International Journal of Systematic Bacteriology (1 999), 49, 1829-1 837 Printed in Great Britain
Pyrococcus glycovorans sp. nov., a hyperthermophilic archaeon isolated from the East Pacific Rise
Georges Barbier,' Anne Godfroy,' Jean-Roch Meunier,'t Joel Querellou,' Marie-Anne Cambon,' Franqoise Lesongeur,' Patrick A. D. Grimont' and Gerard Raguenes'
Author for correspondence: Georges Barbier. Tel: + 33 2 98 22 45 21. Fax: +33 2 98 22 45 45. e-mail: gbarbier(difremer.fr
1 Laboratoire de A hyperthermophilic archaeon, strain AL585T, was isolated from a deep-sea Caracterisation des Micro- hydrothermal vent located on the East Pacific Rise at latitude 13 O N and a organismes Marins, IFREMER, Centre de Brest, depth of 2650 m. The isolate was a strictly anaerobic coccus with a mean cell BP 70, 29280 Plouzane, diameter of 1 pm. The optimum temperature, pH and concentration of sea salt France for growth were 95 "C, 7.5 and 30 g I-'. Under these conditions, the doubling 2 Unite des Enterobacteries, time and cell yield were 0.5 h and 5 x lo8cells ml-l. Strain AL585T grew lnstitut Pasteur, 75724 preferentially in media containing complex proteinaceous carbon sources, Paris cedex 15, France glucose and elemental sulfur. The G+C content of the DNA was 47 molo/o. Sequencing of the 165 rDNA gene showed that strain AL585T belonged to the genus Pyrococcus and was probably a new species. This was confirmed by total DNA hybridization. Consequently, this strain is described as a new species, Pyrococcus glycovorans sp. nov.
Keywords: archaea, Pyrococcus, hyperthermophile, deep-sea, hydrothermal vent ~
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INTRODUCTION woesei are subjective synonyms (Erauso et al., 1993) and their 16s rDNA and DNA polymerases sequences From the beginning of the last decade, the scientific are identical (5. Qukrellou, unpublished data). Both community has been increasingly interested in hyper- were isolated from samples collected from marine thermophiles, i.e. strains with temperature optima for solfataras of Vulcano island, Italy. ' P. abyssi' and ' P. growth above 80 "C (Brock, 1992). Such organisms are horikoshii' were isolated from deep-sea vents of the attractive objects for basic research into the molecular North Fiji Basin (Erauso et al., 1993) and Okinawa basis of thermophily, thermostability and the origin of Trough (Gonzalez et al., 1998), respectively. Pyro- life as well as for applied research (Blochl et al., 1995). coccus species are coccoid marine archaea, strictly At present, the industrial applications of hyper- anaerobic, heterotrophic and sulfur-reducing with thermophiles are related to their ability to produce optimal temperatures for growth in the range 95- thermostable enzymes (Leuschner & Antranikian, 100 "C. Substrates for growth include complex organic 1995). In this regard, the Thermococcales is the most substrates such as yeast extract, peptone, tryptone, frequently studied archaeal order and Pyrococcus is meat extract and peptides (Fiala & Stetter, 1986). P. the most frequently studied genus within the hyper- Jiuiosus can also grow on starch, maltose (Fiala & thermophiles (Leuschner & Antranikian, 1995). At Stetter, 1986; Raven & Sharp, 1997), glycogen, present, the genus Pyrococcus contains only four pullulan (Brown et al., 1990), pyruvate (Schafer & species : Pyrococcus furiosus (Fiala & Stetter, 1986), Schonheit, 1991) and cellobiose (Kengen et al., 1993). Pyrococcus tvoesei (Zillig et al., 1987), ' Pyrococcus P. woesei can grow on glycogen, starch and gellan gum abyssi' (Erauso et al., 1993) and 'Pyrococcus hori- (Zillig et al., 1987; Koch et al., 1991 ;Riidiger et al., koshii' (Gonzalez et al., 1998). P. furiosus and P. 1992, 1995). ' P. abyssi' and ' P. horikoshii' are unable to grow on carbohydrates (Erauso et al., 1993; t Present address: L'OrCal, Recherche avancke, DCSP - Phototoxicitb, 1 Gonzalez et al., 1998). This report describes a novel Avenue Eugene Schueller, 93600 Aulnay-sous-Bois, France. hyperthermophilic species of the genus Pyrococcus The GenBanUEMBL accession number for the 165 rDNA sequence of strain isolated from samples collected on the East Pacific AL585T is 270247. Rise.
