Structure and Function of Piezophilic Hyperthermophilic Pyrococcus yayanosii pApase.

3'-Phosphoadenosine 5'-monophosphate (pAp) is a byproduct of sulfate assimilation and coenzyme A metabolism. pAp can inhibit the activity of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase and sulfotransferase and regulate gene expression under stress conditions by inhibiting XRN family of exoribonucleases. In metazoans, plants, yeast, and some bacteria, pAp can be converted into 5'-adenosine monophosphate (AMP) and inorganic phosphate by CysQ. In some bacteria and archaea, nanoRNases (Nrn) from the Asp-His-His (DHH) phosphoesterase superfamily are responsible for recycling pAp. In addition, histidinol phosphatase from the amidohydrolase superfamily can hydrolyze pAp. The bacterial enzymes for pAp turnover and their catalysis mechanism have been well studied, but these processes remain unclear in archaea. Pyrococcus yayanosii, an obligate piezophilic hyperthermophilic archaea, encodes a DHH family pApase homolog (PyapApase). Biochemical characterization showed that PyapApase can efficiently convert pAp into AMP and phosphate. The resolved crystal structure of apo-PyapApase is similar to that of bacterial nanoRNaseA (NrnA), but they are slightly different in the α-helix linker connecting the DHH and Asp-His-His associated 1 (DHHA1) domains. The longer α-helix of PyapApase leads to a narrower substrate-binding cleft between the DHH and DHHA1 domains than what is observed in bacterial NrnA. Through mutation analysis of conserved amino acid residues involved in coordinating metal ion and binding substrate pAp, it was confirmed that PyapApase has an ion coordination pattern similar to that of NrnA and slightly different substrate binding patterns. The results provide combined structural and functional insight into the enzymatic turnover of pAp, implying the potential function of sulfate assimilation in hyperthermophilic cells.

Biosynthesis of Chuangxinmycin Featuring a Deubiquitinase-like Sulfurtransferase.

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

Induction of a Toxin-Antitoxin Gene Cassette under High Hydrostatic Pressure Enables Markerless Gene Disruption in the Hyperthermophilic Archaeon Pyrococcus yayanosii.

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (P hhp ) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (P hmtB ), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter P gdh The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles.IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.

Identification of a functional toxin-antitoxin system located in the genomic island PYG1 of piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.

Toxin-antitoxin (TA) system is bacterial or archaeal genetic module consisting of toxin and antitoxin gene that be organized as a bicistronic operon. TA system could elicit programmed cell death, which is supposed to play important roles for the survival of prokaryotic population under various physiological stress conditions. The phage abortive infection system (AbiE family) belongs to bacterial type IV TA system. However, no archaeal AbiE family TA system has been reported so far. In this study, a putative AbiE TA system (PygAT), which is located in a genomic island PYG1 in the chromosome of Pyrococcus yayanosii CH1, was identified and characterized. In Escherichia coli, overexpression of the toxin gene pygT inhibited its growth while the toxic effect can be suppressed by introducing the antitoxin gene pygA in the same cell. PygAT also enhances the stability of shuttle plasmids with archaeal plasmid replication protein Rep75 in E. coli. In P. yayanosii, disruption of antitoxin gene pygA cause a significantly growth delayed under high hydrostatic pressure (HHP). The antitoxin protein PygA can specifically bind to the PygAT promoter region and regulate the transcription of pygT gene in vivo. These results show that PygAT is a functional TA system in P. yayanosii, and also may play a role in the adaptation to HHP environment.

Target DNA recognition and cleavage by a reconstituted Type I-G CRISPR-Cas immune effector complex.

CRISPR-Cas immune systems defend prokaryotes against viruses and plasmids. CRISPR RNAs (crRNAs) associate with various CRISPR-associated (Cas) protein modules to form structurally and functionally diverse (Type I-VI) crRNP immune effector complexes. Previously, we identified three, co-existing effector complexes in Pyrococcus furiosus -Type I-A (Csa), Type I-G (Cst), and Type III-B (Cmr)-and demonstrated that each complex functions in vivo to eliminate invader DNA. Here, we reconstitute functional Cst crRNP complexes in vitro from recombinant Cas proteins and synthetic crRNAs and investigate mechanisms of crRNP assembly and invader DNA recognition and destruction. All four known Cst-affiliated Cas proteins (Cas5t, Cst1, Cst2, and Cas3) are required for activity, but each subunit plays a distinct role. Cas5t and Cst2 comprise a minimal set of proteins that selectively interact with crRNA. Further addition of Cst1, enables the four subunit crRNP (Cas5t, Cst1, Cst2, crRNA) to specifically bind complementary, double-stranded DNA targets and to recruit the Cas3 effector nuclease, which catalyzes cleavages at specific sites within the displaced, non-target DNA strand. Our results indicate that Type I-G crRNPs selectively bind target DNA in a crRNA and, protospacer adjacent motif dependent manner to recruit a dedicated Cas3 nuclease for invader DNA destruction.

Cloning, overexpression, and characterization of a thermostable nitrilase from an Antarctic Pyrococcus sp.

Nitriles are important chemical building blocks for the synthesis of intermediates in fine chemical and pharmaceutical industries. Here, we report a new highly thermostable nitrilase from an Antarctic Pyrococcus sp. MC-FB, a hyperthermophilic archaeon. A gene that encoded a nitrilase was identified and subsequently cloned and overexpressed in Escherichia coli. The recombinant nitrilase, named NitMC-FB, is active as a homodimer (60 kDa) with an optimal temperature and pH of 90 °C and 7.0, respectively. NitMC-FB hydrolyzes preferentially aromatic nitriles, being the first aromatic nitrilase from an archaeon described so far. The K M and V max parameters were determined to be 13.9 mM and 3.7 µmol/min*mg, respectively, with 2-cyanopyridine as the substrate. Additionally, the recombinant nitrilase is highly thermostable with a half-life of 8 h at 90 °C.

