Pyrofollis japonicus gen. nov. sp. nov., a novel member of the family Pyrodictiaceae isolated from the Iheya North hydrothermal field.

A novel hyperthermophilic, heterotrophic archaeon, strain YC29T, was isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough, Japan. Cells of strain YC29T were non-motile, irregular cocci with diameters of 1.2-3.0 µm. The strain was an obligatory fermentative anaerobe capable of growth on complex proteinaceous substrates. Growth was observed between 85 and 100 °C (optimum 90-95 °C), pH 4.9-6.4 (optimum 5.1), and in the presence of 1.4-4.0% (w/v) NaCl (optimum 3.0%). Inorganic carbon was required as a carbon source. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the family Pyrodictiaceae. The genome size was 2.02 Mbp with a G+C content of 49.4%. The maximum values for average nucleotide identity (ANI), average amino acid identity (AAI), and in silico DNA-DNA hybridization (dDDH) value of strain YC29T with relatives were 67.9% (with Pyrodictium abyssi strain AV2T), 61.1% (with Pyrodictium occultum strain PL-19T), and 33.8% (with Pyrolobus fumarii strain 1AT), respectively. Based on the phylogenetic, genomic, and phenotypic characteristics, we propose that strain YC29T represents a novel genus and species, Pyrofollis japonicus gen. nov., sp. (type strain YC29T = DSM 113394T = JCM 39171T).

Thermostabilization mechanisms in thermophilic versus mesophilic three-helix bundle proteins.

The engineered three-helix bundle, UVF, is thermostabilized entropically due to heightened, native-state dynamics. However, it is unclear whether this thermostabilization strategy is observed in natural proteins from thermophiles. We performed all-atom, explicit solvent molecular dynamics simulations of two three-helix bundles from thermophilic H. butylicus (2lvsN and 2lvsC) and compared their dynamics to a mesophilic three-helix bundle, the Engrailed homeodomain (EnHD). Like UVF, 2lvsC had heightened native dynamics, which it maintained without unfolding at 100°C. Shortening and rigidification of loops in 2lvsN and 2lvsC and increased surface hydrogen bonds in 2lvsN were observed, as is common in thermophilic proteins. A buried disulfide and salt bridge in 2lvsN and 2lvsC, respectively, provided some stabilization, and addition of a homologous disulfide bond in EnHD slowed unfolding. The transferability and commonality of stabilization strategies among members of the three-helix bundle fold suggest that these strategies may be general and deployable in designing thermostable proteins.

Microbe-Mineral Interaction and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi.

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.

Structure of a hyperthermostable dimeric archaeal Rubisco from Hyperthermus butylicus.

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon Hyperthermus butylicus is presented at 1.8 Šresolution. Previous structures of archaeal Rubisco have been found to assemble into decamers, and this oligomerization was thought to be required for a highly thermally stable enzyme. In the current study, H. butylicus Rubisco is shown to exist as a dimer in solution, yet has a thermal denaturation midpoint of 114°C, suggesting that high thermal stability can be achieved without an increased oligomeric state. This increased thermal stability appears to be due to an increased number of electrostatic interactions within the monomeric subunit. As such, H. butylicus Rubisco presents a well characterized system in which to investigate the role of assembly and thermal stability in enzyme function.

Pyrodictium delaneyi sp. nov., a hyperthermophilic autotrophic archaeon that reduces Fe(III) oxide and nitrate.

A hyperthermophilic, autotrophic iron and nitrate reducer, strain Su06T, was isolated from an active deep-sea hydrothermal vent chimney on the Endeavour Segment in the north-eastern Pacific Ocean. It was obligately anaerobic, hydrogenotrophic and reduced Fe(III) oxide to magnetite and NO3- to N2. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was more than 97 % similar to other species of the genera Pyrodictium and Hyperthermus. Therefore, overall genome relatedness index analyses were performed to establish whether strain Su06T represents a novel species. For each analysis, strain Su06T was most similar to Pyrodictium occultum PL-19T. Relative to this strain, the average nucleotide identity score for strain Su06T was 72 %, the genome-to-genome direct comparison score was 13-19 % and the species identification score at the protein level was 89 %. For each analysis, strain Su06T was below the species delineation cutoff. Based on its whole genome sequence and its unique phenotypic characteristics, strain Su06T is suggested to represent a novel species of the genus Pyrodictium, for which the name Pyrodictium delaneyi is proposed. The type strain is Su06T (=DSM 28599T=ATCC BAA-2559T).

Magnetite formation from ferrihydrite by hyperthermophilic archaea from Endeavour Segment, Juan de Fuca Ridge hydrothermal vent chimneys.

