Novel Intact Polar and Core Lipid Compositions in the Pyrococcus Model Species, P. furiosus and P. yayanosii, Reveal the Largest Lipid Diversity Amongst Thermococcales.

Elucidating the lipidome of Archaea is essential to understand their tolerance to extreme environmental conditions. Previous characterizations of the lipid composition of Pyrococcus species, a model genus of hyperthermophilic archaea belonging to the Thermococcales order, led to conflicting results, which hindered the comprehension of their membrane structure and the putative adaptive role of their lipids. In an effort to clarify the lipid composition data of the Pyrococcus genus, we thoroughly investigated the distribution of both the core lipids (CL) and intact polar lipids (IPL) of the model Pyrococcus furiosus and, for the first time, of Pyrococcus yayanosii, the sole obligate piezophilic hyperthermophilic archaeon known to date. We showed a low diversity of IPL in the lipid extract of P. furiosus, which nonetheless allowed the first report of phosphatidyl inositol-based glycerol mono- and trialkyl glycerol tetraethers. With up to 13 different CL structures identified, the acid methanolysis of Pyrococcus furiosus revealed an unprecedented CL diversity and showed strong discrepancies with the IPL compositions reported here and in previous studies. By contrast, P. yayanosii displayed fewer CL structures but a much wider variety of polar heads. Our results showed severe inconsistencies between IPL and CL relative abundances. Such differences highlight the diversity and complexity of the Pyrococcus plasma membrane composition and demonstrate that a large part of its lipids remains uncharacterized. Reassessing the lipid composition of model archaea should lead to a better understanding of the structural diversity of their lipidome and of their physiological and adaptive functions.

Pyrococcus kukulkanii sp. nov., a hyperthermophilic, piezophilic archaeon isolated from a deep-sea hydrothermal vent.

A novel hyperthermophilic, piezophilic, anaerobic archaeon, designated NCB100T, was isolated from a hydrothermal vent flange fragment collected in the Guaymas basin at the hydrothermal vent site named 'Rebecca's Roost' at a depth of 1997 m. Enrichment and isolation were performed at 100 °C under atmospheric pressure. Cells of strain NCB100T were highly motile, irregular cocci with a diameter of ~1 µm. Growth was recorded at temperatures between 70 and 112 °C (optimum 105 °C) and hydrostatic pressures of 0.1-80 MPa (optimum 40-50 MPa). Growth was observed at pH 3.5-8.5 (optimum pH 7) and with 1.5-7 % NaCl (optimum at 2.5-3 %). Strain NCB100T was a strictly anaerobic chemo-organoheterotroph and grew on complex proteinaceous substrates such as yeast extract, peptone and tryptone, as well as on glycogen and starch. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The fermentation products from complex proteinaceous substrates were CO2 and H2. The G+C content of the genomic DNA was 41.3 %. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain NCB100T belongs to the genus Pyrococcus, showing 99 % similarity with the other described species of the genus Pyrococcus. On the basis of physiological characteristics, DNA G+C content, similarity level between ribosomal proteins and an average nucleotide identity value of 79 %, strain NCB100T represents a novel species for which the name Pyrococcus kukulkanii sp. nov. is proposed. The type strain is NCB100T (=DSM 101590T=Souchothèque de Bretagne BG1337T).

Analysis of the complete genome sequence of the archaeon Pyrococcus chitonophagus DSM 10152 (formerly Thermococcus chitonophagus).

Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-ß-D glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation.

Molecular dynamics simulations of the Nip7 proteins from the marine deep- and shallow-water Pyrococcus species.

