International Journal of Systematic Bacteriology (1 998), 48, 1 181-1 185 Printed in Great Britain

Thermococcus guaymasensis Spm nov. and Thermococcus aggregans sp. nov., two novel thermophilic archaea isolated from the Guaymas Basin hydrothermal vent site

Francesco Canganella,' William J. Jones,' Agata Gambacorta3 and Garabed Antranikian4

Author for correspondence: Francesco Canganella. Tel : + 39 076 I 357282. Fax : + 39 076 1 357242. e-mail : canganel(a unitus.it

1 Department of Thermococcus strains TYST and TVisolated from the Guaymas Basin Agrobiology and hydrothermal vent site and previously described were compared by DNA-DNA Agrochemistry, University of Tuscia, via C. de Lellis, hybridization analysis with the closest Thermococcus species in terms of 01 100 Viterbo, Italy physiology and nutritional aspects. On the basis of the new data and taking * Ecosystem Research into consideration the molecular, physiological and morphological traits Division, US Environmental published previously, it is proposed that strains TYT and TYST should be classified Protection Agency, Athens, as new species named Thermococcus aggregans sp. nov. and Thermococcus GA, USA guaymasensis sp. nov., respectively. The type strain of T. aggregans is strain CNR, Institute for the TV(= DSM 10597T)and the type strain of T. guaymasensis is strain TYST Chemistry of Interesting B io logica I Molecules (= DSM 11113T). (ICMIB), Arc0 Felice (Na), Italy I

Department of ~ Keywords: Thermococcus, archaea, hydrothermal vents Biotechnology I, Technical University of Hamburg, Germany

INTRODUCTION from 85 to 88 "C (75 "C for T. stetteri) and the G+C content ranges approximately between 42 and During the past 3 years, six additional species be- 57 mol %, except for T. litoralis (38 mol %). According longing to the genus Thermococcus have been de- to the most recent investigations on the microbiology scribed. Presently, 10 species represent this taxonomic of hydro thermal vent sites, Thermococcus species group: Tlrcrmococcus celer (Zillig et al., 1983), ' Ther- appear to be present in significant numbers in these mococcus litoralis (Neuner et ul., I990), Tlzermococcus ecosystems (Antoine et al., 1995; Marteinsson et al., stetteri (Miroshnichenko et al., 1989), Thermococcus 1995). profundus (Kobayashi et al., 1994), Thermococcus peptonopliilus (Gonzalez et al., 1993, Thermococcus Two novel deep-sea Thermococcus strains (TYST and chitonophtrgus (Huber et al., 1999, Therrnococcus TYT) previously isolated from the Guaymas Basin alcaliphi1zi.s (Keller et ul., 1999, Thermococcus ,fumi- (Gulf of California) and extensively described (Can- colans (Godfroy et al., 1996), Thermococcus hydro- ganella & Jones, 1994a; Canganella et al., 1997) thermalis (Godfroy et nl., 1997) and ' Thermococcus showed a relatively high similarity of 16s rDNA zilligii, (Ronimus et al., 1997). All species are organo- sequences with other Thermococeus species but ex- trophic, thermophilic anaerobes growing preferen- hibited some different physiological properties (Can- tially on proteinaceous compounds and utilizing carbo- ganella et al., 1997). Compared with the most recently hydrates :is carbon sources rarely. Elemental sulphur described T. hydrothermulis and T. -fumicoluns, our considerably stimulates growth with the concomitant strains are able to grow on starch, there is no absolute formation of H,S. Typical optima temperatures range requirement for elemental sulphur, and they are more sensitive to NaCl concentrations higher than 5.0 %

...... , ...... , ...... , , ...... , .. . (w/v). DNA-DNA hybridization experiments were Abbreviation : TOR, temperature of renaturation. necessary to determine the taxonomic standing of The EMBL accession numbers for the partial 165 rDNA sequences of strains these two strains. Based on the results of these analyses, TYT and TYST are YO8384 and Y08385, respectively. both strains were found to represent new species of