Hyperthermophilic iron reducers are common in hydrothermal chimneys found along the Endeavour Segment in the northeastern Pacific Ocean based on culture-dependent estimates. However, information on the availability of Fe(III) (oxyhydr) oxides within these chimneys, the types of Fe(III) (oxyhydr) oxides utilized by the organisms, rates and environmental constraints of hyperthermophilic iron reduction, and mineral end products is needed to determine their biogeochemical significance and are addressed in this study. Thin-section petrography on the interior of a hydrothermal chimney from the Dante edifice at Endeavour showed a thin coat of Fe(III) (oxyhydr) oxide associated with amorphous silica on the exposed outer surfaces of pyrrhotite, sphalerite, and chalcopyrite in pore spaces, along with anhydrite precipitation in the pores that is indicative of seawater ingress. The iron sulfide minerals were likely oxidized to Fe(III) (oxyhydr) oxide with increasing pH and Eh due to cooling and seawater exposure, providing reactants for bioreduction. Culture-dependent estimates of hyperthermophilic iron reducer abundances in this sample were 1740 and 10 cells per gram (dry weight) of material from the outer surface and the marcasite-sphalerite-rich interior, respectively. Two hyperthermophilic iron reducers, Hyperthermus sp. Ro04 and Pyrodictium sp. Su06, were isolated from other active hydrothermal chimneys on the Endeavour Segment. Strain Ro04 is a neutrophilic (pH opt 7-8) heterotroph, while strain Su06 is a mildly acidophilic (pH opt 5), hydrogenotrophic autotroph, both with optimal growth temperatures of 90-92 °C. Mössbauer spectroscopy of the iron oxides before and after growth demonstrated that both organisms form nanophase (<12 nm) magnetite [Fe3 O4 ] from laboratory-synthesized ferrihydrite [Fe10 O14 (OH)2 ] with no detectable mineral intermediates. They produced up to 40 mm Fe(2+) in a growth-dependent manner, while all abiotic and biotic controls produced <3 mm Fe(2+) . Hyperthermophilic iron reducers may have a growth advantage over other hyperthermophiles in hydrothermal systems that are mildly acidic where mineral weathering at increased temperatures occurs.

Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

Dissection of hydrogen bond interaction network around an iron-sulfur cluster by site-specific isotope labeling of hyperthermophilic archaeal Rieske-type ferredoxin.

The electronic structure and geometry of redox-active metal cofactors in proteins are tuned by the pattern of hydrogen bonding with the backbone peptide matrix. In this study we developed a method for selective amino acid labeling of a hyperthermophilic archaeal metalloprotein with engineered Escherichia coli auxotroph strains, and we applied this to resolve the hydrogen bond interactions with the reduced Rieske-type [2Fe-2S] cluster by two-dimensional pulsed electron spin resonance technique. Because deep electron spin-echo envelope modulation of two histidine (14)N(δ) ligands of the cluster decreased non-coordinating (15)N signal intensities via the cross-suppression effect, an inverse labeling strategy was employed in which (14)N amino acid-labeled archaeal Rieske-type ferredoxin samples were examined in an (15)N-protein background. This has directly identified Lys45 N(α) as providing the major pathway for the transfer of unpaired electron spin density from the reduced cluster by a "through-bond" mechanism. All other backbone peptide nitrogens interact more weakly with the reduced cluster. The extension of this approach will allow visualizing the three-dimensional landscape of preferred pathways for the transfer of unpaired spin density from a paramagnetic metal center onto the protein frame, and will discriminate specific interactions by a "through-bond" mechanism from interactions which are "through-space" in various metalloproteins.

Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota.

Two new autotrophic carbon fixation cycles have been recently described in Crenarchaeota. The 3-hydroxypropionate/4-hydroxybutyrate cycle using acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme has been identified for (micro)aerobic members of the Sulfolobales. The dicarboxylate/4-hydroxybutyrate cycle using oxygen-sensitive pyruvate synthase and phosphoenolpyruvate carboxylase as carboxylating enzymes has been found in members of the anaerobic Desulfurococcales and Thermoproteales. However, Sulfolobales include anaerobic and Desulfurococcales aerobic autotrophic representatives, raising the question of which of the two cycles they use. We studied the mechanisms of autotrophic CO(2) fixation in the strictly anaerobic Stygiolobus azoricus (Sulfolobales) and in the facultatively aerobic Pyrolobus fumarii (Desulfurococcales). The activities of all enzymes of the 3-hydroxypropionate/4-hydroxybutyrate cycle were found in the anaerobic S. azoricus. In contrast, the aerobic or denitrifying P. fumarii possesses all enzyme activities of the dicarboxylate/4-hydroxybutyrate cycle. We conclude that autotrophic Crenarchaeota use one of the two cycles, and that their distribution correlates with the 16S rRNA-based phylogeny of this group, rather than with the aerobic or anaerobic lifestyle.

Biosynthesis of long-chain polyamines by crenarchaeal polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum.

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.

The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota.

BACKGROUND: Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. RESULTS: The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced -- Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. CONCLUSION: The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

Di-myo-inositol phosphate and novel UDP-sugars accumulate in the extreme hyperthermophile Pyrolobus fumarii.

The archaeon Pyrolobus fumarii, one of the most extreme members of hyperthermophiles known thus far, is able to grow at temperatures up to 113 degrees C. Over a decade after the description of this organism our knowledge about the structures and strategies underlying its remarkable thermal resistance remains incipient. The accumulation of a restricted number of charged organic solutes is a common response to heat stress in hyperthermophilic organisms and accordingly their role in thermoprotection has been often postulated. In this work, the organic solute pool of P. fumarii was characterized using 1H, 13C, and 31P NMR. Di-myo-inositol phosphate was the major solute (0.21 micromol/mg protein), reinforcing the correlation between the occurrence of this solute and hyperthermophily; in addition, UDP-sugars (total concentration 0.11 micromol/mg protein) were present. The structures of the two major UDP-sugars were identified as UDP-alpha-GlcNAc3NAc and UDP-alpha-GlcNAc3NAc-(4<--1)-beta-GlcpNAc3NAc. Interestingly, the latter compound appears to be derived from the first one by addition of a 2,3-N-acetylglucoronic acid unit, suggesting that these UDP-sugars are intermediates of an N-linked glycosylation pathway. To our knowledge the UDP-disaccharide has not been reported elsewhere. The physiological roles of these organic solutes are discussed.

The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 degrees C.

Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H(2)S. Two large clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment.