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

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.


Introduction
Pyrococcus species are hyperthermophilic members of the order Thermococcales, with optimal growth temperatures near 100 °C.All known Pyrococcus species grow optimally and relatively rapidly at temperatures above 90 °C and produce H 2 S from elemental sulfur (S °) (Zillig et al. 1987).Two Pyrococcus species, P. furiosus (Fiala and Stetter 1986) and P. woesei (Zillig et al. 1987), were isolated from marine sediments at a Vulcano Island beach site in Italy.They have similar morphological and physiological characteristics: both species are cocci and move by means of a tuft of polar flagella.They are heterotrophic and grow optimally at 95-100 °C, utilizing peptides as major carbon and nitrogen sources.Both species use carbohydrates such as maltose, cellobiose and starch.Growth of P. furiosus on β-glucans such as chitin, laminarin, cellobiose and cellulose has been reported (Kengen et al. 1993, Gueguen et al. 1997, Bauer et al. 1999, Blamey et al. 1999, Gao et al. 2003).Neither species requires S °for growth, but S °appears to stimulate growth via detoxification of H 2 , the end product of metabolism.Compared with P. furiosus, P. woesei may grow at higher concentrations of H 2 produced during cultivation (Mukund and Adams 1991).Pyrococcus furiosus requires tungsten (10-25 µM Na 2 WO 4 ) for optimal growth in S °-free medium (Bryant and Adams 1989).The optimal pH and NaCl concentration for growth of P. furiosus may be slightly higher and cover a broader range than for P. woesei (pH 6-8 and 15-35 g NaCl l -1 for P. furiosus, and pH 6 and 30 g NaCl l -1 for P. woesei) (Fiala andStetter 1986, Zillig et al. 1987), although these differences may also reflect variation among laboratories.
Three Pyrococcus genome sequences (P.furiosus, P. horikoshii and P. abyssi) are available in GenBank.In contrast, only 20 sequence accessions from P. woesei are available in Gen-Bank, and no 16S rDNA sequence was available.Our preliminary studies revealed that the P. woesei genome also contains insertion sequence (IS) elements (GenBank accession nos.AF420277 and AF443788).This study was initiated to determine whether the genomic content and positioning of the IS elements provide a measure of relatedness between P. woesei and P. furiosus.
Pyrococcus furiosus and P. woesei were grown in maltose in a medium identical to Pf except that S °was omitted and maltose (1%, w/v) and sodium tungstate (10-100 µM) were added.Cell densities were determined (from triplicate cultures) by the acridine orange staining technique.Briefly, samples (0.1-1.0 µl) were mixed with 37% formalin saturated with sodium borate (10-25 µl), 0.01% acridine orange (0.2 ml) and diluted with Milli-Q water to a final volume of 1 ml.The solutions were filtered through 0.22-µm polycarbonate membranes (GE Osmonics, Minnetonka, MN) and the membranes mounted on microscopic slides.Cell counting was performed randomly on at least 50 × 50 µm squares.Cell numbers were determined by the following equation: Cell numbers ml -1 = number of cells per 10 × 10 µm squares × 2300 ml -1 /volume in ml.
Polymerase chain reactions (PCRs) were performed in a 50 µl reaction volume.Thermal cycling conditions were: 95 °C for 30 s, 52-55 °C for 40 s and 72 °C for 60-120 s for 30 cycles, depending on the size of the desired product (approximately 60 s per 1 kb fragment).
Nested PCR reactions were performed on dilute samples (1:600).Primers 4 and 7 were employed in negative control reactions.