00983 0 1999 IUMS 1829 G. Barbier and others
METHODS (Difco) was done for microscopic observation of flagella. When many non-microbial particles were observed (for Reference strains. P. furiosus DSM 363ST and Thermotoga instance with starch or at temperatures higher than 100 "C), maritima DSM 3109T were obtained from the Deutsche cells were counted by epifluorescent microscopy (Hobbie et Sammlung von Mikroorganismen und Zellkulturen, al., 1977). Samples were diluted in sterile water containing Braunschweig-Stockheim, Germany. ' P. abyssi' was pro- sea salt (30 gl-l), formalin (2.5%) and acridine orange vided by Gael Erauso (Centre National de la Recherche (0.01 %) and filtered onto black nuclepore polycarbonate Scientifique, Station Biologique, Roscoff, France). membrane filters (pore size 0.2 pm; Costar). The acridine Culture conditions. BHI-S medium contained (1-l) : 9 g brain orange counts were confirmed by staining additional cultures heart infusion (Difco), 23 g NaCl (instead of sea salt, to with the DNA-specific stain 4',6-diamidino-2-phenylindole prevent formation of mineral precipitate), 6-05 g PIPES (Porter & Feig, 1980). buffer (Sigma), 10 g sulfur (Prolabo) and 1 mg resazurin For determination of the optimum pH and salinity for (Sigma). 2216-S medium (Belkin & Jannasch, 1985) con- growth, the turbidity of cultures in 22 16-S medium was also tained (1-l) : 2 g peptone (Difco), 0-5 g yeast extract (Difco), quantified with a spectrophotometer (Spectronic 301 ; 30 g sea salt (Sigma, ref. S-9883), 6.05 g PIPES buffer, 10 g Milton Roy). Before taking measurements, Hungate tubes sulfur and 1 mg resazurin. 20AA-S medium contained (1-l) : were gently mixed by inversion. After 6 min, sulfur particles 30 g sea salt, 6.05 g PIPES buffer, 10 g sulfur, 1 mg resazurin, had settled and OD,,, were measured. Absorbance varied 10 ml of a mineral solution (Balch et al., 1979), 10 ml of a linearly with cell numbers from 2.5 x lo7 to 1.5 x 10' cells vitamin solution (Balch et al., 1979) and each of the 20 ml-1 or OD,,, of 0.05-0.25. A regression through the origin classical amino acids at a concentration of 2 mM. A stock gave the following formula: cells ml-l = 4-5x 10' x OD,,, solution of the 20 amino acids each at a concentration of (n = 7, r2 = 0.98). 2 mM was sterilized separately by 0-22 pm filtration For flow-cytometric analysis, cells were fixed for 15 min at (Nalgene). The pH was adjusted with 5 M NaOH and 5 M room temperature with 1 paraformaldehyde (final con- HC1 (measured at room temperature and atmosphere). The YO media were steam-sterilized at 100 "C (in order to avoid centration). Filtered artificial sea water (Sigma, ref. s-1649) melting of sulfur and solidification in blocks at higher was used to dilute samples if necessary. Nucleic acid staining was achieved by addition of SYBR Green-I (Molecular temperatures) for 30 min on two successive days, transferred into an anaerobic chamber (La Calhene, France) containing Probes) at a final dilution of of the commercial solution and 0.1 % Triton X-100. Samples were incubated for a gas mixture of N,, H, and CO, (90: 5 : 5), reduced by addition of sodium sulfide (Sigma) at a final concentration 15-30 min at room temperature in the dark. Enumeration of cells were performed with a FACSort flow cytometer (Becton of 0.5 g 1-1 and then distributed into Hungate tubes or into 50 ml serum vials that were closed with a butyl rubber Dickinson) equipped with an air-cooled laser providing stopper and an aluminium cap. Unless indicated otherwise, 15 mW at 488 nm and with the standard filter set-up. cultures were grown on BHI-S or 2216-S medium at pH 7.5 Forward and right-angle light scatters (FSC and SSC) as (measured at room temperature) and always incubated at well as the green fluorescence of the complex DNA-SYBR atmospheric pressure (100 kPa) under the anaerobic gas Green were collected on logarithmic signals. Samples were mixture mentioned above at 95 "C. Cultures were inoculated run at a calibrated rate of 50 pl min-l and dilutions of the with previous cultures at 5 (v/v). samples in filtered artificial sea water were adapted such that Yo the event rate was below 800 cells s-l to avoid coincidence. Enrichment cultures and purification. After collection, Data were recorded as list-mode files and processed with samples were stored on board for 2-3 weeks at room custom-designed software CYTOWIN (D. Vaulot, unpub- temperature and then in the laboratory at 4 "C in serum vials lished; available online at http ://www.sb-roscoff.fr), which filled with sterile sea water (sea salt, 30 g 1-I) under anaerobic discriminates cell populations by using a combination of all conditions (N, gas phase and 0.5 g sodium sulfide 1-'). the parameters recorded. Samples from the MVT'90 cruise were ground and used for Electron microscopy. For transmission electron microscopy, inoculation (1 0 YOv/v) of 22 16-S and BHI-S media prepared cells were harvested at the end of the exponential phase of at different pHs (3.5,5.5 and 7.5) and incubated