Assessment of the Carbon Monoxide Metabolism of the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus VC-16 by Comparative Transcriptome Analyses.

The hyperthermophilic, sulfate-reducing archaeon, Archaeoglobus fulgidus, utilizes CO as an energy source and it is resistant to the toxic effects of high CO concentrations. Herein, transcription profiles were obtained from A. fulgidus during growth with CO and sulfate or thiosulfate, or without an electron acceptor. This provided a basis for a model of the CO metabolism of A. fulgidus. The model suggests proton translocation by "Mitchell-type" loops facilitated by Fqo catalyzing a Fd(red):menaquinone oxidoreductase reaction, as the major mode of energy conservation, rather than formate or H2 cycling during respiratory growth. The bifunctional CODH (cdhAB-2) is predicted to play an ubiquitous role in the metabolism of CO, and a novel nitrate reductase-associated respiratory complex was induced specifically in the presence of sulfate. A potential role of this complex in relation to Fd(red) and APS reduction is discussed. Multiple membrane-bound heterodisulfide reductase (DsrMK) could promote both energy-conserving and non-energy-conserving menaquinol oxidation. Finally, the FqoF subunit may catalyze a Fd(red):F420 oxidoreductase reaction. In the absence of electron acceptor, downregulation of F420H2 dependent steps of the acetyl-CoA pathway is linked to transient formate generation. Overall, carboxidotrophic growth seems as an intrinsic capacity of A. fulgidus with little need for novel resistance or respiratory complexes.

Anaerobic oxidation of long-chain n-alkanes by the hyperthermophilic sulfate-reducing archaeon, Archaeoglobus fulgidus.

The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain VC-16 (DSM 4304), which is known to oxidize fatty acids and n-alkenes, was shown to oxidize saturated hydrocarbons (n-alkanes in the range C10-C21) with thiosulfate or sulfate as a terminal electron acceptor. The amount of n-hexadecane degradation observed was in stoichiometric agreement with the theoretically expected amount of thiosulfate reduction. One of the pathways used by anaerobic microorganisms to activate alkanes is addition to fumarate that involves alkylsuccinate synthase as a key enzyme. A search for genes encoding homologous enzymes in A. fulgidus identified the pflD gene (locus-tag AF1449) that was previously annotated as a pyruvate formate lyase. A phylogenetic analysis revealed that this gene is of bacterial origin and was likely acquired by A. fulgidus from a bacterial donor through a horizontal gene transfer. Based on three-dimensional modeling of the corresponding protein and molecular dynamic simulations, we hypothesize an alkylsuccinate synthase activity for this gene product. The pflD gene expression was upregulated during the growth of A. fulgidus on an n-alkane (C16) compared with growth on a fatty acid. Our results suggest that anaerobic alkane degradation in A. fulgidus may involve the gene pflD in alkane activation through addition to fumarate. These findings highlight the possible importance of hydrocarbon oxidation at high temperatures by A. fulgidus in hydrothermal vents and the deep biosphere.

Phenylalanine catabolism in Archaeoglobus fulgidus VC-16.

Evidence is presented for a pathway of phenylalanine catabolism in the hyperthermophilic archaeon Archaeoglobus fulgidus involving the following enzymes-phenylalanine:2-oxoglutarate aminotransferase, phenyllactate dehydrogenase, radical iron-sulphur 3-phenyllactyl-CoA dehydratase, phenylpropionyl-CoA dehydrogenase, aryl pyruvate ferredoxin oxidoreductase, ADP-forming acetyl-CoA synthetase and family III CoA-transferase. Hitherto amino acid degradation pathways involving radical iron-sulphur dehydratases have been characterised only in mesophilic clostridia and related bacteria. The difference here is that the pathway is not fermentative but coupled to sulphate reduction. Initial experiments also show the utilisation of tryptophan as a growth substrate and the decarboxylation of caffeate by cell extracts, suggesting the potential to catabolise different classes of aromatic compounds.

Anaerobic oxidation of fatty acids and alkenes by the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus oxidizes fatty acids (C(4) to C(18)) and n-alk-1-enes (C(12:1) to C(21:1)) in the presence of thiosulfate as a terminal electron acceptor. End products of metabolism were CO(2) and sulfide. Growth on perdeuterated hexadecene yielded C(15)- to C(17)-labeled fatty acids as metabolites, thus confirming the ability of A. fulgidus to oxidize alkyl chains.

Temperature effect on the sulfur isotope fractionation during sulfate reduction by two strains of the hyperthermophilic Archaeoglobus fulgidus.

