Search

Mineral transformations by two hyperthermophilic Fe(III)-reducing crenarchaea

The isotopic composition of lipid biomarkers and biomass in sedimentary environments are widely used to infer microbial metabolisms and constrain carbon cycling processes. It has been observed that metabolic energy availability in the form of H2 impacts the stable carbon isotopes of CH4 during hydrogenotrophic methanogenesis, but it is unknown whether this relationship extends to lipids and amino acids. Since lipids and amino acids are long-lived, they can be used to reconstruct past conditions over geologic timescales. Therefore, the controls on their isotopic signatures are important to constrain to better interpret past environments. In this study, the isotopic distributions of carbon metabolized and reduced by the hyperthermophile Methanocaldococcus jannaschii were quantified following growth at 82 °C in a chemostat with high (80–83 µM) and low (15–27 µM) H2 concentrations. As has been shown previously, the stable carbon isotope fractionation factors for CH4 were >15‰ larger in low H2 experiments than in high H2 experiments. Lipid biomarkers and amino acids were similarly impacted with approximately 10‰ larger fractionation factors under low H2 conditions. The increase in fractionation factors can be related to the lower availability of thermodynamic energy, suggesting that even larger fractionation factors would be observed in methanogens living close to their threshold energy needs, as they do in most environments. The resulting isotopic signatures of long-lived lipid biomarkers synthesized by hydrogenotrophic methanogens may become as ‘superlight’ as those synthesized by archaea carrying out the anaerobic oxidation of methane. These results help to describe the underlying mechanisms that determine the isotopic composition of long-lived biomarkers and provide constraints for interpreting these signatures in the environment.

Diverse microbial communities drive biogeochemical cycles in Earth’s ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents., Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed a remotely operated vehicle (ROV)-powered incubator instrument to carry out and compare results from in situ and shipboard RNA stable isotope probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labeled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes, suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations, suggesting that depressurization induced thermal stress in the metabolically active microbes in these incubations. Together, the results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under nearly native conditions in the ocean. IMPORTANCE Diverse microbial communities drive biogeochemical cycles in Earth’s ocean, yet studying these organisms and processes is often limited by technological capabilities, especially in the deep ocean. In this study, we used a novel marine microbial incubator instrument capable of in situ experimentation to investigate microbial primary producers at deep-sea hydrothermal vents. We carried out identical stable isotope probing experiments coupled to RNA sequencing both on the seafloor and on the ship to examine thermophilic, microbial autotrophs in venting fluids from an active submarine volcano. Our results indicate that microbial communities were significantly impacted by the effects of depressurization and sample processing delays, with shipboard microbial communities being more stressed than seafloor incubations. Differences in metabolism were also apparent and are likely linked to the chemistry of the fluid at the beginning of the experiment. Microbial experimentation in the natural habitat provides new insights into understanding microbial activities in the ocean.

Depressurization and sample processing delays may impact the outcome of shipboard microbial incubations of samples collected from the deep sea. To address this knowledge gap, we developed an ROV-powered incubator instrument to carry out and compare results from in situ and shipboard RNA Stable Isotope Probing (RNA-SIP) experiments to identify the key chemolithoautotrophic microbes and metabolisms in diffuse, low-temperature venting fluids from Axial Seamount. All the incubations showed microbial uptake of labelled bicarbonate primarily by thermophilic autotrophic Epsilonbacteraeota that oxidized hydrogen coupled with nitrate reduction. However, the in situ seafloor incubations showed higher abundances of transcripts annotated for aerobic processes suggesting that oxygen was lost from the hydrothermal fluid samples prior to shipboard analysis. Furthermore, transcripts for thermal stress proteins such as heat shock chaperones and proteases were significantly more abundant in the shipboard incubations suggesting that hydrostatic pressure ameliorated thermal stress in the metabolically active microbes in the seafloor incubations. Together, results indicate that while the autotrophic microbial communities in the shipboard and seafloor experiments behaved similarly, there were distinct differences that provide new insight into the activities of natural microbial assemblages under near-native conditions in the ocean.

Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes., Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae. There was also a >14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals. IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.

Desulfurobacterium sp. strain HR11 was isolated from a hydrothermal vent on the Juan de Fuca Ridge. We present the 1.55-Mb genome sequence of HR11, which contains 1,624 putative protein-coding sequences. Overall genome relatedness index analyses indicate that HR11 is a novel subspecies of D. thermolithotrophum.

The abundance of methane isotopologues with two rare isotopes (e.g., 13CH3D) has been proposed as a tool to estimate the temperature at which methane is formed or thermally equilibrated. It has been shown, however, that microbial methane from surface environments and from laboratory cultures is characterized by low 13CH3D abundance, corresponding to anomalously high apparent 13CH3D equilibrium temperatures. We carried out a series of batch culture experiments to investigate the origin of the non-equilibrium signals in microbial methane by exploring a range of metabolic pathways, growth temperatures, and hydrogen isotope compositions of the media. We found that thermophilic methanogens (Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Methanocaldococcus bathoardescens) grown on H2 + CO2 at temperatures between 60 and 80 °C produced methane with Δ13CH3D values (defined as the deviation from stochastic abundance) of 0.5–2.5‰, corresponding to apparent 13CH3D equilibrium temperatures of 200–600 °C. Mesophilic methanogens (Methanosarcina barkeri and Methanosarcina mazei) grown on H2 + CO2, acetate, or methanol produced methane with consistently low Δ13CH3D values, down to −5.2‰. Closed system effects can explain part of the non-equilibrium signals for methane from thermophilic methanogens. Experiments with M. barkeri using D-spiked water or D-labeled acetate (CD3COO−) indicate that 1.6–1.9 out of four H atoms in methane originate from water, but Δ13CH3D values of product methane only weakly correlate with the D/H ratio of medium water. Our experimental results demonstrate that low Δ13CH3D values are not specific to the metabolic pathways of methanogenesis, suggesting that they could be produced during enzymatic reactions common in the three methanogenic pathways, such as the reduction of methyl-coenzyme M. Nonetheless C H bonds inherited from precursor methyl groups may also carry part of non-equilibrium signals.

Some hyperthermophilic heterotrophs in the genus Thermococcus produce H2 in the absence of S° and have up to seven hydrogenases, but their combined physiological roles are unclear. Here, we show which hydrogenases in Thermococcus paralvinellae are affected by added H2 during growth without S°. Growth rates and steady-state cell concentrations decreased while formate production rates increased when T. paralvinallae was grown in a chemostat with 65 µM of added H2(aq) . Differential gene expression analysis using RNA-Seq showed consistent expression of six hydrogenase operons with and without added H2 . In contrast, expression of the formate hydrogenlyase 1 (fhl1) operon increased with added H2 . Flux balance analysis showed H2 oxidation and formate production using FHL became an alternate route for electron disposal during H2 inhibition with a concomitant increase in growth rate relative to cells without FHL. T. paralvinellae also grew on formate with an increase in H2 production rate relative to growth on maltose or tryptone. Growth on formate increased fhl1 expression but decreased expression of all other hydrogenases. Therefore, Thermococcus that possess fhl1 have a competitive advantage over other Thermococcus species in hot subsurface environments where organic substrates are present, S° is absent and slow H2 efflux causes growth inhibition.