at 60,80,95, growth and centrifuged. The pellet was resuspended and 100 and 110 "C. Positive cultures were identified by the fixed for 1 h at 4 "C in 2.5 YOglutaraldehyde in 0.1 M sodium appearance of turbidity and the presence of cells as revealed cacodylate buffer (pH 7.2,llOO mosmol) and then post-fixed by microscopic observation. Cultures were then purified by for 1 h at 4 "C in 1 YO OsO, in 0-1 M sodium cacodylate streaking enrichment samples onto the same media solidified buffer (pH 7.2, 1100 mosmol). After dehydration with in- with Gelrite (Scott Laboratories) as gelling agent and creasing ethanol concentrations (70, 95 and 100 %, v/v), incubated at 95 "C in glass Petri dishes set down in jars cells were embedded in EMBed 8 12 resin, thin-sectioned, prepared under the gas mixture (Godfroy et al., 1997). A contrasted with 7 % (w/v) uranyl acetate in methanol and a collection of 125 thermophilic strains was obtained from 2.5% (w/v) aqueous solution of lead citrate and examined those samples (Raguknks et al., 1995). with a model lOOCX transmission microscope (JEOL). Storage. Purified isolates were grown in BHI-S liquid culture Determination of growth parameters. In order to determine media. Cultures in exponential growth phase were stored the optimum temperature, pH and salinity for growth, cells anaerobically in cryotubes (useful volume of 1.8 ml) for were grown in Hungate tubes containing 6 ml 2216-S long-term storage, at - 20 "C and - 70 "C, after addition of medium. The headspace gas was N,/H,/CO, (90:5:5) at dimethyl sulfoxide (5 % v/v) (Sigma) as cryoprotectant. 100 kPa below and 200 kPa above 100 "C. Temperatures Cultures in exponential growth phase were also stored at were maintained with aluminium heating blocks (Barnstead) 4 "C and were usable for at least 1 year. and were monitored with temperature probes placed in Optical microscopy and determination of cell densities. Cell control tubes. pH and salinity effects on growth were studied number was determined with an Olympus (Japan) micro- at 95 "C. Optimum pH was determined with 22 16-S medium scope, model BH-2, and a Thoma chamber (depth of modified as follows: pH 2.5, 3.5 and 4.5, no buffer; pH 5.5 0.02 mm). Specific coloration using the Spot Test Flagella and 6.5, MES buffer (Sigma), 1 g 1-l; pH 7-5, HEPES buffer
1830 International Journal of Systematic Bacteriology 49 Pyrococcus glycovorans sp. nov.
(Sigma), 1 g 1-l; and pH 8.5 and 9-5,AMPS0 buffer (Sigma), (Marmur & Doty, 1962) under the conditions reported by 1 g 1-'. pH was adjusted with 5 M NaOH and 5 M HC1 Raguenes et a/. (1997) using DNA from Escherichiu coli, (measured at room temperature and pressure). Salt require- Clostridiurn perfringens and Micrococcus luteus (Sigma) as ment was determined with 2216-S medium modified with standards. different dilutions of sea salt. Three replicates were simul- 165 rDNA sequence analysis. The 16s rDNA was amplified taneously studied at each temperature, pH or salinity. by PCR (Saiki et al., 1988) as described previously (Dauga & Determination of growth requirements. Utilization of Grimont, 1991), except that an archaea-specific primer was different individual carbon sources for growth was tested. used. The primer sequences were 5'-TCCGGTTGATCCT- Individual carbon sources were added to a defined medium GCCGGAC-3' (E. coli 16s rRNA gene, positions 2-21) and that contained (1-') : 30 g sea salt, 0-4g ammonium chloride, 5'-CTTTCGGTCGCCCCTACT-3' ( Therrnococcus celer 6.05 g PIPES buffer, 10 ml mineral solution (Balch et a/., 23s rRNA, positions 257-234). The PCR product was 1979), 10 ml vitamin solution (Balch et al., 1979), 10 g sulfur precipitated and dissolved in 30 p1 distilled water. The PCR and 1 mg resazurin. Carbon sources were added at con- product was cloned into pUC18 by using a SureClone centrations of 5 g 1-' except for starch, chitin, cellulose and ligation kit (Pharmacia) and also subcloned as HindIII- lactose, which were added at 10 g l-', and ethanol, which was EcoRI insertions into M13mp18 and M13mp19 phage added at 5 ml 1-l. Growth on 20AA-S was also tested. vectors. Both strands of one clone were sequenced by the Negative controls were performed with tubes without any dideoxy chain-termination method (Sanger et a/., 1977), addition of nitrogen and carbon substrate. Such tubes, except that we used a HotTub DNA sequencing kit prepared with H,/CO, (80:20) headspace gas mixture as (Amersham) to resolve numerous compressions observed sole carbon source, were used to examine the possibility of during sequencing of genes of hyperthermophilic micro- autotrophic growth. To examine growth without elemental organisms. PCR products were also sequenced directly by sulfur, cells were cultured simultaneously on BHI-S and BHI using a cycle sequencing kit (type CSDS; Gibco-BRL) and (i.e. BHI-S medium without sulfur) media. For this exper- 32P-labelled primers (Amersham). As a control, the DNA iment, the headspace gases on BHI medium were N,/H,/ sequence was also obtained with an automatic DNA