~- ~___ 00737 0 1998 IUMS 1181 F. Canganella and others

Thermococcus, and their complete taxonomic descrip- The DNA concentration was 45 pg ml-I. Owing to the high tion is reported in this paper. G+C content of Thermococcus DNA (mean value of 50 mol YO),20% (v/v) DMSO was added to 2.5 x SSC to depress the DNA melting point (T,) (Escara & Hutton, METHODS 1980). The resulting buffer was 2 x SSC + 20 Y DMSO. The mean T, was determined from the DNA melting curves and Source of micro-organisms. Strains TYST and TY' were the optimal temperature of renaturation (TOR) was cal- isolated from sediments collected at the Guaymas Basin culated as T,+25. hydrothermal vent site (2000m depth) in the Gulf of California. On the basis of nutritional, physiological and Renaturation rates were calculated by regression analysis molecular features, they were previously described as Ther- between 10 and 30 min after the reaction start using the ~ZOCOCCUSspecies by Canganella & Jones (1994a, b) and computer program TRANSFERBAS (Jahnke, 1992). Canganella et al. (1 997). T. celer, ' T. litoralis', T. stetteri and T. projundus strains RESULTS AND DISCUSSION were obtained from DSMZ, Braunschweig, Germany (see Table 1). T. peptonophilus strain OG-lT(= JCM 9653T)was Morphology of Thermococcus strains TYST and TYT kindly provided by Chiaki Kato (JAMSTEC, Yokosuka, Japan) and T. hydrothernialis and T..fumicolans were a gift The morphological features of isolates TYST and TY"' from Anne Godfroy (IFREMER, Plouzane', France). have been reported previously (Canganella & Jones, 1994a; Canganella et al., 1997). Under light micro- Growth conditions. The two novel Theruzococcus strains as scopy, cells of TYST and TYT usually appear as well as the reference micro-organisms were grown on a irregular cocci with a variable width of 0-5-3.0 pm. mineral medium containing the following constituents per litre of distilled water: 0.33 g KC1; 2.7 g MgCl .2H,O; 3.4 g Strain TYST is similar to other Thermococcales species MgSO,. 7H,O; 0.25 g NH,Cl; 0.14 g CaC1,. 2H,O; 0.14 g in that a dark granule often forms on the outer cell K,HPO,; 0.01 mg Na,SeO,; 0.01 mg NiC1,. 6H,O; 1.0 g surface. Strain TYST usually exists as single cells, NaHCO,; 18-0 g NaCl; 0.001 g resazurin; 0.5 g cys- whereas strain TYT typically forms cell aggregates up teine. HC1. H,O; 0.5 g Na,S. 9H,O. The medium also con- to 50-100 cells under optimal growth conditions. Both tained Bacto-yeast extract (0.2 g 1-') and 10 ml 1-1 each of strains are non-motile. trace metal and vitamin solutions (Balch et al., 1979). Anaerobic techniques used to prepare and dispense the Nutritional requirements medium, the enrichment experiments, isolation procedures, as well as the physiological characterization of the new Strains TYST and TYT grew chemo-organotrophically isolates have been described elsewhere (Canganella & Jones, on complex substrates, e.g. yeast extract, trypticase, 1994a, b). casein, starch, dextrose and maltose. In the presence of Phylogenetic analysis. DNA of harvested cells was extracted elemental sulphur, higher growth yields were observed according to the procedure of Marmur (1961), digested to with proteinaceous compounds, accompanied by H,S nucleosides, and the G+C content was determined after production (Canganella & Jones, 1994a). No growth nucleoside analysis by HPLC as described by Mesbah et al. was detected in medium supplemented with chitin, (1989). organic acids (formate, acetate, propionate, butyrate; Genomic DNA extraction, PCR-mediated amplification of 0.5 "/o,w/v), alcohols (methanol, ethanol, propanol; the 16s rDNA and purification of PCR products was carried 0.5 YO,v/v), or Difco Casamino acids (0.4 YO,w/v) as out as described previously (Barns et al., 1994; Rainey et al., primary carbon sources (Canganella & Jones, 1994a). 1992; Rainey & Stackebrandt, 1993).

The 16s rDNA sequence was manually aligned against Phylogenetic analysis and DNA hybridization representative sequences of members of the domain Archaea. The following sequences from the EMBL database were The G+C content of DNA extracted from Thermo- analysed : M21529 (T. celer), 275240 (T. stetteri), D37982 coccus isolates TYT and TYST was 42.0 and (T.peptonophilus), 270252 (' T.litoralis'), 270244 (T. hydro- 46.0 mol %, respectively. thermalis), 270250 (T.funiicolans), 275233 (T. projundus), X99570 (T. chitonophagus) and U20 163 (P.furiosus). Pair- The nearly complete sequences consisting of 130 1 wise evolutionary distances were computed using the cor- bases of the amplified 16s rDNA of isolates TYT and rection factor of Jukes & Cantor (1969). The least-squares TYST were previously determined. The sequences of distance method of De Soete (1983) was used in the both isolates were therefore aligned and compared construction of the phylogenetic dendrogram from distance with all the available 16s rDNA sequences of matrices. Thermo- coccus species, including Pyrococcus furiosus. On the DNA isolation and hybridization. DNA was isolated by basis of these data, the 16s rDNA sequence of strain column chromatography using hydroxyapatite according to TYT showed a similarity of 98.4% with strain TYS', the procedure of Cashion et al. (1977). 96.7% with T. chitinophagus, 97.7% with T. hydro- DNA-DNA hybridization was performed according to the thermalis, 97.0 YOwith T.fumicolans and 93.9 % with methods of De Ley et al. (1970) using a Gilford System 2600 P. furiosus. The 16s rDNA sequence of strain TYST spectrophotometer equipped with a Gilford 2527-R thermo- showed a similarity of 96.6% with T. chitinophagus, programmer and plotter. 98.4% with T. hydrothermalis and 96.0% with T.

