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PubMed1999

Purification and molecular characterization of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus: the third of a putative five-member tungstoenzyme family.

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.

Aldehyde Oxidoreductases↗
PubMed2000

Purification and characterization of a membrane-bound hydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus.

Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 micromol of H(2) evolved/mg at 80 degrees C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (alpha and beta) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained approximately 1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H(2) as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the alpha and beta subunits were located in the genome database within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of P. furiosus and show that the alpha subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the alpha and beta subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospirillum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H(2) metabolism is coupled to energy conservation.

Catalysis↗
PubMed2000

Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine.

The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism. This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms. The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes. In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP. We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon Pyrococcus furiosus and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity. These findings verify the identification of the P. furiosus Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.

Amino Acid Sequence↗
PubMed2000

Characterization of hydrogenase II from the hyperthermophilic archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction.

The fermentative hyperthermophile Pyrococcus furiosus contains an NADPH-utilizing, heterotetrameric (alphabetagammadelta), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H(2) production and the reduction of elemental sulfur to H(2)S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an M(r) of 320,000 +/- 20,000 and contains four different subunits with M(r)s of 52,000 (alpha), 39,000 (beta), 30,000 (gamma), and 24,000 (delta). The heterotetramer contained Ni (0.9 +/- 0.1 atom/mol), Fe (21 +/- 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 +/- 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H(2) production with K(m) values near 70 microM and k(cat)/K(m) values near 350 min(-1) mM(-1). In contrast to hydrogenase I, hydrogenase II catalyzed the H(2)-dependent reduction of NAD (K(m), 128 microM; k(cat)/K(m), 770 min(-1) mM(-1)). Ferredoxin from P. furiosus was not an efficient electron carrier for either enzyme. Both H(2) and NADPH served as electron donors for the reduction of elemental sulfur (S(0)) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H(2) using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the beta subunit and three in the delta subunit) and one [2Fe-2S] cluster (in the gamma subunit), as well as two putative nucleotide-binding sites in the gamma subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S(0) concentrations since it has a higher affinity than hydrogenase I for both S(0) and polysulfide.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of the alanine aminotransferase from the hyperthermophilic Archaeon pyrococcus furiosus and its role in alanine production.

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6. 5 to 7.8 and at a temperature of over 95 degrees C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k(cat)/K(m) values for alanine and pyruvate formation were 41 and 33 s(-1) mM(-1), respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

Alanine↗
PubMed2001

Novel type of glucose-6-phosphate isomerase in the hyperthermophilic archaeon Pyrococcus furiosus.

Glucose-6-phosphate isomerase (phosphoglucose isomerase [PGI]) (EC 5.3.1.9) from the hyperthermophilic archaeon Pyrococcus furiosus was purified 500-fold to homogeneity. The enzyme had an apparent molecular mass of 43 kDa and was composed of a single type of subunit of 23 kDa indicating a homodimeric (alpha(2)) structure. Kinetic constants of the enzyme were determined at the optimal pH 7 and at 80 degrees C. Rate dependence on both substrates followed Michaelis-Menten kinetics. The apparent K(m) values for glucose-6-phosphate and fructose-6-phosphate were 8.7 and 1.0 mM, respectively, and the corresponding apparent V(max) values were 800 and 130 U/mg. The enzyme had a temperature optimum of 96 degrees C and showed a significant thermostability up to 100 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. Based on the N-terminal amino acid sequence of the subunit, a single open reading frame (ORF; Pf_209264) was identified in the genome of P. furiosus. The ORF was characterized by functional overexpression in Escherichia coli as a gene, pgi, encoding glucose-6-phosphate isomerase. The recombinant PGI was purified and showed molecular and kinetic properties almost identical to those of the native PGI purified from P. furiosus. The deduced amino acid sequence of P. furiosus PGI did not reveal significant similarity to the conserved PGI superfamily of eubacteria and eucarya. This is the first description of an archaeal PGI, which represents a novel type of PGI.

Amino Acid Sequence↗
PubMed2001

Characterization of an aminoacylase from the hyperthermophilic archaeon Pyrococcus furiosus.

Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-L-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 +/- 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl(2) (and to 74% by the addition of CoCl(2)). After reconstitution with ZnCl(2), the enzyme contained 2.85 +/- 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolar N-acylated L amino acids (Met, Ala, Val, and Leu), as well as N-formyl-L-methionine. The high K(m) values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity with N-acetyl-L-methionine as the substrate occurred at pH 6.5 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in the P. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7 lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn(2+) ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of the P. furiosus aminoacylase (> or = 50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

Amidohydrolases↗
PubMed2001

Cellobiose uptake in the hyperthermophilic archaeon Pyrococcus furiosus is mediated by an inducible, high-affinity ABC transporter.

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different beta-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K(m) = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosus membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtA gene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

ATP-Binding Cassette Transporters↗
PubMed2001

Regulation and mechanism of action of the small heat shock protein from the hyperthermophilic archaeon Pyrococcus furiosus.