Sulfur isotope fractionation during dissimilatory sulfate reduction by two strains of the thermophilic archaeon Archaeoglobus fulgidus (strains VC-16 and Z) was explored over the entire temperature range of growth. The optimal cell-specific sulfate reduction rate (14 fmol cell(-1) h(-1)) was found at 82-84 degrees C but growth was measured as low as 54 degrees C. The fractionation ranged between 0.52 per thousand and 27 per thousand, with largest fractionations were found at intermediate temperatures and the smallest fractionations at the lowest and highest temperatures. There was an inverse relationship between the cell-specific sulfate reduction rate and fractionation, and the cell-specific rate was a good indicator of the expected fractionations regardless of whether temperature or substrate concentrations controlled the rate. Comparison of the fractionation trend found in this study with similar measurements for seven other sulfate-reducers showed that sulfate-reducing organisms respond to temperature in three different ways and this correlated with their maximum fractionation value, but not with the cell-specific sulfate reduction rate. A sulfur isotope model was used to reproduce the observed variation of fractionation with temperature. This approach predicted the rate of internal sulfur transformations as having the major influence on the observed fractionations in the intermediate temperature range, whereas the exchange of sulfate across the cell membrane controls fractionation at low and high temperatures.

Effect of low sulfate concentrations on lactate oxidation and isotope fractionation during sulfate reduction by Archaeoglobus fulgidus strain Z.

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80 degrees C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO2. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 microM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 microM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO2 produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 microM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 microM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.

[High-temperature microbial sulfate reduction can be accompanied by magnetite formation]. / Vysokotemperaturnaia mikrobnaia sulfatreduktsiia mozhet soprovozhdat'sia obrazovaniem magnetita.

The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was found to be capable of lithoautotrophic growth on medium containing molecular hydrogen, sulfate, and amorphous Fe(III) oxide. During the growth of this microorganism, amorphous Fe(III) oxide was transformed into black strongly magnetic sediment rich in magnetite, as shown by Mossbauer studies. Experiments involving inhibition of microbial sulfate reduction and abiotic controls revealed that magnetite production resulted from chemical reactions proceeding at elevated temperatures (83 degrees C) between molecular hydrogen, amorphous Fe(III) oxide, and sulfide formed enzymatically in the course of dissimilatory sulfate reduction. It follows that magnetite production in this system can be characterized as biologically mediated mineralization. This is the first report of magnetite formation as a result of activity of sulfate-reducing microorganisms.

A novel archaeal alanine dehydrogenase homologous to ornithine cyclodeaminase and mu-crystallin.

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.

ADP-dependent glucokinase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324.

The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. The first enzyme of this pathway, ADP-dependent glucokinase, was purified 600-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of 50 kDa. It had a temperature optimum at 83 degrees C and showed a significant thermostability up to 100 degrees C. The enzyme was highly specific for ADP and glucose as substrates; it did not use ATP, CDP, UDP, or GDP as phosphoryl donors, or mannose, fructose and fructose 6-phosphate as phosphoryl acceptors (at 80 degrees C). Only glucosamine was phosphorylated at significant rates. The apparent K(m) values for ADP and glucose (at 50 degrees C) were 0.07 mM and 0.78 mM, respectively; the apparent V(max) value was about 50 U/mg at 50 degrees C and 350 U/mg at 80 degrees C. Divalent cations were required for maximal activity; Mn(2+), Mg(2+ )and Ca(2+), which were most effective, could be replaced partially by Cu(2+), Ni(2+), Co(2+) and Zn(2+). The N-terminal amino acid sequence (42 amino acids) of ADP-dependent glucokinase was almost identical to that of ADP-dependent glucokinase from Thermococcus litoralis. In the genome of the closely related Archaeoglobus fulgidus strain VC16 a homologous gene for ADP-dependent glucokinase could not be identified.

A variant of the hyperthermophile Archaeoglobus fulgidus adapted to grow at high salinity.

A variant of Archaeoglobus fulgidus VC-16 was isolated from cultures obtained after a stepwise transfer from media containing 1.8-6.3% NaCl by a plating-independent, selected-cell cultivation technique, using a laser microscope. This variant, A. fulgidus VC-16S, had a higher growth rate throughout the salt range of the parental strain, but was also able to grow in media containing NaCl up to 6.3%, whereas the parental strain could not grow above 4.5% NaCl. Diglycerol phosphate (DGP), only encountered in the Archaeoglobales, was the major solute accumulated under supra-optimal salinities, whereas at supra-optimal growth temperatures di-myo-inositol phosphate was the predominant solute. The accumulation of compatible solutes during growth of variant VC-16S was lower than in the parental strain within 1.8-4.5% NaCl, but the levels of compatible solutes, including DGP, increased sharply in the variant at higher salinities (5.5 and 6.0%). This variant represents, at this time, one of the most halophilic hyperthermophiles known, and its ability to grow at high salinity appears to be due to the massive accumulation of DGP.