Nanophase iron (oxyhydr)oxides are ubiquitous on Earth, globally distributed on Mars, and likely present on numerous other rocky solar system bodies. They are often structurally and, therefore, spectrally distinct from iron (oxyhydr)oxide bulk phases. Because their spectra vary with grain size, they can be difficult to identify or distinguish unless multiple analysis techniques are used in tandem. Yet, most literature reports fail to use multiple techniques or adequately parameterize sample morphology, making it difficult to understand how morphology affects spectral characteristics across techniques. Here, we present transmission electron microscopy, Raman, visible and near-infrared, and mid-infrared attenuated total reflectance data on synthetic, nanophase akaganéite, lepidocrocite, goethite, hematite, ferrihydrite, magnetite, and maghemite. Feature positions are tabulated and compared to those for bulk (oxyhydr)oxides and other nanophase iron (oxyhydr)oxides from the literature. The utility and limitations of each technique in analyzing nanophase iron (oxyhydr)oxides are discussed. Raman, mid-infrared, and visible near-infrared spectra show broadening, loss of some spectral features, and shifted positions compared to bulk phases. Raman and mid-infrared spectroscopies are useful in identifying and distinguishing akaganéite, lepidocrocite, goethite, and hematite, though ferrihydrite, magnetite, and maghemite have overlapped band positions. Visible near-infrared spectroscopy can identify and distinguish among ferrihydrite, magnetite, and maghemite in pure spectra, though akaganéite, lepidocrocite, and goethite can have overlapping bands. It is clear from this work that further understanding of variable spectral features in nanophase iron (oxyhydr)oxides must await additional studies to robustly assess effects of morphology. This study establishes a template for future work.

Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (eCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats. IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.

Pyrobaculum arsenaticum is a hyperthermophilic archaeon that thrives at 95°C. This strain encodes a putative GH31 intracellular α-glucosidase (Pars_2044, PyAG) in its genome. The recombinant PyAG (rPyAG) was optimally expressed in Escherichia coli at 37°C for 4 h after IPTG induction. The purified rPyAG is a homotetrameric α-glucosidase that exhibited highly thermostable properties. Maximum p-nitrophenyl-α-D-glucopyranoside (pNPG) hydrolysis activity was observed at 90°C and pH 5.0. The enzyme mainly recognized the non-reducing end of the substrate, releasing the glucose unit. rPyAG also had broad substrate specificity, cleaving maltose (α-1,4-linkage), kojibiose (α-1,2-linkage), and nigerose (α-1,3-linkage) with similar efficiency. Based on these results, rPyAG can be used to modify health-relevant sugar conjugates linked by α-1,2- or α-1,3-bonds.

The gut microbiome plays an important role in immune function and has been implicated in several autoimmune disorders. Here we use 16S rRNA sequencing to investigate the gut microbiome in subjects with multiple sclerosis (MS, n=60) and healthy controls (n=43). Microbiome alterations in MS include increases in Methanobrevibacter and Akkermansia and decreases in Butyricimonas, and correlate with variations in the expression of genes involved in dendritic cell maturation, interferon signalling and NF-kB signalling pathways in circulating T cells and monocytes. Patients on disease-modifying treatment show increased abundances of Prevotella and Sutterella, and decreased Sarcina, compared with untreated patients. MS patients of a second cohort show elevated breath methane compared with controls, consistent with our observation of increased gut Methanobrevibacter in MS in the first cohort. Further study is required to assess whether the observed alterations in the gut microbiome play a role in, or are a consequence of, MS pathogenesis., The gut microbiome has been implicated in several autoimmune disorders. Here, the authors study the gut microbiome of patients with multiple sclerosis, and find correlations between altered abundance of certain gut microorganisms and changes in expression of immune defence genes.

Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments., Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (εCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats. IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.

Fe(III) (oxyhydr)oxides are electron acceptors for some hyperthermophilic archaea in mildly reducing geothermal environments. However, the kinds of iron oxides that can be used, growth rates, extent of iron reduction, and the morphological changes that occur to minerals are poorly understood. The hyperthermophilic iron-reducing crenarchaea Pyrodictium delaneyi and Pyrobaculum islandicum were grown separately on six different synthetic nanophase Fe(III) (oxyhydr)oxides. For both organisms, growth on ferrihydrite produced the highest growth rates and the largest amounts of Fe(II), although P. delaneyi produced four times more Fe(II) (25 mM) than P. islandicum (6 mM). Both organisms grew on lepidocrocite and akaganeite and produced 2 and 3 mM Fe(II). Modest growth occurred for both organisms on goethite, hematite, and maghemite where ≤1 mM Fe(II) was produced. The diameters of the spherical mineral end-products following P. delaneyi growth increased by 30 nm for ferrihydrite and 50-150 nm for lepidocrocite relative to heated abiotic controls. For akaganeite, spherical particle sizes were the same for P. delaneyi-reacted samples and heated abiotic controls, but the spherical particles were more numerous in the P. delaneyi samples. For P. islandicum, there was no increase in grain size for the mineral end-products following growth on ferrihydrite, lepidocrocite, or akaganeite relative to the heated abiotic controls. High-resolution transmission electron microscopy of lattice fringes and selected-area electron diffraction of the minerals produced by both organisms when grown on ferrihydrite showed that magnetite and/or possibly maghemite were the end-products while the heated abiotic controls only contained ferrihydrite. These results expand the current view of bioavailable Fe(III) (oxyhydr)oxides for reduction by hyperthermophilic archaea when presented as synthetic nanophase minerals. They show that growth and reduction rates are inversely correlated with the iron (oxyhydr)oxide crystallinity and that iron (oxyhydr)oxide mineral transformation takes different forms for these two organisms.

