International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1097–1104 DOI: 10.1099/ijs.0.02152-0

Vulcanisaeta distributa gen. nov., sp. nov., and souniana sp. nov., novel hyperthermophilic, rod-shaped crenarchaeotes isolated from hot springs in Japan

1 Japan Collection of Takashi Itoh,1 Ken-ichiro Suzuki1† and Takashi Nakase1,2 Microorganisms, RIKEN (The Institute of Physical and Chemical Research), Author for correspondence: Wako-shi, Saitama Takashi Itoh. Tel: j81 48 467 8440. Fax: j81 48 462 4860. 351-0198, Japan e-mail: ito!jcm.riken.go.jp 2 Laboratory of Microbiology, Department Seventeen strains of rod-shaped, heterotrophic, anaerobic, hyperthermophilic of Applied Biology and crenarchaeotes were isolated from several hot spring areas in eastern Japan, Chemistry, Faculty of Applied Bioscience, Tokyo and eight representative strains were characterized further. Cells of these University of Agriculture, strains were straight to slightly curved rods, 04–06 µm in width. Occasionally, Sakuragaoka 1-1-1, cells were branched or bore spherical bodies at the poles. They grew optimally Setagaya-ku, Tokyo 156-8502, Japan at 85–90 SC and at pH 40–45. They utilized yeast extract, peptone, beef extract, Casamino acids, gelatin, starch, maltose and malate as carbon sources and sulfur and thiosulfate as possible electron acceptors. The DNA GMC contents of the novel isolates were 439–462 mol%. The lipids were mainly cyclic and acyclic tetraether core lipids. Phylogenetic analysis of the 16S rDNA sequences revealed that they represented an independent lineage in the family . Moreover, comparison of the 16S rDNA sequences and a DNA–DNA hybridization study showed that they comprised two , which could also be differentiated by the maximal growth temperature and degrees of NaCl tolerance. Therefore, a new , Vulcanisaeta gen. nov., in the family Thermoproteaceae is proposed to accommodate two novel species, Vulcanisaeta distributa sp. nov. and Vulcanisaeta souniana sp. nov. The type species is V. distributa and the type strains are V. distributa IC-017T (l JCM 11212T l DSM 14429T) and V. souniana IC-059T (l JCM 11219T l DSM 14430T).

Keywords: Vulcanisaeta distributa, Vulcanisaeta souniana, Thermoproteaceae, hyperthermophilic

INTRODUCTION proteaceae and Thermofilaceae (Burggraf et al., 1997). They are strict or facultative anaerobes that metabolize All the known species within the kingdom Crenar- sulfur and\or thiosulfate and are reported to be chaeota are obligate thermophiles with maximum distributed widely in terrestrial hot springs and marine growth temperatures ranging from 75 to 113 mC geothermal habitats (Huber & Stetter, 2001). Until (Stetter, 1998). Using 16S rRNA\DNA-based phylo- recently, only a few genera and species had been genetic analysis, they can be divided into three major described in the order (Bonch-Osmo- clusters that correspond to the orders Desulfurococ- lovskaya et al., 1990; Huber et al., 1987; Vo$ lkl et al., cales, Sulfolobales and Thermoproteales (Ludwig & 1993; Zillig et al., 1981, 1983; Zillig, 1989). However, Klenk, 2001; Reysenbach, 2001). At present, all rod- the recent discoveries of two new genera in the family shaped crenarchaeotes are included in the order Thermoproteaceae (Thermocladium and Caldivirga; Thermoproteales, represented by the families Thermo- Itoh et al., 1998a, 1999) and two novel species [Pyrobaculum arsenaticum (Huber et al., 2000) ...... and Pyrobaculum oguniense (Sako et al., 2001)], as well † Present address: Biological Resource Center, Biotechnology Center, National Institute of Technology and Evaluation, Kazusa-Kamatari, as the detection of several phylotypes related to Kisarazu, Chiba 292-0812, Japan. members of the order Thermoproteales (Barns et al., The DDBJ accession numbers for the 16S rDNA sequences determined in 1994, 1996; Reysenbach et al., 2000; Takai & Hori- this study are AB063630–AB063647, as detailed in Fig. 2. koshi, 1999), suggest that there may be a wider