analysis CO, (90: 5: 5) and H,/CO, (80:20) at a pressure of 100 kPa system (Applied Biosystems); this work was done at Euro and the reductant was titanium nitrilotriacetate (Moensch & Sequence Gene Service (ESGS, France). A total of 1925 bp Zeikus, 1983) at a concentration of 200pM. Growth on was sequenced. The determined sequence was then cystine (10 g 1-') and on polysulfide (10 mM) (Blumentals et aligned with available reference 16s rRNA sequences. Ref- a/., 1990) were also investigated. For all these experiments, erence sequences used were : Thermococcus hydrothermalis the pH was adjusted to 7.5 (measured at room temperature (Z70244), Therrnococcus .fumicolans (Z70250), P. fiiriosus and atmosphere). (U20163), 'P.abyssi' (L19921) and ' P. horikoshii' (D87344). The alignment was performed with 1271 bp. A multiple Growth rates and confidence intervals. Growth rates were sequence file was obtained by using the MEGALIGN program determined by linear regression of semi-logarithmic plots of of the DNASTAR package (Promega), similarities and align- cell concentration against time. The doubling time (t,) was ment were obtained by the CLUSTAL w method with weighted calculated from the slope (p): t, = 10gI0(2)/p. Confidence residues (Thompson et a/., 1994) and phylogenetic recon- intervals were calculated using standard errors of the slope struction was produced using PHYLO-WIN (Galtier et al., (se,) as: p+2 se,. When replicates (n) were available, the 1996)with the following conditions : distance, Jukes-Cantor, mean growth rate and related standard error were calculated and successively the neighbour-joining (Saitou & Nei, 1987), as: p = (Xpi)/n and se, = P(~e,,~)~/n~]~~. maximum-likelihood (Felsenstein, 198 1) and maximum- Anti biotic sensitivity. Sensitivity to antibiotics (chlor- parsimony (Lake, 1987) methods. Bootstrap values were amphenicol, kanamycin, penicillin, rifampicin, streptomycin determined according to Felsenstein (1 985). and vancomycin; all from Sigma) was tested at 80 "C in Quantitative DNA-DNA hybridization. DNA from the ref- BHI-S medium at concentrations of 50, 100 and 150 pg ml-'. erence strains P.furiosus and ' P. abyssi' was used as labelled Thermotoga maritima was used as a bacterial control to test probes in a single hybridization experiment with strain the efficiency of antibiotics at 80 "C. 'P. abyssi' (strain AL585T. Four to five micrograms of each reference-strain ST549) was used as an archaeal control to test the growth of DNA was labelled by incorporation of both ['HIdATP and another archaeon in the presence of antibiotics. [3H]dGTP by using a Megaprime kit (Amersham). The nuclease S 1 method for quantitative DNA-DNA hybrid- DNA extraction and base composition. DNA was prepared ization was carried out as described by Popoff & Coynault from cells harvested at the end of the exponential phase of (1980) by using DE81 filters (Whatman) and a pmatic IV growth. After centrifugation,the cell pellet was suspended in scintillation counter (Kontron Instruments). 5 ml lysis buffer (100 mM Tris/HCl, 100 mM NaC1,0.5 mM EDTA; pH 8) and then 1 Oh (w/v) Sarkosyl, 1 YO(w/v) SDS and 0-4mg proteinase K 1-' were added to achieve cell lysis. RESULTS After 3 h incubation at 40 "C, three extractions with cold Collection of samples phenol/chloroform/isoamyl alcohol (24 :24 : 1) and one ex- traction with chloroform were performed (1 : 1 with lysate). Chimney walls of active, deep-sea vents, frequently Between the first and second phenol/chloroform/isoamyl covered with alvinellid communities, were sampled alcohol extractions, DNA was treated with RNase (5 pg from different sites on the East Pacific Rise at latitudes m1-l) for 1 h at 60 "C. DNA was then precipitated by 11, 13 and 21" N with the deep-submergence vehicle addition of 2 vols ethanol at - 20 "C. The dried DNA pellet during the American oceanographical cruise was resuspended in TE (10 mM Tris/HCl, 2 mM EDTA; Alvin pH 7.4). DNA concentration and purity was tested spectro- MVT'90 in 1990. These samples, chimney wall pieces photometrically at 230, 260, 280 and 320 nm with a and alvinellid tubes were stored in serum vials filled GenQuant I1 spectrophotometer (Pharmacia). DNA base with sterile sea water under anaerobic conditions composition was determined by thermal denaturation obtained by injection of sodium sulfide to a final
International Journal of Systematic Bacteriology 49 1831 G. Barbier and others