BACKGROUND: The identification of the mechanisms of adaptation of protein structures to extreme environmental conditions is a challenging task of structural biology. We performed molecular dynamics (MD) simulations of the Nip7 protein involved in RNA processing from the shallow-water (P. furiosus) and the deep-water (P. abyssi) marine hyperthermophylic archaea at different temperatures (300 and 373 K) and pressures (0.1, 50 and 100 MPa). The aim was to disclose similarities and differences between the deep- and shallow-sea protein models at different temperatures and pressures. RESULTS: The current results demonstrate that the 3D models of the two proteins at all the examined values of pressures and temperatures are compact, stable and similar to the known crystal structure of the P. abyssi Nip7. The structural deviations and fluctuations in the polypeptide chain during the MD simulations were the most pronounced in the loop regions, their magnitude being larger for the C-terminal domain in both proteins. A number of highly mobile segments the protein globule presumably involved in protein-protein interactions were identified. Regions of the polypeptide chain with significant difference in conformational dynamics between the deep- and shallow-water proteins were identified. CONCLUSIONS: The results of our analysis demonstrated that in the examined ranges of temperatures and pressures, increase in temperature has a stronger effect on change in the dynamic properties of the protein globule than the increase in pressure. The conformational changes of both the deep- and shallow-sea protein models under increasing temperature and pressure are non-uniform. Our current results indicate that amino acid substitutions between shallow- and deep-water proteins only slightly affect overall stability of two proteins. Rather, they may affect the interactions of the Nip7 protein with its protein or RNA partners.

A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms.

Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).

Complete genome sequence of the obligate piezophilic hyperthermophilic archaeon Pyrococcus yayanosii CH1.

Pyrococcus yayanosii CH1 is the first obligate piezophilic hyperthermophilic archaeon isolated from the deep-sea hydrothermal site Ashadze on the mid-Atlantic ridge at a depth of 4,100 m. This organism grows within a temperature range of 80 to 108°C and a hydrostatic pressure range of 20 to 120 MPa, with optima at 98°C and 52 MPa, respectively. Here, we report the complete genome sequence (1,716,817 bp, with a G+C content of 51.6%) of the type strain P. yayanosii CH1(T) (= JCM 16557). This genomic information reveals a systematic view of the piezoadaptation strategy and evolution scenario of metabolic pathways in Thermococcales.

Complete genome sequence of hyperthermophilic Pyrococcus sp. strain NA2, isolated from a deep-sea hydrothermal vent area.

Pyrococcus sp. strain NA2, isolated from a deep-sea hydrothermal vent sample, is a novel marine hyperthermophilic archaeon that grows optimally at 93 °C. The complete genome sequence of the strain contains all the genes for the tricarboxylic acid cycle except for succinate dehydrogenase/fumarate reductase, but the genome does not encode proteins involved in polysaccharide utilization.

Pyrococcus yayanosii sp. nov., an obligate piezophilic hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.

An obligate piezophilic anaerobic hyperthermophilic archaeon, designated strain CH1(T), was isolated from a hydrothermal vent site named 'Ashadze', which is located on the Mid-Atlantic Ridge at a depth of 4100 m. Enrichment and isolation of the strain were carried out at 95 °C under a hydrostatic pressure of 42 MPa. Cells of strain CH1(T) were highly motile irregular cocci with a diameter of ~1-1.5 µm. Growth was recorded at 80-108 °C (optimum 98 °C) and at pressures of 20-120 MPa (optimum 52 MPa). No growth was observed under atmospheric pressures at 60-110 °C. Growth was observed at pH 6.0-9.5 (optimum 7.5-8.0) and in 2.5-5.5% (w/v) NaCl (optimum 3.5%). Strain CH1(T) was strictly anaerobic and grew on complex proteinaceous substrates, such as yeast extract, Peptone, and casein, as well as on sucrose, starch, chitin, pyruvate, acetate and glycerol without electron acceptors. The G+C content of the genomic DNA was 49.0±0.5 mol%. Analysis of 16S rRNA gene sequences revealed that strain CH1(T) belongs to the genus Pyrococcus. Based on its physiological properties and similarity levels between ribosomal proteins, strain CH1(T) represents a novel species, for which the name Pyrococcus yayanosii sp. nov. is proposed. The type strain is CH1(T) (=JCM 16557). This strain is also available by request from the Souchothèque de Bretagne (catalogue LMBE) culture collection (collection no. 3310).

A molecular and physiological survey of a diverse collection of hydrothermal vent Thermococcus and Pyrococcus isolates.