Southern blotting and comparison of restriction patterns
Genomic DNAs (5 µg) were digested with the restriction enzyme PstI.Approximately 1 µg of restricted DNA was loaded in each lane and separated in 0.9% agarose gels (11 × 14 cm).Linearized plasmids constructed in this study containing a tnp sequence served as size markers.Electrophoresis was performed at 30 V in TAE buffer (pH 8.5) for 14 to 15 h in a cold room.Transfer of DNAs to a nylon membrane (Immobilon-Ny + Transfer Membrane, Millipore, Bedford, MA) was performed by perfusion according to the manufacturer's recommendations.Briefly, DNA in the agarose gel was depurinated in 0.25 N HCl for 15 min.The agarose gel was rinsed briefly with Milli-Q water, denatured in 0.5 N NaOH + 1.5 M NaCl for 30 min and neutralized with 1 M TrisCl (pH 8.0) + 1.5 M NaCl for 30 min.The DNA was transferred onto nylon membrane with 20× SSC for 6 to 8 h.Labeled DNA probes were prepared with 25 µCi of 32 P-α-labeled dATP and 0.1 ng of purified tnp-I and tnp-II sequences as templates.The probes were boiled for 10 min and immediately added to the pre-warmed hybridization mixture.Hybridization was performed following the standard procedures described by Ausubel et al. (1987).Images were visualized with a Phos-phorImager (Molecular Dynamics, Sunnyvale, CA).Band intensity was measured with the NIH Image 1.61 program (http://rhea.la.asu.edu/dm/data_tools/nih_image).Relative intensity was estimated with the equation: Index (per unit area) = (band intensity -mean background intensity)/(intensity of a band containing one IS -mean background intensity).
PstI restriction fragments containing an IS sequence were deduced from the genome sequence of P. furiosus.

Lambda library construction and screening for insertion sequences
Approximately 5-6 kb PstI restriction fragments from P. woesei genomic DNA were gel purified.The fragments (1 µg) were partially digested with the restriction enzyme Tsp509I (0.1 units) at 56 °C for 15 min to create AATT sticky ends.The restriction enzyme was removed with a spin column (QIAquick Gel Extraction kit, QIAGEN, Valencia, CA).Approximately 50 ng of the sticky fragments were ligated to λZIPLOX, EcoRI Arms vector (Life Technologies, Gaithersburg, MD) with T4 DNA ligase (Promega, Madison, WI) and packaged (Gigapack II Gold Packaging Extract, Stratagene, La Jolla, CA).Propagation of λ phage and excision of plasmid pZL1 were performed following the methods recommended by the manufacturer (Life Technologies).About 1 × 10 -5 positive plaques were obtained.Plaques were excised and screened by standard colony hybridization techniques following the methods recommended for the nylon membrane (Immobilon-Ny + Transfer Membrane, Millipore).

Growth with maltose and tungsten
Pyrococcus woesei showed similar physiological properties to P. furiosus.Cell yields (about 1 × 10 7 cells ml -1 ) from overnight cultures in Pf medium were not significantly different between species.Representative growth curves for P. furiosus and P. woesei in the S °-free medium containing maltose and 100 µM Na 2 WO 4 are shown in Figure 1.One ml of each culture was inoculated into three identical bottles of 100 ml S °-free medium containing 1% maltose and 10-100 µM Na 2 WO 4 to obtain cell densities of about 7-8 × 10 4 cells ml -1 .The cultures were incubated anaerobically at 95 °C.The results in Figure 1 indicate that both P. woesei and P. furiosus use maltose and tolerate Na 2 WO 4 up to 100 µM.
During the first 6 h of incubation, growth of P. furiosus and P. woesei was not significantly different (cell yields were 1.1 × 10 6 ± 3.8 × 10 5 and 1.1 × 10 6 ± 2.3 × 10 5 cells ml -1 , respectively), and both cultures entered stationary phase after 10 h of incubation.However, the maximum cell density obtained for P. woesei (3.6 × 10 6 ± 6.0 × 10 5 cells ml -1 at 10 h) was approximately one log cycle less than for P. furiosus (1.6 × 10 7 ± 2.7 × 10 6 cells ml -1 at 10 h).By late stationary phase, the cell density of P. furiosus had increased slightly (to 3.0 × 10 7 ± 6.6 ×  10 6 cells ml -1 at 19 h).In contrast, the cell density of P. woesei declined about twofold (1.6 × 10 6 ± 4.9 × 10 5 cells ml -1 ) between 10 and 19 h.Figures 2a and 2b show the morphologies of P. furiosus (Figure 2a) and P. woesei (Figures 2b) at 19 h.Similar cell morphologies and arrangements of P. woesei and P. furiosus were observed during the exponential phase; most cells were arranged singly or in pairs (picture not shown).Unlike P. furiosus (Figure 2a), large aggregates of cells were observed in P. woesei (Figure 2b) during the stationary phase, suggesting that P. woesei cells in this phase are more sensitive to stress-induced autolysis than cells of P. furiosus.Therefore, growth phenotypes of P. woesei and P. furiosus in the S °-free medium containing maltose and sodium tungstate are not identical.