Fig. I. Transmission electron micrographs of strain AL585T. (a) Single cells and division by constriction (arrow). Bar, 1 pm. (b) View of the cell wall. Arrows indicate surface layer (SL), periplasmic space (PS) and cytoplasmic membrane (CM). Bar, 0.1 pm. - concentration of 0.5 g 1-'. Samples were brought back stained layer (5 nm), probably corresponding to a to the laboratory at ambient temperature immediately surface-layer protein as already described for other after the cruise. Strain AL585T was isolated from a thermococcales (Huber et al., 1995) (Fig. lb). Cells sample containing pieces of one smoker and its divided by constriction, as visible in Fig. l(a), but covering of alvinellid tubes collected at a site called budding cells were not observed. Clumps of dense, Totem, latitude 13" N, depth 2650 m. spherical particles were frequently present inside the cytoplasm and fibres and dense particles were some- Enrichment and purification times attached externally to the cell envelope. Electron microscopy permitted the study of the diameter dis- Strain AL585T was obtained at 95 "C in BHI-S tribution. The diameters of single cells ranged from 0.5 medium, pH 7.5. Purification of strain AL585T was to 1.5 prn, depending on their size and the axis of performed by successive plating of culture samples measurement. The diameters measured were normally onto solid BHI-S medium prepared with Gelrite. distributed and the mean diameter was 1 pm. Small, translucent, colourless colonies appeared within a few days, indicating that growth was slow in such Growth parameters conditions compared with liquid medium. One isolated colony was transferred to liquid medium and stored. Near atmospheric pressure, strain ALMTgrew slowly at 75 and 104 OC, but growth was not observed at Morphology 107 "C. The maximum growth rate was observed at 95 "C (Fig. 2a). The highest cell yield was also obtained Observations by phase-contrast microscopy revealed at this temperature, which can be considered as that strain AL5MTcells were cocci occurring singly, in optimal. Growth was slow at pH 2-5 and 9-5 and the pairs or in small aggregates. The cells appeared to be optimum pH was between 6.5 and 8-5 (Fig. 2b). The motile and polar flagella were observed by phase- highest cell yield was obtained at pH 7.5, which can be contrast microscopy after specific coloration. Numer- considered as the optimum pH. Growth was observed ous diplococci were observed during the exponential at sea-salt concentrations ranging from 20 to 60 g 1-l. growth phase, indicating that cells probably divided by Calculated growth rates were not significantly different constriction. Under electron microscopy, more or less between 30,40 and 60 g 1-l. Strain ALWTwas unable spherical cocci were observed (Fig. la). These cells to grow without sea salt or at a concentration of 80 g were delimited by a cytoplasmic membrane (5 nm) 1-1 (Fig. 2c). The highest cell concentrations at the end covered by a bilayered cell envelope with an inner of the exponential phase were obtained for 30 g 1-I sea periplasmic space (1 5 nm) and an external, densely salt, which can be considered as optimal. Under the
~~~ ~ 1832 International Journal of Systematic Bacteriology 49 Pyrococcus glyco vorans sp. nov.
0.2 -
*n 0.15 - =8 i? € f 0.1 - 9 5 a L 4 I I 0 1 I I I I 70 80 90 100 110 2 4 6 8 10 Temperature ("C) Time (h)
Fig. 3. Growth curve as determined by flow-cytometry counts.
n
0.1s B e phase was abnormally short. In order to observe the = 0.1 s growth kinetics at relatively low concentrations di- : I rectly, we determined cell numbers against time using 3 0.05 ti E E E € flow cytometry. A volume of 0.1 ml of a culture 0 containing 2 x 10' cells ml-l was inoculated in 30 ml 2 4 6 8 10 12 PH BHI-S medium and cultivated at 95 "C. This culture (c) . was sampled over 8-7 h. Flow cytometry counts are shown in Fig. 3. Three observations were made during
0.2 the exponential phase of growth and the three cor- 1 responding points, from 6 x lo5 to 6-2 x lo7cells ml-', -h were perfectly aligned (r2= 0.9999). From these f 0.15 - 0 points, a doubling time of 27 min was calculated. *e I These results suggested that growth rates for strain 0.1 i 9 B - AL585T calculated at microscopically or densitometri-
ePD cally countable concentrations (from lo7 to 5 x 10' cells ml-l) were suboptimal. This signifies that ' optimal 4 0.05 - I growth' (i.e. maximum growth rate) in serum vials or Hungate tubes was only observable at relatively low Oicell concentrations, not easily compatible with the 0 20 40 60 80 100 techniques commonly used for cell counting. When using these techniques, Thoma chamber counts or OD measurements, we observed a transition phase between Fig. 2. Temperature, pH and sea-salt concentration optima for the exponential growth phase and the stationary phase, growth of strain AL585T on 2216-S medium. (a) Growth rate as a function of temperature (30 g sea salt I-', pH 7.5). (b) Growth during which the growth rate decreased. rate as a function of pH (30 g sea salt I-', 95 "C). (c) Growth rate as a function of sea-salt concentration (95 "C, pH 7.5). Bars indicate confidence intervals. Nutritional requirements Elemental sulfur clearly stimulated growth of strain AL585T in complex medium (maximum concen- supposed optimal conditions (95 "C, pH 7.5, 30 g 1-1 trations greater than or equal to 2 x 10' cells ml-l) sea salt), the mean doubling time from three replicates (Fig. 4). We repeatedly observed that cystine had less was 93 min and the highest cell concentration observed effect (maximum concentrations between 5 x lo' and 2 x 10' cells ml-l) and that polysulfide