Hydrothermal fluids (341°C and 19°C) were collected

Two heterotrophic hyperthermophilic strains, ES1T and CL1T, were isolated from Paralvinella sp. polychaete worms collected from active hydrothermal vent chimneys in the north-eastern Pacific Ocean. Both were obligately anaerobic and produced H2S in the presence of elemental sulfur and H2. Complete genome sequences are available for both strains. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strains are more than 97 % similar to most other species of the genus Thermococcus . Therefore, overall genome relatedness index analyses were performed to establish that these strains are novel species. For each analysis, strain ES1T was determined to be most similar to Thermococcus barophilus MPT, while strain CL1T was determined to be most similar to Thermococcus sp. 4557. The average nucleotide identity scores for these strains were 84 % for strain ES1T and 81 % for strain CL1T, genome-to-genome direct comparison scores were 23 % for strain ES1T and 47 % for strain CL1T, and the species identification scores were 89 % for strain ES1T and 88 % for strain CL1T. For each analysis, strains ES1T and CL1T were below the species delineation cut-off. Therefore, based on their whole genome sequences, strains ES1T and CL1T are suggested to represent novel species of the genus Thermococcus for which the names Thermococcus paralvinellae sp. nov. and Thermococcus cleftensis sp. nov. are proposed, respectively. The type strains are ES1T ( = DSM 27261T = KACC 17923T) and CL1T ( = DSM 27260T = KACC 17922T).

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeonPyrococcussp. ST04. This enzyme shows 7-barrel fold and a C-domain (amino acids 342–597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and β-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and β-glucose-1-phosphate (β-G1P). The Km values for kojibiose and phosphate were determined to be 2.53 ± 0.21 mM and 1.34 ± 0.04 mM, respectively. PsPGM then converts β-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from β-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to β-G1P was 3.51 ± 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea.

Genome organization near cyclomaltodextrinases (CDases) was analyzed and compared for four different hyperthermophilic archaea: Thermococcus, Pyrococcus, Staphylothermus, and Thermofilum. A gene (CL1_0884) encoding a putative CDase from Thermococcus sp. CL1 (tccd) was cloned and expressed in Escherichia coli. TcCD was confirmed to be highly thermostable, with optimal activity at 85℃. The melting temperature of TcCD was determined to be 93oC by both differential scanning calorimetry and differential scanning fluorimetry. A size-exclusion chromatography experiment showed that TcCD exists as a monomer. TcCD preferentially hydrolyzed α-cyclodextrin (α-CD), and at the initial stage catalyzed a ring-opening reaction by cleaving one α-1,4-glycosidic linkage of the CD ring to produce the corresponding single maltooligosaccharide. Furthermore, TcCD could hydrolyze branched CDs (G1-α-CD, G1-β- CD, and G2-β-CD) to yield significant amounts (45%, 40%, and 46%) of isomaltooligosaccharides (panose and 6(2)-α-maltosylmaltose) in addition to glucose and maltose. This enzyme is one of the most thermostable maltogenic amylases reported, and might be of potential value in the production of isomaltooligosaccharides in the food industry.