02152 # 2002 IUMS Printed in Great Britain 1097 T. Itoh, K. Suzuki and T. Nakase diversity of rod-shaped crenarchaeotes in geothermal at atmospheric pressure. Caldivirga maquilingensis JCM habitats. 10307T was cultivated as a reference strain, as described previously (Itoh et al., 1999). In the course of our exploration for novel thermophilic Archaea inhabiting terrestrial geothermal habitats, we Phenotypic and genetic studies. Morphology, growth have isolated a number of thermophilic organisms characteristics, utilization of carbon sources, possible elec- from hot springs in Japan and the Philippines. A tron acceptors, antibiotic sensitivity, lipid composition, preliminary grouping of the rod-shaped isolates by the DNA base composition and DNA–DNA relatedness were ability to grow at 85 mC, DNA base composition and determined as described previously (Itoh et al., 1998a). partial 16S rDNA sequences revealed the existence of Morphology, growth characteristics and utilization of car- another unassigned group belonging to the family bon sources were studied using the medium described above. Thermoproteaceae For electron microscopy, cells were placed on a collodion- , in addition to groups correspond- coated grid, shadowed with platinum\palladium and ex- ing to the genera Caldivirga, Thermocladium and amined with a transmission electron microscope (H-300; (T. Itoh, K. Suzuki, P. C. Sanchez and Hitachi). Possible electron acceptors were identified using a T. Nakase, unpublished). This paper describes the test medium (Itoh et al., 1998a) under a N# atmosphere. isolation and characterization of a novel group of Growth was estimated by fluorescence intensities after organisms in the family Thermoproteaceae and pro- treatment with NanoOrange dye (Molecular Probes) ac- poses a new genus, Vulcanisaeta gen. nov., including cording to the supplier’s protocol or direct counting using a two novel species, Vulcanisaeta distributa sp. nov. and Petroff-Hauser counting chamber (0n02 mm in depth). A Vulcanisaeta souniana sp. nov. good correlation between the two methods was obtained. 16S rDNA sequencing and phylogenetic analysis were METHODS conducted as reported previously (Itoh et al., 1999). 16S rDNA was amplified with primers A-20F (5h-TCCGGTTG- Isolation procedure. Samples of hot-spring water, mud and ATCCTGCCG-3h, corresponding to positions 8–24 in the soil were collected from several hot spring sites in Japan in Escherichia coli numbering system) and A-1530R (5h-GGA- 1993 and 1994 (Table 1). The samples were transported in GGTGATCCAGCCG-3h, positions 1540–1525). Partial 16S sterile plastic tubes without temperature control and were rDNA sequences were determined with a sequencing primer inoculated within 3 days of sampling into an enrichment A-690R (5h-GGATTTCRCCCCTAC-3h, positions 699– medium described previously (Itoh et al., 1998a) at pH 685). In addition, almost complete 16S rDNA sequences of values ranging from 3n5to6n0. The cultures were incubated five novel isolates and Pyrobaculum organotrophum JCM at 85 mC under N# at atmospheric pressure. After 1 week of 9190T were determined in this study. The 16S rDNA cultivation, cultures yielding predominantly rod-shaped cells sequences were aligned first on the   program were purified by the serial dilution technique, as described (Thompson et al., 1997) and edited manually with the aid of previously (Itoh et al., 1998a). the SSU rRNA database (Van de Peer et al., 2000). Culture conditions. The isolates were routinely cultivated in Evolutionary distances were calculated after gaps, ambigu- the salt base solution of the Sulfolobus medium (Brock et al., ous bases and unalignable regions had been eliminated. The " " 1972), containing 0n5 g yeast extract l− ,1n0 mg resazurin l− , phylogenetic tree was constructed by the neighbour-joining −" −" 0n5gNa#S.9H#Ol and 10n0 g sulfur l ,pH4n0–4n5 (adjust- method (Saitou & Nei, 1987) and was evaluated by bootstrap ed at room temperature). N# was used as the gas phase sampling (Felsenstein, 1985).

Table 1. Sources of novel isolates ...... Strains identified as Thermoproteus spp. are underlined. All locations are in east Japan (prefectures are shown after commas). The pH of the samples was determined at room temperature. , Not determined.