failed to was around 5 x 10' cells ml-'. This doubling time was higher than the lowest previously described for other stimulate growth (maximum concentrations less than or equal to 5 x lo7 cells ml-l). Elemental sulfur species: 37 min for (Fiala & Pyrococcus P. furiosus appeared to prolong growth, which was inhibited by Stetter, 1986), 35 min for P. woesei (Zillig et al., 1987), 33 rnin for (Erauso 1993) and 32 rnin metabolic products in its absence. A high concen- 'P. abyssi' et al., tration (80%) of H, in the gas phase also strongly for ' P. horikoshii' (Gonzalez et al., 1998). inhibited growth. In the presence of elemental sulfur, Considering this mean doubling time under optimal cultures produced large amounts of hydrogen sulfide conditions, we observed that the duration of cultures during growth (data not shown). These results suggest made with highly diluted inocula (dilution of v/v, that addition of elemental sulfur prevents H, inhibition for instance) until the end of the exponential growth of growth, as proposed by Fiala & Stetter (1986). lnternational Journal of Systematic Bacteriology 49 1833 G. Barbier and others
*** 0.014 L hydrothermalis
0.002
*** 0.019 I: fumicolans
*** 0.005 ‘p abyssj’ I
I-==*** 0.013 ***
pAL585I 10 15 20 0.002 Time (h)
0.004 bl P Fig. 4. Influence of the presence and absence of sulfur compounds and of the gas phase on growth of strain AL585T in BHI medium. Gas phases tested were N,/H2/C02 (90:5:5), Fig. 5. Unrooted phylogenetic tree of strain AL585T and other unsupplemented (a)or supplemented with sulfur (A),cystine reference species from the Thermococcales. The tree was (0)or polysulfide (m), and H2/C02(80:20) (0)(without added based on an alignment done with CLUSTAL w and established sulfur compounds). using PHYLO-WIN with the following parameters: distance, Jukes-Cantor, and successively neighbour-joining, maximum- likelihood and maximum-parsimony methods. Bootstrap values (encircled numbers) are those of the neighbour-joining method with 500 replicates. ***, All three methods give the same Strain AL585Tappeared to be an obligate heterotroph. branching. Numbers on the branches give the evolutionary No growth was observed in mineral medium, in the distances. absence of a carbon source, with an HJCO, gas phase (80:20), with or without elemental sulfur (data not shown). Yeast extract plus peptone (2216s medium) and brain heart infusion (BHI-S medium) supported 165 rRNA sequence analysis rapid and efficient growth of strain AL585T(maximum The sequence of the 16s rDNA of strain AL585T was concentrations greater than or equal to 2 x 10’ cells determined and revealed that this isolate belonged to ml-l). This strain was also able to grow equally the genus Pyrococcus. Consequently, the sequence was efficiently with peptone or glucose as carbon and aligned with other available 16s rDNA sequences for energy sources. Less efficient growth was observed Pyrococcus species ; sequence similarities were found with starch and maltose as carbon sources (maximum to be 98 YOto P. furiosus, ‘ P. abyssi’ and ‘ P. horikoshii’, concentrations between 5 x lo’ and 2 x 10’ cells ml-l). 96 YOto Thermococcus hydrothermalis and 94 YOto A small amount of growth was obtained with meat Therrnococcus fumicolans. Fig. 5 gives the correspond- extract, yeast extract, chitin, cellobiose and a mixture ing phylogenetic tree. In this tree, strain AL585T of 20 amino acids (maximum concentrations between appeared closer to P. furiosus than to ‘P. abyssi’ and 1-5x lo7 and 5 x lo7 cells ml-l). None of the other P. horikoshii’. substrates tested as carbon sources permitted growth. DNA-DNA hybridization Antibiotic sensitivity Labelled probes prepared from total DNA from P. Strain AL585T was resistant to rifampicin, vanco- furiosus and ‘P. abyssi’, the two Pyrococcus species mycin, penicillin, kanamycin, streptomycin and chlor- previously described, closely related to strain AL585T, amphenicol at concentrations of 50, 100 and 150 pg were hybridized with total DNA from strain AL585T. ml-? Thermotoga maritima exhibited the expected The levels of DNA reassociation measured were 20 pattern of antibiotic sensitivity (Huber et al., 1986) at and 18 YO,respectively. We can observe that the values 80 “C, demonstrating that the antibiotics were active are far below 70 YO,which is the percentage to consider at high temperature. for definition of a new species (Wayne et al., 1987). Those results confirm that strain AL585T is a new DNA composition species of Pyrococcus. The G+C content of the DNA of strain AL585T was DISCUSSION 47a 1 mol%. The G+C contents of P. furiosus and ‘ P. abyssi’, determined under the same conditions, The novel marine hyperthermophilic strain AL58 5T were 38 i 1 and 44 & 1 mol YO.These two values are can be assigned to the archaeal domain (Woese et al., identical to the values published previously (Fiala & 1990) because of its resistance to antibiotics and its 16s Stetter, 1986; Erauso et al., 1993). rDNA sequence. This latter result (Fig. 5) indicates