Identical 16S rDNA sequences
Nucleotide sequences of the 16S rRNA, tRNA Ala and 23S rRNA (partial) cluster from P. woesei were determined (GenBank accession no.AY519654) and found to be identical to sequences of the homologous cluster in P. furiosus (GenBank accession no.AE010139), including the intergenic spacer regions.The two intergenic spacer regions are sensitive sites for differentiation in the Thermococcales, especially species of Pyrococcus, as shown by DiRuggiero et al. (1995).

Analysis of nucleotide sequences
To date, there are 20 GenBank accessions of P. woesei sequences available.These nucleotide sequences (28,857 bp) include 22 genes and one three-gene cluster (X59857).BLAST search results for P. woesei against the genome sequences of P. furiosus, P. horikoshii OT3 and P. abyssi are summarized in Table 1.The nucleotide sequences of P. woesei were highly homologous (99-100% similarities), especially with sequences of P. furiosus.They have uniformly lower similarity (approximately 75%) to the homologous sequences of P. horikoshii and P. abyssi.The genomes of P. horikoshii and P. abyssi contain no amylase genes homologous to the two P. furiosus α-amylase genes (GenBank accession nos.AF240464 and AF177906) and no IS elements, confirming a previous study (DiRuggiero et al. 2000).

Southern blotting and comparison of restriction patterns
The P. woesei genome contains IS elements (GenBank accession nos.AF420277 and AF443788) homologous to the IS elements in the P. furiosus genome.In P. furiosus, the IS elements are grouped into three types according to their nucleotide sequences: type I (11 copies), type II (eight copies) and type III (four copies).BLAST searches of entire IS elements against the complete P. furiosus genome revealed an additional four truncated copies of the ISs (approximately 300-700 bp), which are classified as type D in this study.The IS elements in all classes contained no PstI restriction sites.To simplify subsequent discussions, IS numbers were assigned and the coordinates of the IS elements in the genome of P. furiosus are listed in Table 2.The sizes of fragments containing an IS element between two PstI restriction sites were predicted from the genome sequence of P. furiosus.The predicted Pst I restriction fragment sizes are summarized in Table 2. Southern blot analysis of PstI restriction patterns and DNA hybridization with probes specific to tnp sequences (Figure 3) revealed that the P. woesei genome (lane 1) contains multiple copies of the IS elements shown in Table 2.In the P. furiosus genome (lane 2), band numbers 1-7 and a cluster of large fragments (bracket 8) were mapped according to their relative intensities and predicted PstI restriction fragment sizes.Generally, the mapping correlated well with band intensities of bands 1-6.Bands denoted no. 8 contain large unresolved fragments (> 10 kb) and may contain a mixture of divergent IS elements.The PstI restriction fragments predicted for P. furiosus did not include an 8 kb fragment containing an IS element, because the 8 kb fragment (band 7) observed for P. furiosus may be a divergent IS element and this area was not accounted for in the comparison.The restriction pattern for P. woesei (lane 1) showed the same PstI fragments as P. furiosus (bands 1 and 3-6 of P. furiosus), except for the band corresponding to band 2 in P. furiosus (lane 2).These bands contain 10 IS elements, including two elements of type D in band 3 of P. furiosus and one element in band 5, implying that the genomes of P. furiosus and P. woesei are colinear in dispersed regions throughout the genome.