The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed

[1] Forty-nine hydrothermal sulfide-sulfate rock samples from the Endeavour Segment of the Juan de Fuca Ridge, northeastern Pacific Ocean, were dated by measuring the decay of 226Ra (half-life of 1600 years) in hydrothermal barite to provide a history of hydrothermal venting at the site over the past 6000 years. This dating method is effective for samples ranging in age from ∼200 to 20,000 years old and effectively bridges an age gap between shorter- and longer-lived U-series dating techniques for hydrothermal deposits. Results show that hydrothermal venting at the active High Rise, Sasquatch, and Main Endeavour fields began at least 850, 1450, and 2300 years ago, respectively. Barite ages of other inactive deposits on the axial valley floor are between ∼1200 and ∼2200 years old, indicating past widespread hydrothermal venting outside of the currently active vent fields. Samples from the half-graben on the eastern slope of the axial valley range in age from ∼1700 to ∼2925 years, and a single sample from outside the axial valley, near the westernmost valley fault scarp is ∼5850 ± 205 years old. The spatial relationship between hydrothermal venting and normal faulting suggests a temporal relationship, with progressive younging of sulfide deposits from the edges of the axial valley toward the center of the rift. These relationships are consistent with the inward migration of normal faulting toward the center of the valley over time and a minimum age of onset of hydrothermal activity in this region of 5850 years.

Iron–sulfur clusters are ubiquitous in biology and function in electron transfer and catalysis. They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12 nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300 nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20 nm units containing ∼6,400 Fe atoms and ∼170 IssA monomers. In support of roles in both iron–sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin in vitro., The biosynthesis of iron-sulfur clusters in anaerobic organisms has not been extensively investigated. Here, the authors identify and characterize a multi-subunit protein that stores iron and sulfur in thioferrate for the assembly of the clusters in Pyrococcus furiosus.

Microbial productivity at hydrothermal vents is among the highest found anywhere in the deep ocean, but constraints on microbial growth and metabolism at vents are lacking. We used a combination of cultivation, molecular, and geochemical tools to verify pure culture H 2 threshold measurements for hyperthermophilic methanogenesis in low-temperature hydrothermal fluids from Axial Volcano and Endeavour Segment in the northeastern Pacific Ocean. Two Methanocaldococcus strains from Axial and Methanocaldococcus jannaschii showed similar Monod growth kinetics when grown in a bioreactor at varying H 2 concentrations. Their H 2 half-saturation value was 66 μM, and growth ceased below 17–23 μM H 2 , 10-fold lower than previously predicted. By comparison, measured H 2 and CH 4 concentrations in fluids suggest that there was generally sufficient H 2 for Methanocaldococcus growth at Axial but not at Endeavour. Fluids from one vent at Axial (Marker 113) had anomalously high CH 4 concentrations and contained various thermal classes of methanogens based on cultivation and mcrA / mrtA analyses. At Endeavour, methanogens were largely undetectable in fluid samples based on cultivation and molecular screens, although abundances of hyperthermophilic heterotrophs were relatively high. Where present, Methanocaldococcus genes were the predominant mcrA / mrtA sequences recovered and comprised ∼0.2–6% of the total archaeal community. Field and coculture data suggest that H 2 limitation may be partly ameliorated by H 2 syntrophy with hyperthermophilic heterotrophs. These data support our estimated H 2 threshold for hyperthermophilic methanogenesis at vents and highlight the need for coupled laboratory and field measurements to constrain microbial distribution and biogeochemical impacts in the deep sea.

The discovery of deep-sea hydrothermal vents caused scientists to reconsider their notions about life in the deep sea. In these seemingly inhospitable environments, free-living microbes, as well as microbial-animal symbioses, thrive in the warm waters around vents. The biomass per unit area in this environment is comparable to that of rainforests. Uniquely, these highly productive ecosystems are based on microbial chemoautotrophic metabolism, wherein microbes generate metabolic energy by drawing oxygen or nitrate from surrounding seawater to oxidize reduced chemicals (e.g., sulfide) found in the vent fluids (Sievert and Vetriani, 2012, in this issue). The rapid and voluminous fluid flux through hydrothermal vents replenishes these substrates at a rate sufficient to support this substantial community. The tremendous microbial productivity observed at vents raises the question as to whether these microorganisms are also well suited for bioenergy and biofuel production. Here, we discuss the utility and issues associated with two example approaches: in situ bioelectricity generation and microbially mediated large-scale biofuel production.