The small heat shock protein (sHSP) from the hyperthermophile Pyrococcus furiosus was specifically induced at the level of transcription by heat shock at 105 degrees C. The gene encoding this protein was cloned and overexpressed in Escherichia coli. The recombinant sHSP prevented the majority of E. coli proteins from aggregating in vitro for up to 40 min at 105 degrees C. The sHSP also prevented bovine glutamate dehydrogenase from aggregating at 56 degrees C. Survivability of E. coli overexpressing the sHSP was enhanced approximately sixfold during exposure to 50 degrees C for 2 h compared with the control culture, which did not express the sHSP. Apparently, the sHSP confers a survival advantage on mesophilic bacteria by preventing protein aggregation at supraoptimal temperatures.

Animals↗
PubMed2001

Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus.

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690+/-20 kDa and is assumed to be octomeric, while approximately 30% of the protein is purified as a large ( approximately 1.6 MDa) complex that is not active. The apparent K(m) values and catalytic efficiencies for the substrates pyruvate and ATP (at 80 degrees C, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1). s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) x s(-1), respectively. Maximal activity was measured at pH 9.0 (at 80 degrees C) and at 90 degrees C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k(cat)/K(m) = 32 (mM. s(-1)]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration ( approximately 5% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

Adenosine Monophosphate↗
PubMed2001

Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100 degrees C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S(0)). Growth rates were highest on media containing peptides and S(0), with or without maltose. Growth did not occur on the peptide medium without S(0). S(0) had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S(0) with or without maltose were the same, suggesting that S(0) is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S(0) in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S(0)-reducing enzyme in this organism and the mechanism of the S(0) dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

Culture Media↗
PubMed2001

DNA microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus: evidence for anNew type of sulfur-reducing enzyme complex.

DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S(primary)) on the levels of gene expression in cells grown at 95 degrees C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S(primary) and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S(primary). Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S(primary)-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S(primary)-grown cells, and these are also thought be involved in S(primary) reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S(primary)-grown cells, a finding consistent with the fact that growth on peptides is a S(primary)-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S(primary), although the reason for this is as yet unknown. Of the 20 S(primary)-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S(primary)-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.

Archaeal Proteins↗
PubMed2001

Experimental evolution of enzyme temperature activity profile: selection in vivo and characterization of low-temperature-adapted mutants of Pyrococcus furiosus ornithine carbamoyltransferase.

We have obtained mutants of Pyrococcus furiosus ornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15 degrees C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.

Adaptation, Biological↗
PubMed2001

Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domain Eucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases delta and epsilon, onto primed DNA templates. RFC-like genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia coli cells to determine their roles in DNA synthesis. The P. furiosus RFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) and P. furiosus PCNA (PfuPCNA). The ATPase activity of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II from P. furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved in Archaea and Eucarya.

Adenosine Triphosphatases↗
PubMed2002

Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus.

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

Amino Acid Sequence↗
PubMed2002

Characterization of a fourth tungsten-containing enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus grows optimally near 100 degrees C using peptides and carbohydrates as carbon sources, and it reduces elemental sulfur (S(0)), if present, to H(2)S. Tungsten (W), an element rarely used in biology, is required for optimal growth, and three different tungsten-containing enzymes have been previously purified from this organism. They all oxidize aldehydes of various types and are thought to play primary roles in the catabolism of sugars or amino acids. Here, the purification of a fourth tungsten-containing enzyme, termed WOR 4, from cell extracts of P. furiosus grown with S(0) is described. This was achieved by monitoring through multiple chromatography steps the W that is not associated with the three characterized tungstoenzymes. The N-terminal sequence of WOR 4 and the approximate molecular weight of its subunit determined electrophoretically (69,000) correspond to the product of an ORF (PF1961, wor4) present in the complete genome sequence of P. furiosus. WOR 4 is a homodimer and contains approximately one W, three Fe, three or four acid-labile sulfide, and one Ca atom per subunit. The visible and electron paramagnetic resonance spectra of the oxidized and reduced enzyme indicate the presence of an unusual iron-sulfur chromophore. WOR 4 does not oxidize aliphatic or aromatic aldehydes or hydroxy acids, nor does it reduce keto acids. Consistent with prior microarray data, the protein could not be purified from P. furiosus cells grown in the absence of S(0), suggesting that it may have a role in S(0) metabolism.

Aldehyde Oxidoreductases↗
PubMed2002

Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1.

We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.

Amino Acid Sequence↗
PubMed2003

PAV1, the first virus-like particle isolated from a hyperthermophilic euryarchaeote, "Pyrococcus abyssi".

We describe the first virus-like particle of a hyperthermophilic euryarchaeote which was discovered in a strain of "Pyrococcus abyssi" previously characterized in our laboratory. This particle, named PAV1, is lemon-shaped (120 nm x 80 nm), with a short tail terminated by fibers, and resembles the virus SSV1, the type member of the Fuselloviridae, isolated from Sulfolobus shibatae. Sensitivity of the virus-like particle to organic solvents and detergents suggested that the envelope of PAV1 may contain lipids in addition to proteins. It contains a double-stranded circular DNA of 18 kb which is also present in high copy number in a free form in the host cytoplasm. No integrated form of the PAV1 genome could be detected in the host chromosome. Under standard growth conditions, the host cells continuously release PAV1 particles into the culture supernatant without spontaneous lysis, with a maximum reached in the late stationary phase. UV, gamma irradiation, treatment with mitomycin C, and various physiological stresses had no effect on PAV1 production. Screening of a large number of Thermococcales isolates did not permit to find a sensitive host. These results suggest that PAV1 persists in the host strain in a stable carrier state rather than a prophage.

DNA↗