Mapping the napA locus in P. woesei
Labeled probes specific to tnp sequences were used to locate flanking DNAs at the ends of the IS elements in a λ library containing genomic DNA of P. woesei.Twenty positive plaques were isolated and the inserts in pZL1 were sequenced.An alignment of the 20 clones carries a 78-nucleotide-long sequence at the right end of the IS (corresponding to nucleotide numbers 632-779 of a type III IS) joining with a 22-nucleotide-long sequence aligned perfectly to coordinates 286776-286797 in the P. furiosus genome.In P. furiosus, coordinates 286776-286797 are part of a putative Na + /H + antiporter (napA) gene (coordinates 285783-286922) that has 69% identity to a putative napA-3 (PAB1247) in P. abyssi and a hypothetical Na + /H + antiporter open reading frame (PH0302) in P. horikoshii OT3.The Na + /H + antiporter is a membrane protein that exchanges Na + and H + and plays an important role in osmoregulation (Hellmer et al. 2002, Waditee et al. 2002).
To map the napA locus in P. woesei, sets of primers (Primers 4 to 9) were employed to walk upstream and downstream of the IS sequences mentioned above.Cloning and sequencing of the napA locus in P. woesei confirmed the presence of an type III IS disrupting the relative coordinates 2828675 and 2828676 and revealed a direct repeat sequence (5′-CCAG GAT-3′) flanking the type III IS element.The presence of direct repeats flanking the IS implies that the type III IS element was inserted into the napA of P. woesei by transposition (Mahillon and Chandler 1998).
The nucleotide sequence of the disrupted napA locus (total 1969 bp) was submitted to GenBank (accession no.AF461062).Apart from the type III IS insert and the direct repeat sequences, the nucleotide sequence is 99% similar to the homologous sequence (coordinates 285783-286922) in P. furiosus (Table 1).The nucleotide sequence of the type III IS element is 99.5% similar to the type III IS element 20 in the P. furiosus genome.

