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1 Deep-sea Microorganism Research Group, Japan Marine Science and Technology Center (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan
2 Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
3 Japan Collection of Microorganisms, RIKEN, Wako, Saitama 351-0198, Japan
Correspondence
Takahiko Nagahama
nagahama{at}jamstec.go.jp
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Published online ahead of print on 27 June 2003 as DOI 10.1099/ijs.0.02712-0.
The GenBank/EMBL/DDBJ accession number for the 18S rDNA, D1/D2 region of 26S rDNA and internal transcribed spacer sequences of SY-260T is AB100440.
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; Fell, 1976; Hagler & Ahearn, 1987). This anamorphic genus in the Hymenomycetes is characterized mainly by the assimilation of inositol and D-glucuronate and production of starch (Fell & Statzell-Tallman, 1998), but is known to be polyphyletic (Takashima & Nakase, 1999; Fell et al., 2000). We isolated Cryptococcus strains in a survey of yeasts from various deep-sea floors in the north-west Pacific Ocean (Nagahama et al., 2001a). A strain from sediment collected at a depth of 2406 m in Suruga Bay (34° 36' 55 N, 138° 34' 77 E; temperature, 3 °C) is described here as a novel yeast species of the genus Cryptococcus.
Sample collection and isolation
Samples were collected from Suruga Bay by using an unmanned submersible vessel without contamination by open water, as described previously (Takami et al., 1997; Nagahama et al., 2001a). Yeasts were isolated from fresh deep-sea sediments and cultured on YM agar, 1/5 YM agar, potato dextrose agar, cornmeal agar or marine agar (all from Difco), dissolved in artificial sea water supplemented with 0·01 % chloramphenicol. Agar plates were incubated at low temperature (5–10 °C) for the first 2 weeks and then at 20 °C for 1 month.
Physiological and biochemical characteristics
Strains were characterized morphologically and physiologically by using standard methods with some modifications (Yarrow, 1998). Assimilation of nitrogen compounds was examined on solid media by using a starved inoculum (Nakase & Suzuki, 1986). Vitamin requirements were investigated according to the method of Komagata & Nakase (1967). Ubiquinones were extracted by the method of Yamada & Kondo (1973) with slight modifications and determined by HPLC as described previously (Hamamoto & Nakase, 1995). DNA was extracted and purified following the procedure described by Hamamoto & Nakase (1995) and DNA base composition was determined by using the HPLC method of Tamaoka & Komagata (1984).
Phylogenetic analysis
DNA extraction for PCR was performed by using a QIAamp DNeasy Tissue kit (Qiagen) with some modifications (Nagahama et al., 2001b). Primers used for amplification and sequencing of the 18S rDNA, 5·8S rDNA and internal transcribed spacer (ITS) regions were those described by White et al. (1990); primers for the D1/D2 region of 26S rDNA were those described by Fell et al. (2000). PCR products were purified by using ExoSAP-IT (USB) and sequenced by using a model 4000L (LI-COR) or MegaBACE 1000 (Pharmacia) DNA sequencer.
All sequences were aligned by using CLUSTAL W 1.81 (Thompson et al., 1994) and adjusted manually. Positions where one or more species contained a length mutation and a region that was aligned ambiguously were not included in subsequent phylogenetic analysis. Phylogenetic trees were constructed by utilizing the maximum-likelihood (ML) method (Felsenstein, 1981) with the optimal Ts/Tv ratio of the HKY85 model (Hasegawa et al., 1985), estimated from the neighbour-joining tree (Saitou & Nei, 1987) in PAUP 4.0b8 (Swofford, 1998). This tree was derived by a heuristic search with random stepwise addition of 100 replicates. Robustness of branches in the tree was evaluated by bootstrap analysis (Felsenstein, 1985) with 100 resamplings.
Sequences of the 18S rDNA, 26S rDNA, 5·8S rDNA and ITS regions of SY-260T were deposited in GenBank/DDBJ under accession number AB100440.
Phylogenetic position of strain SY-260T isolated from deep-sea sediment
We sequenced a DNA fragment of strain SY-260T that included 18S rDNA, ITS1, 5·8S rDNA, ITS2 and the D1/D2 region of 26S rDNA. This sequence had some affinity to that of species in the Cryptococcus luteolus lineage (Takashima & Nakase, 1999) or Luteolus clade (Scorzetti et al., 2002).
The relationship between members of this group and strain SY-260T was estimated, based on 2077 bp in the 18S rDNA, 5·8S rDNA and ITS regions (Fig. 1). Overall, this branching order was statistically robust and consistent with those based on 18S rDNA in the phylogenetic tree drawn by Bai et al. (2001a, b). Species used in this study were divided into the subclades Luteolus, Mrakii and Dioszegia (Fig. 1), which were supported strongly by bootstrap values. Strain SY-260T was obviously placed into the subclade Luteolus. Strain SY-260T and two Bullera species, Bullera coprosmaensis and Bullera oryzae, formed a cluster that was separate from the other members of the subclade Luteolus. The subclade Mrakii, which consists of eight Bullera species, is relatively coherent with the subclade Luteolus. These two subclades appeared to be related to each other and were separate from the subclade Dioszegia.