Isolate(s) Location Sample Temperature (mC) pH Date

IC-017T, IC-019 Ohwakudani, Kanagawa Water  2n0–2n509\1993 IC-058 Ohwakudani, Kanagawa Soil 85 2n702\1994 IC-051, IC-064 Ohwakudani, Kanagawa Soil 90 1n402\1994 IC-052, IC-065 Ohwakudani, Kanagawa Soil 90 2n102\1994 IC-059T, IC-060, IC-061, IC-062 Sounzan, Kanagawa Water  3n0–3n202\1994 IC-029 Okushiobara, Tochigi Soil 90 1n211\1993 IC-030 Okushiobara, Tochigi Soil 90 3n011\1993 IC-031 Okushiobara, Tochigi Water 75 2n911\1993 IC-032 Okushiobara, Tochigi Water 85 3n411\1993 IC-135 Tamagawa, Akita Water 90 1n611\1994 IC-141 Tamagawa, Akita Water 65 1n811\1994 IC-124 Fukenoyu, Akita Water 90 2n411\1994 IC-136 Gosyokake, Akita Soil 65 2n511\1994 IC-140 Ohnuma, Akita Mud 75 3n311\1994

1098 International Journal of Systematic and Evolutionary Microbiology 52 Vulcanisaeta gen. nov., with two species

(a) 55–100 mC; pH range 2n2–6n9). Among the rod-shaped strains isolated, three strains were later identified as Thermoproteus spp. by DNA base composition and partial 16S rDNA sequences (data not shown). Fol- lowing comparison of the partial 16S rDNA sequences of the remaining 17 strains (see Fig. 2a), eight representative strains (IC-017T, IC-029, IC-059T,IC- 064, IC-065, IC-124, IC-135 and IC-141) were selected for further characterization.

Morphology Cells of the eight strains were rigid, straight or slightly curved rods. They were 0n4–0n6 µm in width and highly (b) variable in length, mostly ranging from 3 to 7 µm. Cells longer than 20 µm were rarely observed. Bran- ched cells (Fig. 1c) or cells with terminal spheres (resembling the ‘golf clubs’ of Thermoproteus tenax; Zillig et al., 1981) were commonly observed. Occasion- ally, the cells were arranged in a V- or X-shape (Fig. 1b), and some strains (e.g. IC-029 and IC-135) formed cell aggregates 10–50 µm in diameter. Under the electron microscope, cells showed a single pilus or a few pili attached to the poles or at the sides, as shown in Fig. 1. The pili were usually shorter than 7 µm, although long pili of 15–20 µm were occasionally observed. No motile cells were observed. Bursting of (c) the cell envelope at the terminals was observed in some cells (Fig. 1a, c).

Growth characteristics The eight strains grew under stringent anaerobic conditions with N#,H#\CO# (4:1, v\v; 100 kPa) or N#\CO# (4:1, v\v; 100 kPa) as the gas phase. Unlike Thermocladium or Caldivirga strains (Itoh et al., 1998a, 1999), they grew well even in the absence of a vitamin mixture or archaeal cell-extract solution in the me- dium. They did not grow in air. In a low-oxygen atmosphere (5% air in N#), there was initially no ...... growth, but some strains were capable of growth after Fig. 1. Transmission electron micrographs of Vulcanisaeta prolonged incubation (more than 2 weeks) accom- strains. (a) Cell of strain IC-059T with pili attached terminally or panied by the reduction of the culture medium, as laterally to the cell. (b) Paired cells of strain IC-017T arranged in indicated by decolorization of resazurin. When three a V-shape. (c) Branching cell of strain IC-124. Bars, 1 µm. strains (IC-017T, IC-059T, IC-124) were cultured in medium in which sulfur was replaced by sodium −" thiosulfate (1n0gl ), they showed weak growth in a RESULTS AND DISCUSSION low-oxygen atmosphere, but not in air. Growth tem- Isolation perature and pH ranges for growth are shown in Table 2. When strains IC-017T, IC-059T and IC-124 were The novel rod-shaped, hyperthermophilic strains were cultured in the presence of 10 mM trisodium citrate T isolated from samples collected directly from solfataric under optimal growth conditions (IC-017 ,90mC, pH T fields or piped hot spring water (Sounzan), as shown in 4n5; IC-059 and IC-124, 85 mC, pH 4n5) with reciprocal Table 1. The samples yielded predominantly rod- shaking at 100 r.p.m., they grew with respective shaped cells under the enrichment conditions described doubling times of 6 5, 4 7 and 5 5 h and the cultures at n n n ( in Methods, while coccoid cells were often the pre- stationary phase contained approximately 4n2i10 , ( ( −" dominant organisms grown in enrichment cultures at 5n3i10 and 5n6i10 cells ml . Hydrogen sulfide was lower pH values (2n0–2n5) or under air or a H#\CO# gas detected in cultures of the three strains. The eight mixture (4:1, v\v; 100 kPa). After isolation by serial isolates showed no or negligible growth under auto- dilution, the purity of the rod-shaped strains was trophic conditions of a H#\CO# (4:1, v\v; 100 kPa) confirmed by microscope observation of the cultures gas mixture. All strains except IC-059T failed to grow grown under various conditions (temperature range in the presence of more than 1n0% (w\v) NaCl, while http://ijs.sgmjournals.org 1099 T. Itoh, K. Suzuki and T. Nakase