Calibration of sulfate levels in the archean ocean.

The size of the marine sulfate reservoir has grown through Earth's history, reflecting the accumulation of oxygen into the atmosphere. Sulfur isotope fractionation experiments on marine and freshwater sulfate reducers, together with the isotope record, imply that oceanic Archean sulfate concentrations were <200 microM, which is less than one-hundredth of present marine sulfate levels and one-fifth of what was previously thought. Such low sulfate concentrations were maintained by volcanic outgassing of SO2 gas, and severely suppressed sulfate reduction rates allowed for a carbon cycle dominated by methanogenesis.

Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii.

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus(Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.

Room-temperature synthesis of L-alanine using the alanine dehydrogenase of the hyperthermophilic archaeon Archaeoglobus fulgidus.

Alanine dehydrogenase from the hyperthermophilic archaeon Archaeoglobus fulgidus was used at room temperature for batch synthesis of L-alanine by the reductive amination of pyruvate. The reaction mixture included yeast formate dehydrogenase for regeneration of NADH with formate as electron donor. The synthesis of L-alanine at room temperature was accompanied by no detectable loss of alanine dehydrogenase activity over 139 h and > or =99% consumption of pyruvate. The total number of enzyme turnovers was 5.1 million. This work demonstrates the potential utility of novel hyperthermostable enzymes that can be both very active and highly stable at moderate temperature.

Sugar utilization in the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324: starch degradation to acetate and CO2 via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming).

The hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324, rather than the type strain VC16, was found to grow on starch and sulfate as energy and carbon source. Fermentation products and enzyme activities were determined in starch-grown cells and compared to those of cells grown on lactate and sulfate. During exponential growth on starch, 1 mol of glucose-equivalent was incompletely oxidized with sulfate to approximately 2 mol acetate, 2 mol CO2 and 1 mol H2S. Starch-grown cells did not contain measurable amounts of the deazaflavin factor F420 (<0.03 nmol/mg protein) and thus did not show the F420-specific green-blue fluorescence. In contrast, lactate (1 mol) was completely oxidized with sulfate to 3 mol CO2 by strain 7324, and lactate-grown cells contained high amounts of F420 (0.6 nmol/mg protein). In extracts of starch-grown cells, the following enzymes of a modified Embden-Meyerhof pathway were detected: ADP-dependent hexokinase (ADP-HK), phosphoglucose isomerase, ADP-dependent 6-phosphofructokinase (ADP-PFK), fructose-1,6-phosphate aldolase, glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAP:FdOR), phosphoglycerate mutase, enolase, and pyruvate kinase (PK). Specific activities of ADP-HK, ADP-PFK, GAP:FdOR, and PK were significantly higher in starch-grown cells than in lactate-grown cells, indicating induction of these enzymes during starch catabolism. Pyruvate conversion to acetate involved pyruvate:ferredoxin oxidoreductase and ADP-forming acetyl-CoA synthetase. The findings indicate that the archaeal sulfate reducer A. fulgidus strain 7324 converts starch to acetate via a modified Embden-Meyerhof pathway and acetyl-CoA synthetase (ADP-forming). This is the first report of growth of a sulfate reducer on starch, i.e. on a polymeric sugar.

Characterization and sequence comparison of temperature-regulated chaperonins from the hyperthermophilic archaeon Archaeoglobus fulgidus.

We have cloned and sequenced the genes encoding two chaperonin subunits (Cpn-alpha and Cpn-beta), from Archaeoglobus fulgidus, a sulfate-reducing hyperthermophilic archaeon. The genes encode proteins of 545 amino acids with calculated Mr of 58 977 and 59 683. Both proteins have been identified in cytoplasmic fractions of A. fulgidus by Western analysis using antibodies raised against one of the subunits expressed in Escherichia coli, and by N-terminal amino acid sequencing of chaperonin complexes purified by immunoprecipitation. The chaperonin genes appear to be under heat shock regulation, as both proteins accumulate following temperature shift-up of growing A. fulgidus cells, implying a role of the chaperonin in thermoadaptation. Canonical Box A and Box B archaeal promoter sequences, as well as additional conserved putative signal sequences, are located upstream of the start codons. A phylogenetic analysis using all the available archaeal chaperonin sequences, suggests that the alpha and beta subunits are the results of late gene duplications that took place well after the establishment of the main archaeal evolutionary lines.