Strains of hyperthermophilic anaerobic hydrothermal vent archaea maintained in the culture collection assembled by Holger Jannasch at the Woods Hole Oceanographic Institution between 1984 and 1998 were identified and partially characterized by Denaturing Gradient Gel Electrophoresis, 16S rRNA gene sequencing, and by growth tests at different temperatures and on different organic carbon and nitrogen sources. All strains were members of the genera Thermococcus and Pyrococcus. The greatest phylogenetic diversity was found in strains from a single Guaymas Basin core isolated by serial dilution from four different depth horizons of heated sediment incubated at the corresponding in situ temperatures. In contrast, geographically distinct vent locations and sample materials yielded a lower diversity of isolates when enriched under uniform temperature regimes and without prior dilution of the source material.

Pyrococcus CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life.

A novel hydrothermal site was discovered in March 2007, on the mid-Atlantic ridge during the cruise 'Serpentine'. At a depth of 4100 m, the site 'Ashadze' is the deepest vent field known so far. Smoker samples were collected with the ROV 'Victor 6000' and processed in the laboratory for the enrichment of anaerobic heterotrophic microorganisms under high-temperature and high-hydrostatic pressure conditions. Strain CH1 was successfully isolated and assigned to the genus Pyrococcus, within the Euryarchaeota lineage within the Archaea domain. This organism grows within a temperature range of 80 to 108 degrees C and a pressure range of 20 to 120 MPa, with optima for 98 degrees C and 52 MPa respectively. Pyrococcus CH1 represents the first obligate piezophilic hyperthermophilic microorganism known so far. Comparisons of growth yields obtained under high-temperature/high-pressure conditions for relative organisms isolated from various depths, showed clear relationships between depth at origin and responses to hydrostatic pressure.

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus Pyrococcus from an Algerian hydrothermal hot spring.

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80-85 degrees C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.

Compatible solutes of the hyperthermophile Palaeococcus ferrophilus: osmoadaptation and thermoadaptation in the order thermococcales.

The effect of salinity and growth temperature on the accumulation of intracellular organic solutes was examined in the hyperthermophilic archaeon Palaeococcus ferrophilus. The genus Palaeococcus represents a deep-branching lineage of the order Thermococcales, which diverged before Thermococcus and Pyrococcus. Palaeococcus ferrophilus accumulated mannosylglycerate, glutamate, and aspartate as major compatible solutes. Unlike members of the genera Pyrococcus and Thermococcus, Palaeococcus ferrophilus did not accumulate di-myo-inositol phosphate, a canonical solute of hyperthermophiles. The level of mannosylglycerate increased in response to both heat and salt stress; glutamate increased at supraoptimal growth temperatures, whereas aspartate increased at supraoptimal salt concentration. Proline, alanine, and trehalose were also found in lesser amounts, but their levels did not respond significantly to any of the stresses imposed. Additionally, the genes involved in the synthesis of mannosylglycerate in Palaeococcus ferrophilus and Thermococcus litoralis were identified. In both organisms the synthesis proceeds via the two-step pathway comprising mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The mpgS and mpgP genes of Palaeococcus ferrophilus were expressed in Escherichia coli and the proteins were characterized. MPGS had maximal activity at 90 degrees C and pH near 7.0, and was strictly dependent on Mg2+. MPGP had optimal activity at 90 degrees C and pH 6.0 and was barely dependent on Mg2+. The half-life values for inactivation of MPGS and MPGP at 83 degrees C were 18 and 25 min, respectively. A comparative discussion of the osmo- and thermoadaptation strategies in these three genera of the Thermococcales is presented.

Molecular diversity of new Thermococcales isolates from a single area of hydrothermal deep-sea vents as revealed by randomly amplified polymorphic DNA fingerprinting and 16S rRNA gene sequence analysis.

Members of the Thermococcales are anaerobic Archaea belonging to the kingdom Euryarchaea that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new Thermococcales isolates collected from six different chimneys of a single hydrothermal field located in the 13 degrees N 104 degrees W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of Thermococcus hydrothermalis, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to Pyrococcus strains, whereas all the other isolates are Thermococcus strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of Thermococcales: About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.

Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1.

