Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus

Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H₂S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of ³⁵S-labeled elemental sulfur was detected. However, [³⁵S]cysteine and [³⁵S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS⁻, which in turn opens up the S₈ elemental sulfur ring. In addition to H₂S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion.     

Production of hydrogen from α-1,4- and β-1,4-linked saccharides by marine hyperthermophilic Archaea

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism Pyrococcus furiosus, were examined for their ability to grow and produce H₂ on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H₂ even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only P. furiosus had a gene for an extracellular β-1,4-endoglucanase. P. furiosus was serially adapted for growth on CF11 cellulose and H₂ production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H₂ production rates were 29 fmol, 37 fmol, and 54 fmol of H₂ produced cell⁻¹ doubling⁻¹ on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H₂ production rate came from growth on starch (2.6 mM H₂ produced h⁻¹). Hyperthermophilic heterotrophs may serve as an important alternate source of H₂ for hydrogenotrophic microorganisms in low-H₂ hydrothermal environments, and some are candidates for H₂ bioenergy production in bioreactors.     

Effect of Cultivation Conditions on the Growth and Activities of Sulfur Metabolism Enzymes and Carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes Strain 41

The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold–arsenic concentrate and elemental sulfur as a source of energy. Growth in the presence of S⁰ under auto- or mixotrophic conditions was less stable than in media containing iron monoxide. The enzymes involved in the oxidation of sulfur inorganic compounds—thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodanase, adenylyl phosphosulfate reductase, sulfite oxidase, and sulfur oxygenase—were determined in the cells of the sulfobacilli grown in mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle—ribulose bisphosphate carboxylase—and several other enzymes involved in the heterotrophic fixation of carbon dioxide. Activities of carboxylases depended on the composition of the cultivation media.     

An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO₂, and H₂, together with minor amounts of ethanol. The organism also generates H₂S in the presence of elemental sulfur (S⁰). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S⁰. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (~1.0 g-atom/subunit) and zinc (~1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent K_ms (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.     

Influence of tungsten on metabolic patterns in Pyrococcus furiosus,a hyperthermophilic archaeon

Pyrococcus furiosus is a strictly anaerobic heterotroph that grows optimally around 100 °C. It can be cultured in an artificial seawater-based medium with either peptides or maltose as the carbon source. Significant stimulation of cell yields were observed when trace levels of tungsten (as tungstate) were added to an energy-limited chemostat culture of P. furiosus when maltose is present, but not when peptides were the sole carbon source. The addition of tungsten also led to dramatic increases in the specific activities within cell-free extracts of aldehyde ferredoxin oxidoreductase (AOR), which is a tungsten-iron-sulfur protein. Moreover, the addition of tungsten to cells growing in maltose/peptide medium dramatically reduced the specific activity of intracellular proteases, suggesting a preference for the utilization of maltose over peptides as the carbon and energy source in the presence of tungsten.Non-standard abbreviations EPPS N-[2-Hydroxyethyl]-piperazine-N-[3-propane-sulfonic acid] - VFA volatile fatty acids - LNA 1-Lys-p-nitroaniline - MeOSAPTNA MeO-Suc-Arg-Pro-Tyr-p-nitroaniline - AOR aldehyde ferredoxin oxidoreductase     

Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis

Enzymatic characterization of the four group 3 pyridine nucleotide disulfide oxidoreductase (PNDOR) homologues TK1299, TK0304, TK0828, and TK1481 from Thermococcus kodakarensis was performed, with a focus on their CoA-dependent NAD(P)H: elemental sulfur (S⁰) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H₂S from S⁰ and free CoA–SH was observed, and the addition of enzyme and NADPH enhanced H₂S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA–SH acted to solubilize S⁰ by forming CoA persulfides, followed by reduction of an enzyme–S–S–CoA intermediate produced after both enzymatic and non-enzymatic evolution of H₂S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA–SH, implying a direct reaction with S⁰. TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA–SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S⁰-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S⁰, suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S⁰ was absent.     

