Archaea 1, 277–283 © 2004 Heron Publishing—Victoria, Canada A proposal to rename the hyperthermophile Pyrococcus woesei as Pyrococcus furiosus subsp. woesei WIROJNE KANOKSILAPATHAM,1 JUAN M. GONZÁLEZ,1,2 DENNIS L. MAEDER,1 1,3 1,4 JOCELYNE DIRUGGIERO and FRANK T. ROBB 1 Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202, USA 2 Present address: IRNAS-CSIC, P.O. Box 1052, 41080 Sevilla, Spain 3 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20274, USA 4 Corresponding author ([email protected]) Received April 8, 2004; accepted July 28, 2004; published online August 31, 2004 Summary Pyrococcus species are hyperthermophilic mem- relatively rapidly at temperatures above 90 °C and produce ° bers of the order Thermococcales, with optimal growth tem- H2S from elemental sulfur (S ) (Zillig et al. 1987). Two Pyro- peratures approaching 100 °C. All species grow heterotrophic- coccus species, P. furiosus (Fiala and Stetter 1986) and ° ally and produce H2 or, in the presence of elemental sulfur (S ), P. woesei (Zillig et al. 1987), were isolated from marine sedi- H2S. Pyrococcus woesei and P.furiosus were isolated from ma- ments at a Vulcano Island beach site in Italy. They have similar rine sediments at the same Vulcano Island beach site and share morphological and physiological characteristics: both species many morphological and physiological characteristics. We re- are cocci and move by means of a tuft of polar flagella. They port here that the rDNA operons of these strains have identical are heterotrophic and grow optimally at 95–100 °C, utilizing sequences, including their intergenic spacer regions and part of peptides as major carbon and nitrogen sources. Both species the 23S rRNA. Both species grow rapidly and produce H2 in the presence of 0.1% maltose and 10–100 µM sodium tungstate in use carbohydrates such as maltose, cellobiose and starch. β S°-free medium. However, P. woesei shows more extensive Growth of P. furiosus on -glucans such as chitin, laminarin, autolysis than P. furiosus in the stationary phase. Pyrococcus cellobiose and cellulose has been reported (Kengen et al. 1993, furiosus and P.woesei share three closely related families of in- Gueguen et al. 1997, Bauer et al. 1999, Blamey et al. 1999, sertion sequences (ISs). A Southern blot performed with IS Gao et al. 2003). Neither species requires S° for growth, but S° probes showed extensive colinearity between the genomes of appears to stimulate growth via detoxification of H2, the end P. woesei and P. furiosus. Cloning and sequencing of ISs that product of metabolism. Compared with P. furiosus, P. woesei were in different contexts in P. woesei and P. furiosus revealed may grow at higher concentrations of H2 produced during cul- that the napA gene in P.woesei is disrupted by a type III IS ele- tivation (Mukund and Adams 1991). Pyrococcus furiosus re- ment, whereas in P.furiosus, this gene is intact. A type I IS ele- quires tungsten (10–25 µM Na2WO4) for optimal growth in ment, closely linked to the napA gene, was observed in the S°-free medium (Bryant and Adams 1989). The optimal pH same context in both P.furiosus and P.woesei genomes. Our re- and NaCl concentration for growth of P. furiosus may be sults suggest that the IS elements are implicated in genomic re- slightly higher and cover a broader range than for P. woesei arrangements and reshuffling in these closely related strains. (pH 6–8 and 15–35 g NaCl l–1 for P. furiosus, and pH 6 and We propose to rename P. woesei a subspecies of P. furiosus 30gNaCll–1 for P. woesei) (Fiala and Stetter 1986, Zillig et based on their identical rDNA operon sequences, many com- mon IS elements that are shared genomic markers, and the ob- al. 1987), although these differences may also reflect variation servation that all P. woesei nucleotide sequences deposited in among laboratories. GenBank to date are > 99% identical to P.furiosus sequences. Three Pyrococcus genome sequences (P.furiosus, P.horiko- shii and P. abyssi) are available in GenBank. In contrast, only Keywords: hyperthermophile, sulfur reduction, transposon. 