A hyperthermophilic archaeal strain, KOD1, isolated from a solfatara on Kodakara Island, Japan, has previously been reported as Pyrococcus sp. KOD1. However, a detailed phylogenetic tree, made possible by the recent accumulation of 16S rRNA sequences of various species in the order Thermococcales, indicated that strain KOD1 is a member of the genus Thermococcus. We performed DNA-DNA hybridization tests against species that displayed high similarity in terms of 16S ribosomal DNA sequences, including Thermococcus peptonophilus and Thermococcus stetteri. Hybridization results and differences in growth characteristics and substrate utilization differentiated strain KOD1 from T. peptonophilus and T. stetteri at the species level. Our results indicate that strain KOD1 represents a new species of Thermococcus, which we designate as Thermococcus kodakaraensis KOD1 sp. nov.

A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei.

Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures approaching 100 degrees C. All species grow heterotrophically and produce H2 or, in the presence of elemental sulfur (S(o)), H2S. Pyrococcus woesei and P. furiosus were isolated from marine sediments at the same Vulcano Island beach site and share many morphological and physiological characteristics. We report here that the rDNA operons of these strains have identical sequences, including their intergenic spacer regions and part of the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10-100 microM sodium tungstate in S(o)-free medium. However, P. woesei shows more extensive autolysis than P. furiosus in the stationary phase. Pyrococcus furiosus and P. woesei share three closely related families of insertion sequences (ISs). A Southern blot performed with IS probes showed extensive colinearity between the genomes of P. woesei and P. furiosus. Cloning and sequencing of ISs that were in different contexts in P. woesei and P. furiosus revealed that the napA gene in P. woesei is disrupted by a type III IS element, whereas in P. furiosus, this gene is intact. A type I IS element, closely linked to the napA gene, was observed in the same context in both P. furiosus and P. woesei genomes. Our results suggest that the IS elements are implicated in genomic rearrangements and reshuffling in these closely related strains. We propose to rename P. woesei a subspecies of P. furiosus based on their identical rDNA operon sequences, many common IS elements that are shared genomic markers, and the observation that all P. woesei nucleotide sequences deposited in GenBank to date are > 99% identical to P. furiosus sequences.

Expanding tRNA recognition of a tRNA synthetase by a single amino acid change.

Aspartyl-tRNA synthetase (AspRS) occurs in two types: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), whereas the nondiscriminating enzyme (ND-AspRS) also synthesizes Asp-tRNA(Asn), which is a required intermediate for protein synthesis in many organisms. We attempted to expand the tRNA recognition of the discriminating Thermococcus kodakaraensis AspRS to that of a ND-AspRS by in vitro mutagenesis. An alignment of 26 archaeal AspRS proteins revealed two positions (26 and 85 in the T. kodakaraensis sequence) whose amino acid identity changes according to the enzymes' tRNA specificity. In their anticodon-binding domain, D-AspRS proteins contain W26 (or Q26) and K85, compared with H26 and P85 in the ND-AspRSs. T. kodakaraensis AspRS gained the ability to form Asp-tRNA(Asn) in vitro when the W26H or K85P changes were introduced independently or in combination. In the aminoacylation of tRNA(Asn) or tRNA(Asp) transcripts, the mutant enzymes displayed at least a 100- to 500-fold change in tRNA specificity, as judged by the ratio of the k(cat)K(m) values of Asp-tRNA(Asp) vs. Asp-tRNA(Asn) formation. That T. kodakaraensis mutant AspRSs mischarge tRNA(Asn) was also manifested in the higher level (1.7%) of aspartylation of unfractionated Pyrococcus tRNA compared with that achieved by the wild-type enzyme (0.9%). Northern blot analysis of the Asp-tRNA separated by acidurea gel electrophoresis confirmed the in vitro synthesis of Asp-tRNA(Asn). A structure-based model points to a direct interaction of K85 in T. kodakaraensis AspRS with the anticodon nucleotide C36 of tRNA(Asp). Thus, a switch between D-AspRS and ND-AspRS enzymes could have evolved with only limited amino acid changes.

Archaeal fructose-1,6-bisphosphate aldolases constitute a new family of archaeal type class I aldolase.