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.

Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.

The hyperthermophile Pyrococcus yayanosii CH1 is the only high-pressure-requiring microorganism obtained thus far within the archaea domain or among all non-psychrophiles in any domain. In this study, we developed a genetic manipulation system for P. yayanosii after first isolating a facultatively piezophilic derivative strain, designated P. yayanosii A1. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene was overexpressed in strain P. yayanosii A1 and was demonstrated to confer host cell resistance against simvastatin. Furthermore, using simvastatin as a selection marker, the endogenous pyrF of P. yayanosii A1 was disrupted through homologous recombination, thus generating the additional host strain P. yayanosii A2 (ΔpyrF). A markerless gene disruption vector was constructed by incorporating a pyrF-sim (R) cassette that enables the combined use of simvastatin resistance for positive selection and 5-FOA for counter selection. The utility of this versatile disruption system was demonstrated by deleting the carbon-nitrogen hydrolase of P. yayanosii strain A1. These results demonstrate that a variety of genetic tools are now in place to study unknown gene function and the molecular mechanisms of piezophilic adaptation in P. yayanosii.

Heterologous expression of hyperthermophilic cellulases of archaea Pyrococcus sp. by fungus Talaromyces cellulolyticus.

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the high cellulolytic enzyme-producing fungi. T. cellulolyticus exhibits the potential ability for high amount production of enzyme proteins. Using the homologous expression system under the control of a glucoamylase promoter, some kinds of cellulases of T. cellulolyticus can be expressed by T. cellulolyticus. On the other hand, hyperthermophilic cellulase has been expected to be useful in the industrial applications to biomass. The hyperthermophilic archaea Pyrococcus horikoshii and P. furiosus have GH family 5 and 12 hyperthermophilic endocellulase, respectively. The two kinds of hyperthermophilic endocellulases were successfully produced by T. cellulolyticus using the above expression system under the control of a glucoamylase promoter of T. cellulolyticus. These recombinant cellulases exhibited the same characteristics as those of the recombinant cellulases prepared in E. coli. The productions of the recombinant enzymes were estimated to be over 100 mg/L. In this study, we first report the overexpression of the hyperthermophilic enzymes of archaea using the fungal expression system.

Identification and characterization of an archaeal kojibiose catabolic pathway in the hyperthermophilic Pyrococcus sp. strain ST04.

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 ß-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and ß-glucose-1-phosphate (ß-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 ß-G1P into G6P in the presence of 6 mM MgCl2. Conversion activity from ß-G1P to G6P was 46.81 ± 3.66 U/mg, and reverse conversion activity from G6P to ß-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.

Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04.

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 <13% sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 2.4.1.25), amylopullulanase (EC 3.2.1.41), and branching enzyme (EC 2.4.1.18). This gene was cloned and expressed in Escherichia coli, and the recombinant product (Pyrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-ß-cyclodextrin (G1-ß-CD) and ß-cyclodextrin (ß-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (Km) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-ß-cyclodextrin (G2-ß-CD) was 0.3 mM. The kcat values were 381.0 min(-1) for G3 and 1,545.0 min(-1) for G2-ß-CD. The enzyme was inhibited competitively by the reaction product G2, and the Ki constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.

Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes.

Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (α4ß4 and αßγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the ß subunit (PDHß) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHßs from α4ß4- and αßγδ-type enzyme complexes, eight PDHßs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α4ß4-type PDHßs showed greater thermostability than most of the αßγδ-type PDHßs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α4ß4-type PDHßs utilized ferricyanide as the most preferable electron acceptor, whereas αßγδ-type PDHßs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHßs likely reflect whether they were from an αßγδ- or α4ß4-type complex, though the wider divergence observed within αßγδ-type PDHßs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHßs did not reflect the complex type. Interestingly, the k cat value for free α4ß4-type PDHß from P. horikoshii was much larger than the value for the same subunit within the α4ß4-complex. This indicates that the isolated PDHß could be a useful element for an electrochemical system for detection of L-proline.

An evolutionary analysis of the GH57 amylopullulanases based on the DOMON_glucodextranase_like domains.

Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α-1,4 and α-1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON_glucodextranase_like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα-amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination.

Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge.

Pyrococcus sp. strain ST04 is a hyperthermophilic, anaerobic, and heterotrophic archaeon isolated from a deep-sea hydrothermal sulfide chimney on the Endeavour Segment of the Juan de Fuca Ridge in the northeastern Pacific Ocean. To further understand the distinct characteristics of this archaeon at the genome level (polysaccharide utilization at high temperature and ATP generation by a Na(+) gradient), the genome of strain ST04 was completely sequenced and analyzed. Here, we present the complete genome sequence analysis results of Pyrococcus sp. ST04 and report the major findings from the genome annotation, with a focus on its saccharolytic and metabolite production potential.

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins.

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (approximately 12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication.

Modulating the pH profile of the pullulanase from Pyrococcus yayanosii CH1 by synergistically engineering the active center and surface.

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5-5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (PulPY2) was successfully shifted from 6.4 to 5 with a 2-fold increase in the specific activity based on synergistic engineering of the active center and surface. Synergistic engineering was performed by introducing histidine within 6 Å of the active sites, and by enhancing negative charges on the enzymatic surface. Two single-site mutants of PulPY2-Q13H and PulPY2-I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant PulPY2-Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to PulPY2. These results not only obtained an applicable pullulanase for industrial application, but also provided a strategy for shifting the optimal pH of the enzyme based on synergistic engineering of the active center and surface.

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).