Identification of type I IS as a genetic marker
The P. furiosus genome contains a type I IS element located at coordinates 284983-285764 (Table 2) upstream of the napA locus (coordinates 285783-286922).This raises the question whether the type I IS is also present in P. woesei.A PCR-based strategy with Primers 9 and 6 was employed (see also relative binding coordinates in Figures 4a and 4b) to amplify DNAs of P. woesei and P. furiosus.As anticipated, a fragment of about 1079 bp was obtained from the positive control.From P. woesei, two fragments (60 and 1858 bp) should have been amplified with Primers 9 and 6; however, only the short fragment was produced because of the blocking of PCR reactions by the distal distant primer pair, Primers 9 and 6 (see map in Figure 4b.Nested PCR amplifications on a diluted (1:600) primary reaction from P. woesei using a set of primer pairs (Primers 4 and 7 as negative control, Primers 4 and 3, Primers 9 and 5, and Primers 9 and 6) that bind internally or externally, or both, to the large fragment resulted in a trace amount of PCR fragment containing type I IS (results not shown).These results suggest that a trace amount of the large fragment was produced in the primary amplification reaction with Primers 9 and 6 from the genomic DNA of P. woesei and imply that the genome of P. woesei contains the type I IS element in the same context as identified upstream of the napA locus in the P. furiosus genome.Sequencing of the PCR products con-firmed that the large fragments were identical.Maps comparing the napA loci of P. furiosus and P. woesei are shown in Fig- ures 4a and 4b.
In conclusion, the physiological properties of P. woesei are similar to those of P. furiosus.Both species grow rapidly in the presence of 0.1% maltose and 10-100 µM Na 2 WO 4 in S °-free medium.However, the cell density of P. woesei cultures decreased faster than that of P. furiosus in stationary phase, because of more extensive autolysis.This demonstrates a phenotypic difference between the two isolates that is probably related to differences in membrane integrity and osmotic function.We speculate that this could be due, in part, to the defective napA gene in P. woesei.The Na + /H + antiporter encoded by napA is a membrane protein that exchanges Na + and protons and often plays an important role in osmoregulation (Hellmer et al. 2002, Waditee et al. 2002).
The 16S rRNA, tRNA Ala and 23S rRNA (partial) gene cluster from P. woesei (GenBank accession no.AY519654) was sequenced in this study for the first time.The results revealed complete identity with the P. furiosus operon (GenBank accession no.AE010139).Comparisons of the 20 P. woesei sequences publicly available (Table 1) confirmed identity or high similarity to homologous nucleotide sequences of P. furiosus.Restriction patterns of fragments containing IS element (Figure 3) markers support colinearity between the genomes of both species.The presence of two IS elements in the Na + /H + antiporter (napA) gene in P. woesei, including one in the same context as the single type I IS in P. furiosus, not only supports our contention that the ISs continue to be active,  but provides evidence that they contribute to the divergence of the two strains.It has been proposed that the divergence of the P. furiosus genome from the genomes of P. abyssi and P. horikoshii is mediated largely by IS movement or recombination between ISs in P. furiosus (Maeder et al. 1999, Lecompte et al. 2001, Zivanovic et al. 2002).We suggest that P. woesei and P. furiosus are closely related, certainly in comparison with the many E. coli strains that continue to be recognized as species, and the new designation P. furiosus subsp.woesei is proposed.

Figure 1 .
Figure 1.Growth of P. furiosus and P. woesei in sulfur-free medium containing 1% maltose and 100 µM sodium tungstate.Triplicate cultures of P. furiosus and P. woesei were incubated anaerobically at 95 °C.Cells were counted in at least 50 of the 50 × 50 µm squares.

Figure 2 .
Figure 2. Micrographs of acridine orange stained cells of Pyrococcus furiosus and P. woesei growing in sulfur-free medium containing 1% maltose and 100 µM sodium tungstate.The photographs were taken at identical magnification with an oil immersion objective lens.Numbers in parentheses are the volumes of the cultures used to prepare the microscopic slides.(a) Pyrococcus furiosus at 19 h.The cell density corresponds to the value shown in Figure 1 (about 3 × 10 7 cells ml -1 ).Most cells were single, in pairs or in short chains and rarely in aggregated morphologies.(b) Pyrococcus woesei at 19 h.The cell density of the value for the mid-exponential growth phase shown in Figure 1 (about 1.6 × 10 6 cells ml -1 ).

Figure 3 .
Figure 3. Restriction patterns from a Southern blot probed with 32 P-labeled tnp DNAs.The DNA from P. woesei (lane 1) and P. furiosus (lane 2) were completely digested with PstI restriction enzyme.Size markers in kilobases (kb) are shown on the left of the Figure.Band numbers were assigned only to the bands from P. furiosus and are shown on the right of the figure.Intensities of bands and indices were determined.

Figure 4 .
Figure 4. Maps of insertion sequence (IS) elements in the napA loci of (a) P. furiosus and (b) P. woesei.Parallel lines represent doublestranded DNAs.Boxes with thick arrows represent IS elements.Thick arrows represent genes.Arrowheads on the thin lines represent forward (pointing right) and reverse primers (pointing left) (see binding coordinates in Materials and methods).Coordinates in the P. furiosus genome are shown by the vertical dotted lines.Direct repeat sequences are boxed.The napA gene in P. woesei contains two IS elements, including the type I IS in the same context as in the genome of P. furiosus.