1834 International Journal of Systematic Bacteriology 49 Pyrococcus glycovorans sp. nov.
Table 1. Comparison of characteristics of strain AL585T and other Pyrococcus species
Data were taken from Fiala & Stetter (1986), Brown et al. (1990), Schafer & Schonheit (1991), Kengen et al. (1993) and Raven & Sharp (1997) (P.furiosus); Zillig et al. (1987), Koch et al. (1991) and Rudiger et al. (1992, 1995) (P. woesei); Erauso et al. (1993) ('P. abyssi'); and Gonzalez et al. (1998) ('P. horikoshii'). ND, Not determined.
Characteristic P. furiosus Vcl' P. woesei DSM 3773' ' P. abyssi' GE5 ' P. horikoshii' JCM 9974 Strain AL585' (= DSM 363gT) (= CNCM 1-1302)
G+C content (mol%) 37-38 37.5 4445 44 47 Growth temperature ("C): Optimum 100 100-103 96 98 9s Range 70-103 up to 1os* 67-102 80-102 75-104 pH : Optimum 7 ND 6.8 7.0 Range 5-9 ND 4.0-8.5 5-8 NaCl concentration (g I-'): Optimum 20 30 30 24 26 Range 5-50 ND 7-SO 10-50 17-52 Carbon sources used Yeast extract, peptone, Yeast extract, tryptone, Brain heart infusion, yeast Peptone, yeast extract, beef Brain heart infusion, yeast tryptone, meat extract, glycogen. starch, gellan extract, meat extract, extract, tryptone, casein, extract, peptone, meat casein, starch, maltose, peptone, tryptone, 20 20 amino acids extract, starch, chitin, cellobiose, glycogen, amino acids maltose, cellobiose, pullulan. pyruvate glucose. 20 amino acids * No minimum temperature for growth was given. that this strain belongs to the order Thermococcales already described for P. furiosus and P. woesei. This is (Zillig et al., 1987), which is so far represented by two in line with 16s rDNA sequence comparisons (Fig. 5), genera, Pyrococcus (Fiala & Stetter, 1986) and Therm- which separated Pyrococcus species in two groups ococcus (Zillig et al., 1983), and the strain is included in corresponding with these phenotypic differences. the genus Pyrococcus. The new strain has a mor- However, strain AL585T is able to use glucose as a phology similar to the previously described species of carbon source for its growth, a feature that appears to Pyrococcus and, similar to other strains of Pyrococcus, be unique in the genus Pyrococcus. This ability to strain AL585T has an optimal growth temperature ferment proteins, amino acids and carbohydrates in above 90 "C, while Thermococcus species grow the presence of sulfur appears to correspond to the optimally below 90 "C (Godfroy et al., 1996, 1997; characteristics of the original ecosystem surrounding Miroshnichenko et al., 1998; Canganella et al., this isolate. Strain AL585T was isolated from pieces of 1998; Duffaud et al., 1998). Characteristics of strain an active smoker covered with an alvinellid colony, AL585T and other species of the genus Pyrococcus are made mainly of Alvinella pompejuna individuals. Cur- shown in Table 1. Strain AL585T has a G + C content rent knowledge of the ecology of this species higher than other strains of Pyrococcus, and this (Desbruyeres et al., 1998) clearly indicates that all the criterion distinguishes the abyssal strains (' P. abyssi', major elements we used to prepare culture media (sea 'P. horikoshii' and strain AL585T) very clearly from salt, sulfur, proteins and carbohydrates) are present in the coastal strains (P.furiosus and P. woesei). How- the immediate vicinity of this animal. ever, the growth responses to temperature, pH and On the basis of its physiological characteristics, its 16s salinity do not differentiate strain AL585T from the rDNA sequence and DNA-DNA hybridization data, other species of Pyrococcus. The maximum growth strain ALWTrepresents an new species of rate observed for strain AL585T corresponds to a Pyrococcus, and we propose to name it Pyrococcus glycovorans sp. doubling time of 0.5 h, which is similar to that observed nov. for other Pyrococcus species. However, in the case of strain AL585T, the maximum growth rate was much lower (doubling time of 1-5h) at the end of the Descr ip t ion of Pyrococcus glyco vorans Barb ier, exponential phase of growth, in the range of cell Godfroy, Meunier and Raguhes sp. nov. concentrations usually studied. We can hypothesize that this transition phase could reveal the existence of Pyrococcus glycovorans (gly.co.vo'rans. Gr. adj. glykos a limiting factor for growth or an inhibiting effect of sweet, referring to glucose; L. part. pres. vorans eating, metabolic products when growth occurs in closed devouring; M.L. part. adj. glycovorans eating glu- vessels. This last hypothesis will have to be tested cose). further by studying the behaviour of strain AL585T Cells are cocci with a mean diameter of 1 pm (range when grown in a fermenter. 