Table 2. Characters of the novel isolates ...... All of the isolates utilized maltose, starch, malate, yeast extract, peptone, beef extract, Casamino acids and gelatin as carbon sources. None of the isolates utilized -arabinose, -, lactose, sucrose, -xylose, acetate, butyrate, formate, fumarate, propionate, pyruvate, succinate, methanol, formamide, methylamine or trimethylamine as carbon sources. All of the isolates tested utilized sulfur and thiosulfate as electron acceptors. None of the isolates tested utilized fumarate, malate or nitrate as electron acceptors. , Not determined.

Character IC-017T IC-064 IC-065 IC-029 IC-124 IC-135 IC-141 IC-059T

Growth temperature (mC): Maximum 99 99 99 97 92 99 99 89 Minimum 70 70 75 75 65 75 75 65 Growth pH:* Maximum 5n66n06n05n65n65n65n65n0 Minimum 3n12n73n13n13n13n53n13n5 Utilization of carbon sources: Galactose j jkkkkk k Glucose k kjkkkk k Mannose j kjkkkk k Citrate k kjkkkk k Possible electron acceptors:† Cystine j\jj\j \kk\kj\k \k \ k\k FeCl$ j\kk\k \kk\kk\j \k \ k\k Oxidized glutathione k\jj\j \kk\kk\j \k \ k\k Sulfate j\kj\k \kk\kk\k \k \ k\j

* Media contained 10 mM trisodium citrate as buffer. † Media were reduced with titanium(III) citrate\Na#S ; 9H#O.

Table 3. DNA–DNA relatedness between the novel isolates

Strain Source Relatedness (%) with biotinylated DNA from:

12345678

1. IC-017T Ohwakudani 100 73 86 85 44 54 49 30 2. IC-064 Ohwakudani 103 100 94 90 55 66 63 52 3. IC-065 Ohwakudani 93 90 100 87 55 65 60 49 4. IC-029 Okushiobara 85 79 90 100 55 67 61 47 5. IC-124 Fukenoyu 41 52 50 55 100 67 63 37 6. IC-135 Tamagawa 60 62 62 68 68 100 97 39 7. IC-141 Tamagawa 56 59 59 64 67 94 100 37 8. IC-059T Sounzan 42 52 52 54 38 38 35 100 C. maquilingensis JCM 10307T 24352323

T strain IC-059 grew in up to 1n25% (w\v) NaCl (but ether core lipids. The core lipid fractions contained not 1n5%). acyclic or various cyclized glycerol–bisphytanyl–gly- cerol tetraethers. A trace amount of diether core lipid Other phenotypic studies was detected in strains IC-017T and IC-059T.

