Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.     

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re-annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.     

Defining components of the chromosomal origin of replication of the hyperthermophilic archaeon Pyrococcus furiosus needed for construction of a stable replicating shuttle vector

We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.     

Evidence of recent lateral gene transfer among hyperthermophilic archaea

A total of 153 nucleotide differences were found over a contiguous 16 kb region between two hyperthermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis. The 16 kb region in P. furiosus is flanked by insertion sequence (IS) elements with inverted and direct repeats. Both IS elements contain a single open reading frame (ORF) encoding a putative protein of 233 amino acids identified as a transposase. This 16 kb region has the features of a typical bacterial composite transposon and represents a possible mechanism for lateral gene transfer between Archaea or possibly between Archaea and Bacteria. A total of 23 homologous IS elements was found in the genome sequence of P. furiosus, whereas no full-length IS elements were identified in the genomes of Pyrococcus abyssi and Pyrococcus horikoshii. Only one IS element was found in T. litoralis. In P. furiosus and T. litoralis, the 16 kb region contains an ABC transport system for maltose and trehalose that was characterized biochemically for T. litoralis. Regulation of expression studies showed that the malE gene, located on the transposon, and the encoded trehalose/maltose-binding protein (TMBP) are induced in the presence of maltose and trehalose in both P. furiosus and T. litoralis. The implications of transposition as a mechanism for lateral gene transfer among Archaea are discussed.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea<Emphasis Type="Italic"> Pyrococcus abyssi</Emphasis> and<Emphasis Type="Italic"> P. furiosus</Emphasis>

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.     

Cloning, expression, and structure analysis of carbamate kinase-like carbamoyl phosphate synthetase from Pyrococcus abyssi

Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100 degrees C. Carbamoyl phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form carbamoyl phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.     

Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii

Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes auto-adenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90 degrees C and nick-joining activity at 70 to 90 degrees C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.     

Comparison between Pyrococcus horikoshii and Pyrococcus abyssi genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms

Recent work suggests that restriction-modification gene complexes are mobile genetic elements that insert themselves into the genome and cause various genome rearrangements. In the present work, the complete genome sequences of Pyrococcus horikoshii and Pyrococcus abyssi, two species in a genus of hyperthermophilic archaeon (archaebacterium), were compared to detect large genome polymorphisms linked with restriction-modification gene homologs. Sequence alignments, GC content analysis, and codon usage analysis demonstrated the diversity of these homologs and revealed a possible case of relatively recent acquisition (horizontal transfer). In two cases out of the six large polymorphisms identified, there was insertion of a DNA segment with a modification gene homolog, accompanied by target deletion (simple substitution). In two other cases, homologous DNA segments carrying a modification gene homolog were present at different locations in the two genomes (transposition). In both cases, substitution (insertion/deletion) in one of the two loci was accompanied by inversion of adjacent chromosomal segment. In the fifth case, substitution by a DNA segment carrying type I restriction, modification, and specificity gene homologs was likewise accompanied by adjacent inversion. In the last case, two homologous DNA segments, were found at different loci in the two genomes (transposition), but only one of them had insertion of a modification homolog and an unknown ORF. The possible relationship of these polymorphisms to attack by restriction enzymes on the chromosome will be discussed.     

Holliday junction-resolving enzymes from eight hyperthermophilic archaea differ in reactions with cruciform DNA

Holliday junction-resolving enzymes have been identified in a broad variety of organisms and tissues. In this study, six new Holliday junction-cleaving enzymes (Hjcs) were obtained from hyperthermophilic crenarchaeal and euryarchaeal species, including Pyrococcus horikoshii, Pyrococcus abyssi, Methanococcus jannaschii, Methanobacterium thermautotrophicum, Archaeoglobus fulgidus, and Aeropyrum pernix. The genes were cloned and overexpressed in Escherichia coli, and the respective proteins were purified from crude extracts to homogeneity. For an initial characterization of the enzymatic activities, synthetic heat-stable fixed and mobile cruciform DNA substrates were used at 75 degrees C. The Hjcs from Pyrococcus furiosus, Sulfolobus solfataricus, and the archaeal virus SIRV2 were included in the study for comparison. Despite their sequence homology, the enzymes showed marked differences in their reactions with individual cruciform DNAs. While the fixed cruciform structure was cleaved by all enzymes at only one major position, the mobile cruciform structure displayed different cleavage patterns for individual Hjcs, each with several cleavage positions. Furthermore, a strong bias for cleavage of one direction across the junction was observed with the fixed cruciform DNA for all enzymes. In contrast, the mobile cruciform DNA displayed different preferences, depending on the enzyme used.     

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.     

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.     

In vitro replication slippage by DNA polymerases from thermophilic organisms

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.     

The GINS complex from Pyrococcus furiosus stimulates the MCM helicase activity

Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.     

CRISPR elements in the Thermococcales: evidence for associated horizontal gene transfer inPyrococcus furiosus

The presence and distribution of CRISPR (clustered regularly interspaced short palindrome repeat) elements in the archaeal order Thermococcales were analyzed. Four complete genome sequences from the speciesPyrococcus abyssi, P. furiosus, P. horikoshii, andThermococcus kodakaraensis were studied. A fragment of the genome ofP. furiosus was flanked by CRISPR elements upstream and by a single element downstream. The composition of the gene sequences contained in this genome fragment (positions 699013 to 855319) showed significant differences from the other genes in theP. furiosus genome. Differences were observed in the GC content at the third codon positions and the frequency of codon usage between the genes located in the analyzed fragment and the other genes in theP. furiosus genome. These results represent the first evidence suggesting that repeated CRISPR elements can be involved in horizontal gene transfer and genomic differentiation of hyperthermophilic Archaea.     

Metabolic and evolutionary relationships among Pyrococcus Species: genetic exchange within a hydrothermal vent environment

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.     

A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii

A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii. These two genes were cloned and the corresponding expressed proteins were characterized. The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity. The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178). The protein from PH1426 was a typical, homodimeric flavoprotein. These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P. horikoshii. The redox protein and thioredoxin reductase retained their full activities for over 1h at 100 degrees C. The redox potential of the redox protein was similar to that of thioredoxin from E. coli and lower than that of glutathione. Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence.     

Bacterial start site prediction.

With the growing number of completely sequenced bacterial genes, accurate gene prediction in bacterial genomes remains an important problem. Although the existing tools predict genes in bacterial genomes with high overall accuracy, their ability to pinpoint the translation start site remains unsatisfactory. In this paper, we present a novel approach to bacterial start site prediction that takes into account multiple features of a potential start site, viz., ribosome binding site (RBS) binding energy, distance of the RBS from the start codon, distance from the beginning of the maximal ORF to the start codon, the start codon itself and the coding/non-coding potential around the start site. Mixed integer programing was used to optimize the discriminatory system. The accuracy of this approach is up to 90%, compared to 70%, using the most common tools in fully automated mode (that is, without expert human post-processing of results). The approach is evaluated using Bacillus subtilis, Escherichia coli and Pyrococcus furiosus. These three genomes cover a broad spectrum of bacterial genomes, since B.subtilis is a Gram-positive bacterium, E.coli is a Gram-negative bacterium and P. furiosus is an archaebacterium. A significant problem is generating a set of 'true' start sites for algorithm training, in the absence of experimental work. We found that sequence conservation between P. furiosus and the related Pyrococcus horikoshii clearly delimited the gene start in many cases, providing a sufficient training set.