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) had few branches that were supported strongly by bootstrap values and appeared to be relatively ambiguous for the 18S–ITS tree drawn above. Two species with identical sequences, Bullera derxii and Bullera sinensis var. sinensis, differed from B. sinensis var. lactis at only one base, although these three species were well-differentiated based on 18S rDNA and ITS sequences. The relationship between strain SY-260T, B. coprosmaensis and B. oryzae was uncertain in this tree, in contrast to that in the 18S–ITS tree. This disagreement seemed to be caused by the remarkably long lineage of Cryptococcus sp. CBS 8369, as a tree that excluded CBS 8369 supported the cluster of strain SY-260T with the two Bullera species. In the nine strains of undescribed Cryptococcus species, CBS 8356 and CBS 8367 were identical to Bullera kunmingensis. The seven remaining strains were divided into three groups and each appeared to qualify as a novel species.
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; Fell et al., 2000; Bai et al., 2001a, 2001b; Hamamoto et al., 2002; Nagahama et al., 2003).
Comparison of physiological and biochemical characteristics between strain SY-260T and related species
Phylogenetically, the species most closely related to strain SY-260T was considered to be B. oryzae (Fig. 1
) but their DNA G+C contents had differences of >10 %, whereas B. coprosmaensis had a DNA G+C content similar to that of strain SY-260T. The evolutionary distances among these three species appear to be too great to estimate relationships based on G+C contents. Strain SY-260T can be discriminated physiologically from B. coprosmaensis and B. oryzae, based on the assimilation of L-sorbose and soluble starch. As ballistoconidia and sexual reproduction were not observed in strain SY-260T, we describe this strain as a novel species of the genus Cryptococcus.
Latin diagnosis of Cryptococcus surugaensis sp. nov. Nagahama, Hamamoto et Nakase
In medio liquido YM post 3 dies ad 25 °C, cellulae ovoideae vel ellipsoidae (2–4x2–7 µm), singulae aut binae. Post unum mensem pellicula fragilis et sedimentum formantur. Cultura in agaro YM ad 25 °C, subflava, nitida, mollis et margine glabra. Hyphae et pseudohyphae non formantur. Fermentatio nulla. Glucosum, galactosum, L-sorbosum, saccharosum, maltosum, cellobiosum, trehalosum, melibiosum, raffinosum, melezitosum, D-xylosum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, glycerolum, erythritolum, ribitolum (exiguum), galactitolum, D-mannitolum, D-glucitolum (exiguum), methyl-
-D-glucosidum, salicinum (exiguum), glucono-
-lactonum, acidum 2-ketogluconicum, acidum 5-ketogluconicum, acidum DL-lacticum, acidum succinicum, acidum citricum et inositolum assimilantur, at non lactosum, inulinum, amylum solubile, ethanolum, acidum D-glucuronicum nec acidum D-galacturonicum. Ethylaminum, lysinum et cadaverinum assimilantur at non kalium nitricum nec natrium nitrosum. Maxima temperatura crescentiae: 31–34 °C. Ad crescentiam vitaminum non necessarium est. Materia amyloidea iodophila formantur. G+C acidi deoxyribonucleati 48·3 mol% (per HPLC). Ubiquinonum majus Q-10.
Typus stirps SY-260T ex sedimentum, Suruga Bay, Japan, isolata est. In collectionibus culturarum quas Japan Collection of Microorganisms, Wako, Saitama, Japan, sustentant, no. JCM 11903T (=CBS 9426T) deposita est.
Description of Cryptococcus surugaensis sp. nov. Nagahama, Hamamoto & Nakase
Cryptococcus surugaensis (su.ru.ga.en'sis. N.L. masc. adj. surugaensis referring to the geographical origin of the species).
In YM broth (Difco) after 3 days culture at 25 °C, cells are ovoidal to ellipsoidal (2–4x2–7 µm) and occur singly or in parent-bud pairs. A sediment and fragile pellicle are formed after 1 month. After 1 month on YM agar at 25 °C, streak culture is light yellow, glistening, soft and has an entire margin. In Dalmau plate cultures on cornmeal agar (Difco), no branching hyphae or pseudohyphae are formed. Fermentation ability is negative. The following carbon compounds are assimilated: D-glucose, galactose, L-sorbose, sucrose, maltose, cellobiose, trehalose, melibiose, raffinose, melezitose, D-xylose, L-arabinose, D-arabinose, D-ribose, L-rhamnose, glycerol, erythritol, ribitol (weak), galactitol, D-mannitol, D-glucitol (weak), methyl -D-glucoside, salicin (weak), glucono--lactone, 2-ketogluconic acid, 5-ketogluconic acid, DL-lactic acid, succinic acid, citric acid and inositol; no growth occurs on lactose, inulin, soluble starch, ethanol, D-glucuronic acid or D-galacturonic acid. The nitrogen compounds ethylamine, lysine and cadaverine are assimilated. No growth occurs on potassium nitrate or sodium nitrite. Maximum temperature for growth is 31–34 °C. Vitamins are not required for growth. No growth occurs on 50 % glucose/yeast extract agar. Growth occurs in the presence of 100 p.p.m. cycloheximide. Growth in the presence of 10 % sodium chloride is negative. Starch-like substances are produced. Diazonium blue B reaction is positive. Urease activity is positive. G+C content of nuclear DNA is 48·3 mol% (by HPLC). Major ubiquinone is Q-10.
The type strain of C. surugaensis, SY-260T, was isolated from sediments collected from the deep-sea floor of Suruga Bay, Japan. This strain has been deposited in the Japan Collection of Microorganisms, Saitama, Japan, as JCM 11903T (=CBS 9426T).
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ACKNOWLEDGEMENTS |
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