Results of utilization of carbon sources and possible DNA base composition and DNA–DNA hybridization electron acceptors are listed in Table 2. In antibiotic sensitivity testing, the eight strains were susceptible to The DNA GjC contents of the 17 strains, determined erythromycin, novobiocin and rifampicin, while they by the HPLC method of Tamaoka (1994), ranged from T were resistant to ampicillin, chloramphenicol, kana- 43n9to46n2 mol%. The values for IC-017 and IC- mycin, oleandomycin, streptomycin and vancomycin 059T were respectively 45 4 and 44 9 mol%. The results " n n (all at 100 µgml− ). Lipid composition studies showed of the DNA–DNA hybridization study are shown in that strains IC-017T, IC-059T and IC-124 had tetra- Table 3. All eight strains showed more than 30%

1100 International Journal of Systematic and Evolutionary Microbiology 52 Vulcanisaeta gen. nov., with two species

the phylogenetic tree (Fig. 2a), strains IC-059T and IC- 060, both isolates from Sounzan, are clearly separated from the remaining strains, with 2n5–3n6% sequence difference (100% bootstrap confidence). The remain- ing strains cluster with 98n2% sequence similarity and can be divided into two subclusters, one including the isolates from Ohwakudani and Okushiobara and the other including the isolates from Fukenoyu, Ohnuma, Gosyokake and Tamagawa, with 0n9–1n8% sequence difference between the two subclusters. The phylogenetic positions of the isolates within the kingdom were deduced from compari- son of almost entire 16S rDNA sequences of rep- resentative isolates (IC-017T, IC-059T, IC-065, IC-124 and IC-135) with known crenarchaeotes, including P. organotrophum. The sequences determined in this study were 1476 bp for IC-017T, 1477 bp for IC-059T, IC-124 and IC-135 and 1426 bp for P. organotrophum JCM 9190T. The PCR-amplified fragment of strain IC- 065 was about 2n2 kbp. Subsequent sequence analysis of the fragment showed that it consisted of 1476 nucleotides of the 16S rDNA exon (excluding the primer annealing regions) and a putative intron of 691 bp after position 1391 (E. coli numbering system). These 16S rDNA (exon) sequences were different from chimeric artefacts, according to the I program of the Ribosomal Database Project (Maidak ...... et al., 1999). Alignment of the 16S rDNA sequences Fig. 2. Phylogenetic trees of Vulcanisaeta strains. (a) Tree based showed that the sequences of all five isolates contained on partial 16S rDNA sequences (561 base positions) of all isolates. The selected strains are shown in bold. (b) Tree based all of the small-subunit rRNA signature sequences that define the crenarchaeotes (Woese, 1993), except for a on almost entire 16S rDNA sequences (1317 base positions) of T representative isolates and members of the Thermoproteales. base at position 1083 in strain IC-059 (C instead of T Numbers at nodes indicate bootstrap values of 1000 trails found in other crenarchaeotes). (values " 70% are shown). Accession numbers are given in parentheses. Bars indicate evolutionary distance. A phylogenetic tree constructed showed that the five isolates were incorporated in the family Thermopro- teaceae (tree not shown). Next, a 16S rRNA\DNA relatedness to each other, suggesting that they belong phylogenetic tree was reconstructed with the five to a single genus. Among the eight strains, strain IC- strains and known members of the family Thermopro- T 059 showed lower levels of relatedness to all of the teaceae, by comparing 1317 base positions (Fig. 2b). other strains (%54%), indicating that it represents a The five isolates formed a distinct and shorter lineage separate species. The remaining strains may be divided with 100% bootstrap confidence. The sequence simi- into three genetic groups when strains with more than larities among the isolates were 98 8%, while the T n 70% similarity are grouped (group 1, strains IC-017 , values between the isolates and known species of the IC-029, IC-064 and IC-065; group 2, strain IC-124; Thermoproteaceae ranged from 92n8to95n0%. On group 3, strains IC-135 and IC-141). The DNA–DNA the phylogenetic tree, the isolates were affiliated with relatedness between genetic groups ranged from 41 to the Thermoproteus\Pyrobaculum clade as a sister 68%. Groups 1 and 2 shared low DNA–DNA related- group, although the bootstrap value was low (51%). ness (41–55%), while strains in group 3 showed relatively high relatedness to some strains in groups 1 and 2 (for example, 61–68% to IC-029, 63–68% to IC- Proposal of a new genus and two novel species 124). The eight strains showed low similarity values (5%) to C. maquilingensis JCM 10307T. The 17 isolates are rigid rods, 0n4–0n6 µm in width, and often form terminal spheres (the ‘golf club’). They are 16S rDNA analysis anaerobic, weakly acidophilic, heterotrophic and hy- perthermophilic, growing optimally at 85–90 mC. They In order to examine the phylogenetic relationships gain energy by reducing sulfur and possess cyclic and among the isolates, the partial 16S rDNA sequences acyclic tetraether core lipids. In addition to these were determined (569–610 bp) and a phylogenetic tree phenotypic properties, 16S rDNA sequence analysis was constructed by comparing 561 base positions demonstrates that they belong to the family Thermo- (positions 64–666 in the E. coli numbering system). On proteaceae, which currently comprises four genera, http://ijs.sgmjournals.org 1101 T. Itoh, K. Suzuki and T. Nakase