20 sequence accessions from P. woesei are available in Gen- Bank, and no 16S rDNA sequence was available. Our prelimi- nary studies revealed that the P. woesei genome also contains insertion sequence (IS) elements (GenBank accession nos. Introduction AF420277 and AF443788). This study was initiated to deter- Pyrococcus species are hyperthermophilic members of the or- mine whether the genomic content and positioning of the IS el- der Thermococcales, with optimal growth temperatures near ements provide a measure of relatedness between P. woesei 100 °C. All known Pyrococcus species grow optimally and and P. furiosus. ARCHAEA ONLINE at http://archaea.ws 278 KANOKSILAPATHAM ET AL. Materials and methods Nucleotide sequences of the three primers specific for tnp are: 5′-ATG AAG GCT GAG AGC ATT CTA TAC TC-3′, 5′-ATG Organisms and growth in maltose AAG TCT GAA ACC ATT ATT TAC TGG-3′ and 5′-TTA ′. Pyrococcus furiosus DSM 3638 and P. woesei DSM 3773 GGA TAA CTG GGG CAT CAC-3 These primers bind spe- were grown under anaerobic conditions at 95 °C in Pf medium cifically to tnp at coordinates 1–26, 1–27 and 682–702, re- spectively. Primers 4 to 8 were designed to bind specifically to containing (per liter): 24 g NaCl, 4 g Na2SO4, 0.7 g KCl, 0.2 g the napA locus of the P. furiosus genome (binding coordi- NaHCO3, 0.1 g KBr, 0.03 g H3BO3, 10.8 g MgCl2·6H2O, 1.5 g nates 286645–286663, 286694–286716, 286776–286797, CaCl2·2H2O, 0.025 g SrCl2·6H2O, 5 g tryptone, 1 g yeast ex- tract, 1 ml resazurin (0.2 g l–1 solution), and 5–10 g sulfur 286922–286947 and 285765–285786, respectively). The nu- ′ powder; the pH was adjusted to 6.8 (González et al. 1998). cleotide sequences of these primers are: 5 -TCC ATG CTA ′, 5′ Cells were harvested by centrifugation and DNA was ex- GTC TTT TTGC-3 -CGA GTG AAC ATC CGA CAA ′ ′ ′, tracted as described by Charbonnier and Forterre (1995). TCT TA-3 ,5-TTA AGG CAA CTT CCA TCC TTGG-3 5′ ′ Pyrococcus furiosus and P.woesei were grown in maltose in -AGA ATC ATT AGA ACC AGA AGA GTA GC-3 and ′ ′ a medium identical to Pf except that S° was omitted and malt- 5 -TAG CAA GGG GAT GAT AAC ATGG-3 . An outward ′ ose (1%, w/v) and sodium tungstate (10–100 µM) were added. and conserved primer sequence (5 -CTT CAA GAG GTG ′ Cell densities were determined (from triplicate cultures) by ATA CCC CAG TTA TCC-3 ) was designed to bind specifi- ′ the acridine orange staining technique. Briefly, samples cally to the 3 end of tnp (binding coordinates 672–699). (0.1–1.0 µl) were mixed with 37% formalin saturated with so- Polymerase chain reactions (PCRs) were performed in a dium borate (10–25 µl), 0.01% acridine orange (0.2 ml) and 50 µl reaction volume. Thermal cycling conditions were: diluted with Milli-Q water to a final volume of 1 ml. The solu- 95 °C for 30 s, 52–55 °C for 40 s and 72 °C for 60–120 s for tions were filtered through 0.22-µm polycarbonate mem- 30 cycles, depending on the size of the desired product branes (GE Osmonics, Minnetonka, MN) and the membranes (approximately 60 s per 1 kb fragment). mounted on microscopic slides. Cell counting was performed Nested PCR reactions were performed on dilute samples randomly on at least 50 × 50 µm squares. Cell numbers were (1:600). Primers 4 and 7 were employed in negative control re- determined by the following equation: Cell numbers ml–1 = actions. number of cells per 10 × 10 µm squares × 2300 ml–1/volume in ml. Southern blotting and comparison of restriction patterns Phylogenetic analyses of nucleotide sequences Genomic DNAs (5 µg) were digested with the restriction en- zyme PstI. Approximately 1 µg of restricted DNA was loaded Nucleotide sequences of P. woesei were compared by BLAST in each lane and separated in 0.9% agarose gels (11 × 14 cm). against genomic sequences of P. furiosus, P. horikoshii Linearized plasmids constructed in this study containing a tnp OT3 and P. abysii available at the GeneMate Web site sequence served as size markers. Electrophoresis was per- (http://comb5-156.umbi.umd.edu/genemate/). All available formed at 30 V in TAE buffer (pH 8.5) for 14 to 15 h in a cold nucleotide sequences of P. woesei were obtained from room. Transfer of DNAs to a nylon membrane (Immo- GenBank. The accession nos. are: X15329, AY443493, bilon-Ny+ Transfer Membrane, Millipore, Bedford, MA) was AF443788, AF420277, AF043283, U84155, Y09481, performed by perfusion according to the manufacturer’s rec- AF240464, AF177906, AF239672, X59857, X73527, ommendations. Briefly, DNA in the agarose gel was de- X60161, X70668, X67205, U56247, U10285, M83988, purinated in 0.25 N HCl for 15 min. The agarose gel was M83987 and AF461062. 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 16S rDNA of P. woesei 8.0) + 1.5 M NaCl for 30 min. The DNA was transferred onto Ala Sequencing of the 16S rRNA-tRNA -23S rRNA gene cluster nylon membrane with 20× SSC for 6 to 8 h. Labeled DNA ′ in P. woesei was performed with primers 344F (5 -ACGG probes were prepared with 25 µCi of 32P-α-labeled dATP and ′ ′ GGC GCA GCA GGC GCGA-3 ), 345R (5 -CCCT TCC 0.1 ng of purified tnp-I and tnp-II sequences as templates.