0-5-1.5 pm), motile with polar flagella. Cell division As with other Pyrococcus species, strain AL5UTgrows occurs by constriction. Obligately anaerobic. Grows efficiently in the presence of proteinaceous substrates. optimally at 30 g 1-1 sea salt and pH 7.5. At atmos- Unlike ' P.abyssi' and ' P. horikoshii', strain AL585Tis pheric pressure, growth occurs between 75 and 104 "C. also able to use different carbohydrates, an ability Obligately chemo-organotrophic. Ferments protein
International JournaI of Systematic Bacteriology 49 1835 G. Barbier and others and proteolytic products and carbohydrates, especially from the Guaymas Basin hydrothermal vent site. Int J Syst glucose. Sulfur is not necessary for growth but sig- BacterioZ48, 1181-1 185. nificantly enhances final cell concentrations when Dauga, P. A. D. & Grimont, P. A. D. (1991). Nucleotide sequence grown in closed culture vessels. Sulfur is reduced to of 16s rRNA from ten Serratia species. Res kIicrobiol 141, H,S. At low cell concentrations, under optimal con- 1139-1 149. ditions and at atmospheric pressure, doubling time is Desbruyhes, D., Chevaldonnb, P., Alayse, A.-M. & 15 other 0.5 h and maximal concentrations at the beginning of authors (1998). Biology and ecology of the 'Pompeii worm' the stationary phase can reach 5 x lo8cells m1-I. G + C (Alvinella pompejana Desbruyeres and Laubier), a normal content is 47 mol Yo.16s rDNA sequence comparisons dweller of an extreme deep-sea environment: a synthesis of locate Pyrococczis glycovorans within the Thermo- current knowledge and recent developments. Deep-sea Res coccales, in the archeal domain. The GenBank/EMBL Part 11 Top Stud Oceanogr 45, 383-422. accession number for the 16s rDNA sequence is Duffaud, G. D., d'Hennezel, 0. B., Peek, A. S.,Reysenbach, A.-L. & 270247. Kelly, R. M. (1998). Isolation and characterization of Thermo- coccus barossii, sp. nov., a hyperthermophilic archaeon isolated The type strain, AL585' (= CNCM I-2120T), was from a hydrothermal vent flange formation. Syst Appl Microbiol isolated from pieces of a deep-sea smoker and its 21, 4049. covering of alvinellid tubes collected at 13" N at a Erauso, G., Reysenbach, A. L., Godfroy, A. & 8 other authors depth of 2650 m on the East Pacific Rise. (1993). Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Arch Microbioll60, 338-349. ACKNOWLEDGEMENTS Felsenstein, J. (1981). Evolutionary trees from DNA sequences: We thank the chief scientists of the American oceano- a maximum likelihood approach. J Mol Evol17, 368-376. graphical cruise MVT'90, Richard A. Lutz and Robert Felsenstein, J. (1985). Confidence limits on phylogenies : an Vriejenhoek (Institute of Marine and Coastal Sciences, approach using the bootstrap. Evolution 30, 783-79 1. Rutgers University, New Brunswick, NJ, USA) and our Fiala, G. & Stetter, K. 0. (1986). sp. nov. colleagues Anne-Marie Alayse and Daniel Desbruyeres Pyrococcus fiuiosus (IFREMER, Centre de Brest, France) for collection of represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 "C. 56-61. samples. We thank the captain and crew of N.0. Atlantis II Arch Microbioll45, and the D.S. V.Alvin pilots and support crew. We also thank Galtier, N., Gouy, M. & Gautier, C. (1996). SEAVIEW and PHYLO- Dominique Marie (CNRS, Station Biologique, Roscoff, WIN: two graphic tools for sequence alignment and molecular France) and Germaine Dorange (Universite de Bretagne phylogeny. Comput Appl Biosci 12, 543-548. Occidentale, UFR Sciences, Brest, France) for their tech- Godfroy, A., Meunier, J.-R., Guezennec, J., Lesongeur, F., nical support, respectively for flow cytometric analysis and Raguhes, G., Rimbault, A. & Barbier, G. (1996). Thermococcus transmission electron microscopy. We thank Pascale
1836 International Journal of Systematic Bacteriology 49 Pyrococcus glycovorans sp. nov.
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