Fructose-1,6-bisphosphate (FBP) aldolase activity has been detected previously in several Archaea. However, no obvious orthologs of the bacterial and eucaryal Class I and II FBP aldolases have yet been identified in sequenced archaeal genomes. Based on a recently described novel type of bacterial aldolase, we report on the identification and molecular characterization of the first archaeal FBP aldolases. We have analyzed the FBP aldolases of two hyperthermophilic Archaea, the facultatively heterotrophic Crenarchaeon Thermoproteus tenax and the obligately heterotrophic Euryarchaeon Pyrococcus furiosus. For enzymatic studies the fba genes of T. tenax and P. furiosus were expressed in Escherichia coli. The recombinant FBP aldolases show preferred substrate specificity for FBP in the catabolic direction and exhibit metal-independent Class I FBP aldolase activity via a Schiff-base mechanism. Transcript analyses reveal that the expression of both archaeal genes is induced during sugar fermentation. Remarkably, the fbp gene of T. tenax is co-transcribed with the pfp gene that codes for the reversible PP(i)-dependent phosphofructokinase. As revealed by phylogenetic analyses, orthologs of the T. tenax and P. furiosus enzyme appear to be present in almost all sequenced archaeal genomes, as well as in some bacterial genomes, strongly suggesting that this new enzyme family represents the typical archaeal FBP aldolase. Because this new family shows no significant sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I FBP aldolases (FBP aldolase Class IA).

The evolution of lipids.

Living organisms on the Earth which are divided into three major domains--Archaea, Bacteria, and Eucarya, probably came from a common ancestral cell. Because there are many thermophilic microorganisms near the root of the universal phylogenetic tree, the common ancestral cell should be considered to be a thermophilic microorganism. The existence of a cell is necessary for the living organisms; the cell membrane is the essential structural component of a cell, so its amphiphilic property is vital for the molecule of lipids for cell membranes. Tetraether type glycerophospholipids with C40 isoprenoid chains are major membrane lipids widely distributed in archaeal cells. Cyclization number of C40 isoprenoid chains in thermophilic archaea influences the fluidity of lipids whereas the number of carbons and degree of unsaturation in fatty acids do so in bacteria and eucarya. In addition to the cyclization of the tetraether lipids, covalent bonding of two C40 isoprenoid chains was found in hyperthermophiles. These characteristic structures of the lipids seem to contribute to their fundamental physiological roles in hyperthermophiles. Stereochemical differences between G-1-P archaeal lipids and G-3-P bacterial and eucaryal lipids might have occurred by the function of some proteins long after the first cell was developed by the reactions of small organic molecules. We propose that the structure of lipids of the common ancestral cell may have been similar to those of hyperthermophilic archaea.

Pyrococcus glycovorans sp. nov., a hyperthermophilic archaeon isolated from the East Pacific Rise.

A hyperthermophilic archaeon, strain AL585T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at latitude 13 degrees N and a depth of 2650 m. The isolate was a strictly anaerobic coccus with a mean cell diameter of 1 micron. The optimum temperature, pH and concentration of sea salt for growth were 95 degrees C, 7.5 and 30 g l-1. Under these conditions, the doubling time and cell yield were 0.5 h and 5 x 10(8) cells ml-1. Strain AL585T grew preferentially in media containing complex proteinaceous carbon sources, glucose and elemental sulfur. The G + C content of the DNA was 47 mol%. Sequencing of the 16S rDNA gene showed that strain AL585T belonged to the genus Pyrococcus and was probably a new species. This was confirmed by total DNA hybridization. Consequently, this strain is described as a new species, Pyrococcus glycovorans sp. nov.

Archaeon Pyrococcus kodakaraensis KOD1: application and evolution.

Archaea is the third domain which is phylogenetically differentiated from the other two domains, bacteria and eucarya. Hyperthermophile within the archaea domain has evolved most slowly retaining many ancestral features of higher eukaryotes. Pyrococcus kodakaraensis KOD1, which grows at 95 degrees C optimally, is a newly isolated hyperthermophilc archaeon. The KOD1 strain possesses a circular genome, whose size is estimated to be approximately 2,036 kb. KOD1 enzymes involved in the genetic information processing system, such as DNA polymerase, Rec protein, aspartyl tRNA synthetase and molecular chaperonin, share features of eukaryotic enzymes. Rapid and accurate PCR method by KOD1 DNA polymerase and enzyme stabilization system by KOD1 chaperonin are also introduced in this article.