. Methionine is more difficult to oligomerize than glycine and methionine dimer was formed in the incubation of dry powder of methionine. Methionine oligomers as long as trimers, as well as methionylglycine and glycylmethionine, were formed under every condition with these additional compounds. Among the compounds tested, the oligomerization reaction rate was accelerated by the presence of water and by an increase in pH. Ammonia also increased the oligomerization rate but consumed methionine by side reactions and resulted in the rapid degradation of methionine and its peptides. Similarly, glycine accelerated the oligomerization rate of methionine and the degradation of methionine, producing water, ammonia, and bicarbonate through its decomposition. With Gly, heterogeneous dimers (methionylglycine and glycylmethionine) were formed in greater amounts than with other additional compounds although smaller amount of these heterogeneous dimers were formed with other additional compounds. These results suggest that accelerated reaction rates induced by water and co-existing reactive compounds promote the oligomerization of less reactive amino acids during diagenesis and enhance the formation of peptides composed of multiple amino acids.

Evaporite Borate-Containing Mineral Ensembles Make Phosphate Available and Regiospecifically Phosphorylate Ribonucleosides: Borate as a Multifaceted Problem Solver in Prebiotic Chemistry.

RNA is currently thought to have been the first biopolymer to support Darwinian natural selection on Earth. However, the phosphate esters in RNA and its precursors, and the many sites at which phosphorylation might occur in ribonucleosides under conditions that make it possible, challenge prebiotic chemists. Moreover, free inorganic phosphate may have been scarce on early Earth owing to its sequestration by calcium in the unreactive mineral hydroxyapatite. Herein, it is shown that these problems can be mitigated by a particular geological environment that contains borate, magnesium, sulfate, calcium, and phosphate in evaporite deposits. Actual geological environments, reproduced here, show that Mg2+ and borate sequester phosphate from calcium to form the mineral lüneburgite. Ribonucleosides stabilized by borate mobilize borate and phosphate from lüneburgite, and are then regiospecifically phosphorylated by the mineral. Thus, in addition to guiding carbohydrate pre-metabolism, borate minerals in evaporite geoorganic contexts offer a solution to the phosphate problem in the "RNA first" model for the origins of life.

Effects of Silicate, Phosphate, and Calcium on the Stability of Aldopentoses.

Ribose is an important constituent of RNA: ribose connects RNA bases and forms a strand of sugar phosphates. Accumulation of ribose on prebiotic Earth was difficult because of its low stability. Improvement in the yield of ribose by the introduction of borate or silicate in a formose-like reaction has been proposed. The effects of borates have been further analyzed and confirmed in subsequent studies. Nonetheless, the effects of silicates and phosphates remain unclear. In the present study, we incubated aldopentoses in a highly alkaline aqueous solution at a moderate temperature to determine the effects of silicate or phosphate on the degradation rates of ribose and its isomeric aldopentoses. The formation of a complex of silicate (or phosphate) with ribose was also analyzed in experiments with (29)Si and (31)P nuclear magnetic resonance (NMR). We found that silicate or phosphate complexes of ribose were not detectable under our experimental conditions. The stability of ribose and lyxose improved after addition of 40-fold molar excess (relative to a pentose) of sodium silicate or sodium phosphate to the alkaline solution. The stability was not improved further when an 80-fold molar excess of sodium silicate or sodium phosphate was added. Calcium was removed from these solutions by precipitation of calcium salts. The drop in Ca(2+) concentration might have improved the stability of ribose and lyxose, which are susceptible to aldol addition. The improvement of ribose stability by the removal of Ca(2+) and by addition of silicate or phosphate was far smaller than the improvement by borate. Furthermore, all aldopentoses showed similar stability in silicate- and phosphate-containing solutions. These results clearly show that selective stabilization of ribose by borate cannot be replaced by the effects of silicate or phosphate; this finding points to the importance of borate in prebiotic RNA formation.

Occurrence of Tourmaline in Metasedimentary Rocks of the Isua Supracrustal Belt, Greenland: Implications for Ribose Stabilization in Hadean Marine Sediments.

Abiotic formation of RNA was important for the emergence of terrestrial life, but the acknowledged difficulties of generating and stabilizing ribose have often raised questions regarding how the first RNA might have formed. Previous researchers have proposed that borate could have stabilized ribose; however, the availability of borate on the early Earth has been the subject of intense debate. In order to examine whether borate was available on the early Earth, this study examined metasedimentary rocks from the Isua Supracrustal Belt. Garnet, biotite, and quartz comprise the major constituents of the examined rocks. Field relationships and the chemical compositions of the examined rocks suggest sedimentary origin. The present study found that garnet crystals contain a number of inclusions of tourmaline (a type of borosilicate mineral). All tourmaline crystals are Fe-rich and categorized as schorl. Both garnet and tourmaline often contain graphite inclusions and this close association of tourmaline with garnet and graphite has not been recognized previously. Garnet-biotite and graphite geothermometers suggest that the tourmaline in garnet experienced peak metamorphic conditions (~500 °C and 5 kbar). The mineralogical characteristics of the tourmaline and the whole rock composition indicate that the tourmaline formed authigenically in the sediment during diagenesis and/or early metamorphism. Clay minerals in modern sediments have the capability to adsorb and concentrate borate, which could lead to boron enrichment during diagenesis, followed by tourmaline formation under metamorphic conditions. Clay minerals, deposited on the early Archean seafloor, were the precursors of the garnet and biotite in the examined samples. The studied tourmaline crystals were most likely formed in the same way as modern tourmaline in marine sediments. Therefore, boron enrichment by clays must have been possible even during the early Archean. Thus, similar enrichment could have been possible during the Hadean, providing a stabilization agent for ribose.

Survivability and reactivity of glycine and alanine in early oceans: effects of meteorite impacts.

Prebiotic oceans might have contained abundant amino acids, and were subjected to meteorite impacts, especially during the late heavy bombardment. It is so far unknown how meteorite impacts affected amino acids in the early oceans. Impact experiments were performed under the conditions where glycine was synthesized from carbon, ammonia, and water, using aqueous solutions containing (13)C-labeled glycine and alanine. Selected amino acids and amines in samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). In particular, the (13)C-labeled reaction products were analyzed to distinguish between run products and contaminants. The results revealed that both amino acids survived partially in the early ocean through meteorite impacts, that part of glycine changed into alanine, and that large amounts of methylamine and ethylamine were formed. Fast decarboxylation was confirmed to occur during such impact processes. Furthermore, the formation of n-butylamine, detected only in the samples recovered from the solutions with additional nitrogen and carbon sources of ammonia and benzene, suggests that chemical reactions to form new biomolecules can proceed through marine impacts. Methylamine and ethylamine from glycine and alanine increased considerably in the presence of hematite rather than olivine under similar impact conditions. These results also suggest that amino acids present in early oceans can contribute further to impact-induced reactions, implying that impact energy plays a potential role in the prebiotic formation of various biomolecules, although the reactions are complicated and depend upon the chemical environments as well.

Selective stabilization of ribose by borate.

In this study, borate was found to selectively increase the stability of ribose over other aldopentoses. Ribose is the only sugar present in both early RNA-based biochemistry and contemporary DNA-based life, and the stability of ribose is of fundamental concern for determining the origin of early RNA-based biochemistry. The formose reaction is a potential process in the prebiotic synthesis of ribose and its stereoisomers arabinose, xylose, and lyxose. Ribose is the least stable of these aldopentoses, raising the fundamental question of whether it was originally a component of primitive RNA or was selected through biotic processes. Borate is known to increase the stability of aldopentoses, but the specific differences in the stabilization achieved among different stereoisomers remain unclear. In this study, it was found that the stabilities of all of the tested pentoses increased with the concentration of added borate, but notably, the stability of ribose increased the most. The predominant formation of complexes between borate and ribose was verified, in agreement with previous studies. This borate complex formation might have sequestered ribose from the isomerization and decomposition reactions, resulting in its selective stabilization. These findings indicate that ribose could have accumulated in borate-rich environments on the early Earth and suggest that ribose-based nucleotides combined with phosphate and nucleobases formed abiotically.

Abiotic formation of valine peptides under conditions of high temperature and high pressure.

We investigated the oligomerization of solid valine and the stabilities of valine and valine peptides under conditions of high temperature (150-200 °C) and high pressure (50-150 MPa). Experiments were performed under non-aqueous condition in order to promote dehydration reaction. After prolonged exposure of monomeric valine to elevated temperatures and pressures, the products were analyzed by liquid chromatography mass spectrometry comparing their retention times and masses. We identified linear peptides that ranged in size from dimer to hexamer, as well as a cyclic dimer. Previous studies that attempted abiotic oligomerization of valine in the absence of a catalyst have never reported valine peptides larger than a dimer. Increased reaction temperature increased the dissociative decomposition of valine and valine peptides to products such as glycine, ß-alanine, ammonia, and amines by processes such as deamination, decarboxylation, and cracking. The amount of residual valine and peptide yields was greater at higher pressures at a given temperature, pressure, and reaction time. This suggests that dissociative decomposition of valine and valine peptides is reduced by pressure. Our findings are relevant to the investigation of diagenetic processes in prebiotic marine sediments where similar pressures occur under water-poor conditions. These findings also suggest that amino acids, such as valine, could have been polymerized to peptides in deep prebiotic marine sediments within a few hundred million years.

Stability of amino acids and their oligomerization under high-pressure conditions: implications for prebiotic chemistry.

The polymerization of amino acids leading to the formation of peptides and proteins is a significant problem for the origin of life. This problem stems from the instability of amino acids and the difficulty of their oligomerization in aqueous environments, such as seafloor hydrothermal systems. We investigated the stability of amino acids and their oligomerization reactions under high-temperature (180-400°C) and high-pressure (1.0-5.5 GPa) conditions, based on the hypothesis that the polymerization of amino acids occurred in marine sediments during diagenesis and metamorphism, at convergent margins on early Earth. Our results show that the amino acids glycine and alanine are stabilized by high pressure. Oligomers up to pentamers were formed, which has never been reported for alanine in the absence of a catalyst. The yields of peptides at a given temperature and reaction time were higher under higher-pressure conditions. Elemental, infrared, and isotopic analyses of the reaction products indicated that deamination is a key degradation process for amino acids and peptides under high-pressure conditions. A possible NH(3)-rich environment in marine sediments on early Earth may have further stabilized amino acids and peptides by inhibiting their deamination.

Biogeography and biodiversity in sulfide structures of active and inactive vents at deep-sea hydrothermal fields of the Southern Mariana Trough.

The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and microbial ecosystems in marine environments.

Abundance of Zetaproteobacteria within crustal fluids in back-arc hydrothermal fields of the Southern Mariana Trough.

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.