Involvement of Intermediate Sulfur Species in Biological Reduction of Elemental Sulfur under Acidic,Hydrothermal Conditions

The thermoacidophile and obligate elemental sulfur (S₈⁰)-reducing anaerobe Acidilobus sulfurireducens 18D70 does not associate with bulk solid-phase sulfur during S₈⁰-dependent batch culture growth. Cyclic voltammetry indicated the production of hydrogen sulfide (H₂S) as well as polysulfides after 1 day of batch growth of the organism at pH 3.0 and 81°C. The production of polysulfide is likely due to the abiotic reaction between S₈⁰ and the biologically produced H₂S, as evinced by a rapid cessation of polysulfide formation when the growth temperature was decreased, inhibiting the biological production of sulfide. After an additional 5 days of growth, nanoparticulate S₈⁰ was detected in the cultivation medium, a result of the hydrolysis of polysulfides in acidic medium. To examine whether soluble polysulfides and/or nanoparticulate S₈⁰ can serve as terminal electron acceptors (TEA) supporting the growth of A. sulfurireducens, total sulfide concentration and cell density were monitored in batch cultures with S₈⁰ provided as a solid phase in the medium or with S₈⁰ sequestered in dialysis tubing. The rates of sulfide production in 7-day-old cultures with S₈⁰ sequestered in dialysis tubing with pore sizes of 12 to 14 kDa and 6 to 8 kDa were 55% and 22%, respectively, of that of cultures with S₈⁰ provided as a solid phase in the medium. These results indicate that the TEA existed in a range of particle sizes that affected its ability to diffuse through dialysis tubing of different pore sizes. Dynamic light scattering revealed that S₈⁰ particles generated through polysulfide rapidly grew in size, a rate which was influenced by the pH of the medium and the presence of organic carbon. Thus, S₈⁰ particles formed through abiological hydrolysis of polysulfide under acidic conditions appeared to serve as a growth-promoting TEA for A. sulfurireducens.     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.     

Characterization of elemental sulfur in isolated intact spinach chloroplasts

Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of ³⁵SO₄²⁻ resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S₈) of elemental sulfur (S⁰, valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S⁰) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S₈ under the experimental conditions used for analysis.     

Isolation, Characterization, and In Situ Detection of a Novel Chemolithoautotrophic Sulfur-Oxidizing Bacterium in Wastewater Biofilms Growing under Microaerophilic Conditions

We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S⁰), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 μm) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H₂S and S⁰ to SO₄²⁻ under oxic conditions.     

Somatic embryogenesis and plant regeneration in yakutanegoyou, <Emphasis Type="Italic">Pinus armandii</Emphasis> Franch. var. <Emphasis Type="Italic">amamiana</Emphasis> (Koidz.) Hatusima,an endemic and endangered species in Japan

Induction of somatic embryogenesis in Pinus armandii var. amamiana, an endemic and endangered species in Japan, was initiated from megagametophytes containing immature zygotic embryos on both media with and without plant growth regulators. Across nine open-pollinated families initiation frequency ranged from 0 to 20%, with an average of 1.5%. Embryogenic cultures were maintained and proliferated on a medium supplemented with 2,4-dichlorophenoxyacetic acid (3 μM) and 6-benzylaminopurine (1 μM). Maturation of somatic embryos occurred on medium containing maltose (50 g l⁻¹), activated charcoal (2 g l⁻¹), abscisic acid (100 μM), and polyethylene glycol (100 g l⁻¹). The frequencies of germination and plant conversion of somatic embryos differed among the embryogenic lines from 16 to 51% and from 12 to 40%, respectively. Growth of regenerated somatic plants has been monitored in the field.     

Growth of the haploid phase of the myxomycete Physarum flavicomum in defined minimal medium

The haploid phase (myxamoebae-swarm cells) of the myxomycete Physarum flavicomum grew readily in chemically defined liquid media. The minimal medium contained salts, glucose, biotin, thiamine, hematin, glycine, l-arginine and l-methionine. Cell yields of 1.4x10⁷ cells/ml were obtained in this medium in aerobic shake culture. These cells consumed about 35 μliters of oxygen/mg protein·hr in the minimal medium. The morphology of cells maintained in this medium appeared to be “normal”. l-valine replaced either glycine or l-methionine in the minimal medium but the growth rates and cell yields were reduced. Growth rates increased in media containing four, seven, or fourteen amino acids.     