Table 4. Differential characteristics of genera of the Thermoproteaceae

Characteristic Vulcanisaeta Thermoproteus* Thermoproteus Pyrobaculum Caldivirga Thermocladium neutrophilus

Motility kk kjkk Maximum growth 89–99 96–102 97 97–104 92 82 temperature (mC) Optimum pH for growth 4n0–4n55n0–5n66n86n0–7n04n04n0 Growth on starch jj\kkk† kj Stimulation of growth kk kkjj by a vitamin mixture DNA GjC content (mol%) 44–46 55–56 60 48–58 43 52

* Except for Thermoproteus neutrophilus. † P. oguniense utilizes starch weakly (Sako et al., 2001).

Thermoproteus (Bonch-Osmolovskaya et al., 1990; and further studies are required to evaluate whether or Zillig et al., 1981; Zillig, 1989), Pyrobaculum (Huber et not they should be separated into species or subspecies. al., 1987, 2000; Sako et al., 2001; Vo$ lkl et al., 1993), Thermocladium (Itoh et al., 1998a) and Caldivirga On the basis of the comparison of the phenotypic and (Itoh et al., 1999). The novel isolates, however, can be phylogenetic properties of the isolates described above, differentiated phenotypically from the four known we propose a new genus, Vulcanisaeta gen. nov. and genera, as shown in Table 4. Moreover, 16S rDNA- two novel species, Vulcanisaeta distributa sp. nov. and based phylogenetic analysis supports the distinction of Vulcanisaeta souniana sp. nov. these isolates from the known genera of the Thermo- proteaceae. Therefore, the novel isolates should be There are several volcanic zones in Japan, with nearly assigned to a new genus in the family Thermopro- 80 active volcanoes. Solfataric fields, which are nor- teaceae. mally sulfur-rich, acidic and heated areas, are formed by volcanic activities (Kristjansson & Stetter, 1993). Among the isolates, the strains from Sounzan (strains So far, we have successively isolated members of the IC-059T and IC-060) were clearly separated from the genera Thermoproteus (Itoh et al., 1998b) and Thermo- other strains by the DNA–DNA hybridization study cladium (Itoh et al., 1998a) from hot spring areas in and 16S rDNA sequence comparison. The distinction east Japan. Moreover, several species of the genus of IC-059T and IC-060 from the other isolates is Pyrobaculum have been newly isolated or detected further supported by the relatively lower growth from geothermal areas in Kyushu Island, Japan (Sako temperature range (up to 89 mC) and slightly higher et al., 2001; Takai & Horikoshi, 1999). Along with NaCl tolerance (up to 1n25%) of the two strains (data these genera in the family Thermoproteaceae, the new not shown for IC-060). For the remaining strains, the genus Vulcanisaeta may also be distributed widely in DNA–DNA hybridization data implies the presence hot springs in Japan. As exemplified by the samples of three genetic groups, one for the isolates from collected from Sounzan and Okushiobara, different Ohwakudani and Okushiobara (represented by strains genera of the family Thermoproteaceae may co-exist in IC-017T, IC-029, IC-064 and IC-065), one for strains one sampling site. Yet, each of the sampling sites from Fukenoyu, Gosyokake and Ohnuma (repre- yielded only one type of phylogenetically closely sented by strain IC-124) and another for strains from related strains of the genus Vulcanisaeta. Since all the Tamagawa (represented by strains IC-135 and IC- strains were isolated in the same manner, geographical 141). Such a relationship is partly illustrated in the separation may have affected the diversification of phylogenetic tree based on the partial 16S rDNA the genus. Interestingly, the V. souniana strains were sequences. Nevertheless, we have not found any isolated from piped hot spring water, while the V. phenotypic properties to distinguish these three groups distributa strains were isolated from solfataric fields. properly. Strain IC-124 has a slightly lower growth The former is a rather artificial habitat, and changes in temperature range than the others (maximum 92 mC); environmental factors (such as reduction of tempera- however, strains IC-136 and IC-140, which have ture or volatile chemical components) may have partial 16S rDNA sequences identical to that of strain promoted the diversification. In spite of the fact that IC-124, grow at 95 mC (data not shown). There are the genus Vulcanisaeta was the most common rod- several differences in the capacity to use carbon sources shaped crenarchaeote among our isolates from hot or electron acceptors, but these differences do not springs in Japan, it has not been isolated or detected agree well with the grouping by DNA–DNA related- from other countries. In contrast, the genera Thermo- ness. Thus, all of the strains other than IC-059T and proteus and Pyrobaculum are distributed worldwide, IC-060 should be included in a single species at present, including the Azores, Iceland, Indonesia, Italy, Japan,