Hydrothermal vents are among the most biologically active regions of the deep ocean. However, our understanding of the limits of life in this extreme environment, the extent of biogeochemical transformation that occurs in the crust and overlying ocean, and the impact of vent life on regional and global ocean chemistry is in its infancy. Recently, scientific studies have expanded our view of how vent microbes gain metabolic energy at vents through their use of dissolved chemicals and minerals contained in ocean basalts, seafloor sulfide deposits, and hydrothermal plumes and, in turn, how they catalyze chemical and mineral transformations. The scale of vent environments and the difficulties inherent in the study of life above, on, and below the deep seafloor have led to the development of geochemical and bioenergetic models. These models predict habitability and biological activity based on the chemical composition of hydrothermal fluids, seawater, and the surrounding rock, balanced by the physiological energy demand of cells. This modeling, coupled with field sampling for ground truth and discovery, has led to a better understanding of how hydrothermal vents affect the ocean and global geochemical cycles, and how they influence our views of life on the early Earth and the search for life beyond our own planet.

Articles in this special issue of Oceanography represent a compendium of research that spans the disciplinary and thematic breadth of the National Science Foundation's Ridge 2000 Program, as well as its geographic focal points. The mid-ocean ridge (MOR) crest is where much of Earth's volcanism is focused and where most submarine volcanic activity occurs. If we could look down from space at our planet with the ocean drained, the MOR's topography and shape, along with its intervening fracture zones, would resemble the seams on a baseball, with the ocean basins dominating our planetary panorama. The volcanic seafloor is hidden beneath the green-blue waters of the world's ocean, yet therein lie fundamental clues to how our planet works and has evolved over billions of years, something that was not clearly understood 65 years ago—witness the following quote from H.H. Hess (1962) in his essay on "geopoetry" and commentary on J.H.F. Umbgrove's (1947) comprehensive summary of Earth and ocean history: The birth of the oceans is a matter of conjecture, the subsequent history is obscure, and the present structure is just beginning to be understood. Fascinating speculation on these subjects has been plentiful, but not much of it predating the last decade [the 1950s] holds water.

Endeavour Segment of the Juan de Fuca Ridge is one of three Integrated Study Sites for the Ridge 2000 Program. It is a remarkable, dynamic environment hosting five major hydrothermal fields, numerous smaller fields, and myriad diffuse-flow sites; magma chambers underlie all fields. Over 800 individual extinct and active chimneys have been documented within the central ~ 15 km portion of the ridge, with some edifices reaching 50 m across and up to 45 m tall. Fluid flow is focused along faults within the rift zone, and seismically active faults along the western axial valley wall have been used by both magmas and upwelling hydrothermal fluids. There is significant chemical heterogeneity in basalt compositions within the axial rift valley, with the greatest diversity occurring near the base of the western axial valley wall where normal, transitional, and enriched type mid-ocean ridge basalts occur within tens of meters of each other. Endeavour is the only site where seismic intensity has been linked directly to heat flux at the individual vent field scale. Installation of the world's first high-power and high-bandwidth cabled observatory at Endeavour via NEPTUNE Canada ensures that new discoveries along the Juan de Fuca Ridge will continue into the future.