Elemental sulfur production during mixotrophic growth on hydrogen and thiosulfate of thermophilic hydrogen-oxidizing bacteria

In this study we measured the exogenous production and the intracellular content of elemental sulfur (S⁰) in the thermophilic sulfur-oxidizing bacteriaHydrogenobacter spp. andBacillus schlegelii during mixolithoautotrophic growth on hydrogen and thiosulfate. Under these conditions, all strains produced and released white-yellow hydrophilic S⁰ particles into the growth medium. Hydrophilic S⁰ was separated from cells by a differential low-speed centrifugation procedure. The S⁰ pellets were dried, and the S⁰ was purified by column chromatography and by thin-layer chromatography (TLC). The S⁰ TLC-band could be stained with triphenyltetrazolium chloride and piperidine procedure. Determination of intracellular S⁰ content was performed from fresh cells absolutely free of exogenous S⁰ particles. quantitation of S⁰ was performed by high-performance liquid chromatography, colorimetric thiocyanate procedure, and by UV-spectra analyses. All the strains studied, in particularB. schlegelii strains, released significant quantities of S⁰ into the growth media. In contrast, the intracellular S⁰ content was very low. Significant rhodanese activity in the presence of thiosulfate was measured with toluenepermeabilized cells and cell-free extracts ofB. schlegelii (type strain) andHydrogenobacter spp. (strain T3).     

Metabolism in hyperthermophilic microorganisms

Hyperthermophilic microorganisms grow at temperatures of 90 °C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified asArchaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H₂S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H₂ and CO₂, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hyperthermophiles known that can assimilate CO₂ do so via a reductive citric acid cycle. Two S^o-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without S^o. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S^o has been proposed. However, the mechanisms by which S^o reduction is coupled to energy conservation in this organism and in obligate S^o-reducing hyperthermophiles is not known.Abbreviations ADH alcohol dehydrogenase (ADH) - AOR aldehyde ferredoxin oxidoreductase - FMOR formate ferredoxin oxidoreductase - FOR formaldehyde ferredoxin oxidoreductase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glutamate dehydrogenase - GluOR glucose ferredoxin oxidoreductase - KGOR 2-ketoglutarate ferredoxin oxidoreductase - IOR indolepyruvate ferredoxin oxidoreductase - LDH lactate dehydrogenase - MPT molybopterin - POR pyruvate ferredoxin oxidoreductase - PLP pyridoxal-phosphate - PS polysulfide - TPP thiamin pyrophosphate - S^o elemental sulfur - VOR isovalerate ferredoxin oxidoreductase     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

Sulfur accumulation in eelgrass (Zostera marina) and effect of sulfur on eelgrass growth

Eelgrass (Zostera marina) was grown under exposure to high levels of sediment sulfides to examine their ability to reoxidize sulfides intruding into the plants. The plants were kept under full light (control and high sulfide level) and at 10% of light saturation (high sulfide level) for 3 weeks and growth and accumulation of elemental sulfur (S⁰) in the plants were examined. The growth rate was reduced with ∼75% in the low light treatment, whereas there was no significant difference between the rates at full light saturation. S⁰ was accumulating in the below-ground structures of the plants exposed to high sulfide concentrations with highest concentration in the youngest roots and oldest internodes. There was no accumulation of S⁰ in the leaves, suggesting that the intruding sulfides were reoxidized in the below-ground structures before reaching the leaves. The accumulation of S⁰ was higher in the roots of the low light treatment (up to two times) suggesting a larger intrusion of sulfides. These plants also appeared highly affected by the treatment with rotting meristems and increased mortality after the 3-week growth period. These results are the first to show an accumulation of sulfur compounds internally in seagrasses as a result of reoxidation of sulfides. The reoxidation is facilitated by the internal transport of oxygen and is an example of the advantage of the internal lacunae system in seagrasses.     

Factors influencing the uptake and loss of radiosulfate by rat renal cortical tissue in vitro

Rat kidney cortical slices, during incubation in vitro, lose previously accumulated radiosulfur when exposed to conditions (e.g. addition to the medium of metabolic inhibitors) which normally depress the uptake of S³⁵. The extent of this loss is not affected significantly by the presence of phlorhizin, an agent which enhances markedly radiosulfate accumulation. On the other hand, when tissues are chilled to 1°C., loss is slight or negligible even in the presence of metabolic inhibitors. These data, and observations on the effect of pre-incubation of kidney slices in S³⁵-free media before the addition of radiosulfate, have been interpreted as evidence that S³⁵ accumulation in vitro may be resolved into at least two processes, namely (a) entrance of the isotope-labelled anion into the cells, by diffusion and/or active transport, and (b) complexing of S³⁵ (in ionic or other form) with an intracellular component. The postulated complex is stabilized, perhaps through inactivation of a specific enzyme, by chilling the tissue to 1°C. Possible relationships are discussed among the observations noted above, sulfur metabolism in general, and aspects of the known in vivo transport mechanism for sulfate ion; i.e., renal tubular reabsorption.