1102 International Journal of Systematic and Evolutionary Microbiology 52 Vulcanisaeta gen. nov., with two species the Philippines, Russia and the USA. Our isolation Description of Vulcanisaeta souniana sp. nov. procedures may have a selective effect for Vulcanisaeta Vulcanisaeta souniana (so.un.i.a na. N.L. adj. souniana strains, although we cannot rule out the possibility h of Hojo Soun, a historical feudal lord after whom that the genus Vulcanisaeta has a restricted distribution Sounzan, the place of isolation, was named). that includes Japan. Grows anaerobically and tolerates low levels of oxygen (1%) when sodium thiosulfate is supplemented as an Description of Vulcanisaeta gen. nov. electron acceptor. Heterotrophic. Growth occurs from 65 to 89 mC and between pH 3n5 and 5n0 when buffered Vulcanisaeta (Vul.ca.ni.saehta. L. adj. vulcanicus vol- with 10 mM trisodium citrate. Under optimal growth canic; L. n. saeta stiff hair; N.L. fem. n. Vulcanisaeta conditions, doubling time is 4n7 h. Growth occurs at a rigid rod inhabiting volcanic hot springs). low salinity (%1n25% NaCl). Utilizes proteinaceous substances such as yeast extract, peptone, beef extract, Cells are rigid, straight to slightly curved rods that Casamino acids and gelatin, as well as maltose, starch occasionally bend, branch out or bear spherical bodies and malate as carbon sources. Requires sulfur or at the terminals (‘golf clubs’). Most cells are 0n4– thiosulfate as an electron acceptor. Archaeal cell 0n6i3–7 µm. Pili arise terminally or laterally. No extract or a vitamin mixture has little effect on growth. Produces sulfide during growth. DNA GjC content motility is shown. Prefers extremely high temperature T (85–90 mC) and weakly acidic conditions (pH 4n0–4n5) is 44n9–45n4 mol% (44n9 mol% for IC-059 ). T T for growth. Grows anaerobically. Resistant to chlor- The type strain is IC-059 (l JCM 11219 l DSM amphenicol, kanamycin, oleandomycin, streptomycin 14430T), which was isolated from a hot spring in and vancomycin. Sensitive to erythromycin, novobio- Sounzan, Kanagawa, Japan. Strain IC-060 is also cin and rifampicin. Possesses cyclic and acyclic tetrae- included in this species. ther core lipids. DNA GjC content ranges from 43n9 to 46n2 mol%. The 16S rDNA is typical of a crenar- chaeote in sequence signature analysis. Phylogene- REFERENCES tically, represents an independent lineage in the family Barns, S. M., Fundyga, R. E., Jeffries, M. W. & Pace, N. R. (1994). Thermoproteaceae. Inhabits volcanic acidic hot Remarkable archaeal diversity detected in a Yellowstone National Park springs. The type species is Vulcanisaeta distributa. hot spring environment. Proc Natl Acad Sci U S A 91, 1609–1613. Barns, S. M., Delwiche, C. F., Palmer, J. D. & Pace, N. R. (1996). 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