1182 International Journal of Systematic Bacteriology 48 Two novel Therrnococcus species

T chitonophagus terial systematics (Wayne et al., 1987). Strains TYST and TYT are named Thermococcus guuymusensis sp. nov. and Thermococcus aggregans sp. nov., respect- T aggregans (strain TYT) ively. T. alcaliphilus was not included in this analysis because its primary physiological feature (optimal I- growth at elevated pH) represents a unique taxo- nomical trait ; moreover, its 16s rDNA sequence is not available on GenBank. 'T. zilligii' was also not IT: fumicolans included in the present work because its isolation source (terrestrial freshwater hot pool), its inability to grow at NaCl concentrations higher than 1.2% - T: guaymasensis (strain TYST) (w/v) and its unusual membrane lipid composition clearly differentiate it from the other Thermococcus T: celer species.

T: hydro thermalis k Description of Thermococcus aggregans sp. nov. Thermococcus aggregans (ag'gre.gans. L. v. uggregure to stick together, to aggregate; L. pres. part. aggregans assembling, aggregating). Coccoid archaeon with cell diameters of 1.0-1-5 pm. Cells are Gram-positive, non-motile and occur as single cells, diploid forms or (particularly when cul- tivated on yeast extract/tryptone plus sulphur) P. furiosus aggregates of many cells (up to 50). Colonies are circular, convex and pale-grey in colour, and colony diameters range from 0.5 to 1.0 mm after 48-72 h 0.05 incubation at 70 "C under strictly anoxic conditions. Chemo-organotrophic and strictly anaerobic. Utilizes ...... preferably proteinaceous substrates such as yeast Fig. 1. Unrooted phylogenetic tree indicating the phylogenetic extract and trypticase in the presence of 0.5-1.0 YO position of T. guaymasensis and T. aggregans compared with other Thermococcus species and P. furiosus. (w/v) elemental sulphur. Casein, dextrose, maltose and starch are also utilized. The end products of metabolism on medium containing yeast extract plus tryptone and sulphur are acetate, propionate, iso- fumicolun Y.The phylogenetic tree constructed from all butyrate, isovalerate, CO, and H,S, whereas, after similarity data is shown in Fig. 1. growth on dextrose or maltose plus sulphur, only Thermococcus strains TYST and TYT are distinct acetate, CO, and H,S are formed. The pH range for species as indicated by a DNA hybridization value of growth is 5-6-7-9; the optimum pH is 7.0. Grows 41.2 YO(Table 1). They also exhibit DNA hybridization between 60 and 94 "C, with no growth occurring at values less than 70% with the other Thermococcus 95 "C. The optimum temperature is 88 "C. The NaCl species, indicating that they are new and distinct range for growth is 1-0-3.0 YO(w/v) with the optimum species in accordance with the recommendations of the at 2.5 YO.The G + C content of the DNA is 42-0 mol 70. Committee on reconciliation of approaches to bac- Lipids are mainly represented by one phospholipid

Table 1. DNA hybridization (%) between T, guaymasensis strain TYST, T. aggregans strain TYT and other Thermococcus species

Strain 1 2 2 4 5 6 7 8 9

~~ 1 T. gz.~u.i~n~ase~sis(DSM 1 1 1 13T) 100 2 T. aggregans (DSM 10597T) 42.1 100 3 T. cc.lei* (DSM 2476T) 44.4 33.9 100 4 ' T. litorulis' (DSM 5474) 37.1 52.4 38-2 100 5 T. srcrteri (DSM 5262"') 48.7 41.5 44.9 39.9 100 6 T. peptonopttilus (JCM 9653T) 48.3 31.1 49.5 33.9 47.3 100 7 T. prolundus (DT 5432") 45.9 36.8 48.4 32.7 46.8 50.7 100 8 T.fiiin~~ofan~ (CIP 104680') 50.5 55.6 49.9 40.9 49.2 59.9 44-8 100 9 T. hjdrotherrnalis (CNCMI 13 19') 51.6 43.4 53.9 39.8 39.3 43.6 40-3 57.2 100

___.~~~~ international Journal of Systematic Bacteriology 48 1183 F. Canganella and others -~ - and some minor components; core lipids are charac- REFERENCES teristically archaeol and caldarchaeol compounds in Antoine, E., Guezennec, G., Meunier, 1. R., Lesongeur, F. & equal amounts. Isolated from marine sediments at the Barbier, G. (1995). Isolation and characterization of extremely Guaymas Basin hydrothermal vent site (2000 m depth) thermophilic archaebacteria related to the genus T~ier~lilococci~s in the Gulf of California. Type strain TYT has been from deep-sea hydrothermal Guaymas basin. Curr Microhiol deposited in the DSMZ-German Collection of Micro- 31, 186-192. organisms and Cell Cultures, Braunschweig, Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R. & Wolfe, Germany, as strain DSM 10597' and in the Japanese W. S. (1979). Methanogens: re-evaluation of a unique biological Collection of Microorganisms as JCM 10 137'. group. Microhiol Rcv 43, 260-296. Barns, 5. 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~- lnterna tional Journal of Systematic Bacteriology 48 1185