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

Hydrothermal fluids (341°C and 19°C) were collected 1 m apart from a black smoker chimney and a tubeworm mound on the Boardwalk edifice at the Endeavour Segment in the northeastern Pacific Ocean to study anaerobic microbial growth in hydrothermal mineral deposits. Geochemical modelling of mixed vent fluid and seawater suggests the mixture was anoxic above 55°C and that low H2 concentrations (79 μmol kg(-1) in end-member hydrothermal fluid) limit anaerobic hydrogenotrophic growth above this temperature. A thermophilic, hydrogenotrophic sulfur reducer, Desulfurobacterium strain HR11, was isolated from the 19°C fluid raising questions about its H2 -dependent growth kinetics. Strain HR11 grew at 40-77°C (Topt 72-75°C), pH 5-8.5 (pHopt 6-7) and 1-5% (wt vol(-1) ) NaCl (NaClopt 3-4%). The highest growth rates occurred when S2 O3 (2-) and S° were reduced to H2 S. Modest growth occurred by NO3 (-) reduction. Monod constants for its growth were Ks of 30 μM for H2 and Ks of 20 μM for S2 O3 (2-) with a μmax of 2.0 h(-1) . The minimum H2 and S2 O3 (2-) concentrations for growth were 3 μM and 5 μM respectively. Possible sources of S2 O3 (2-) and S° are from abiotic dissolved sulfide and pyrite oxidation by O2 .

The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95°C autotrophically, heterotrophically, and mixotrophically with acetate, H 2 , and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H 2 and 0.001% yeast extract with and without 0.05% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001% yeast extract was not present, and addition of H 2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14 C-labeled acetate in the presence of H 2 and yeast extract with an efficiency of 55%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.

The combined effect of hydrostatic pressure and heat shock on thermotolerance was examined in the deep-sea hyperthermophilic archaeon Pyrococcus strain ES4. Pressure equivalent to the depth of isolation (22 MPa) enhanced ES4's survival at super-optimal temperatures (101–108°C) relative to low pressure (3 MPa). Pressure also raised the temperature at which a putative heat-shock protein (98 kDa) accumulated. ES4 grown at 95°C and 3 MPa displayed immediate enhanced thermotolerance to 105°C after being shifted to 22 MPa. Cultures grown at 95°C and 22 MPa and then heat shocked at 105°C and 3 MPa retained enhanced thermotolerance after decompression. These results suggest that this deep-sea hyperthermophile has developed pressure-induced responses that include increased survival to hyperthermal conditions.

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

Marine hydrothermal microorganisms respond rapidly to changes in the concentrations and availability of metals within their environment. Hyperthermophilic archaea appear to possess novel mechanisms for metal detoxification, dissimilatory metal reduction and metal assimilation that may be absent in their mesophilic and bacterial counterparts. For example, tungsten was found in high concentrations in a hydrothermal sulfide deposit where hyperthermophiles were also most abundant, consistent with the unique requirement of these organisms for this element. Furthermore, newly isolated genera of iron-reducing hyperthermophiles expand the scope of carbon cycling in hydrothermal environments. The advent of genome sequences and new molecular techniques will facilitate our further understanding of microbe–mineral interactions in these environments.

Methane is a key component in the global carbon cycle with a wide range of anthropogenic and natural sources. Although isotopic compositions of methane have traditionally aided source identification, the abundance of its multiply-substituted “clumped” isotopologues, e.g., 13CH3D, has recently emerged as a proxy for determining methane-formation temperatures; however, the impact of biological processes on methane’s clumped isotopologue signature is poorly constrained. We show that methanogenesis proceeding at relatively high rates in cattle, surface environments, and laboratory cultures exerts kinetic control on 13CH3D abundances and results in anomalously elevated formation temperature estimates. We demonstrate quantitatively that H2 availability accounts for this effect. Clumped methane thermometry can therefore provide constraints on the generation of methane in diverse settings, including continental serpentinization sites and ancient, deep groundwaters., National Science Foundation (U.S.) (EAR-1250394), National Science Foundation (U.S.) (EAR-1322805), Deep Carbon Observatory (Program), Natural Sciences and Engineering Research Council of Canada, Deutsche Forschungsgemeinschaft (Gottfried Wilhelm Leibniz Program), United States. Dept. of Defense (National Defense Science and Engineering Graduate Fellowship), Neil & Anna Rasmussen Foundation, Grayce B. Kerr Fund, Inc. (Fellowship), MIT Energy Initiative (Shell-MITEI Graduate Fellowship), Shell International Exploration and Production B.V. (N. Braunsdorf and D. Smit of Shell PTI/EG grant)