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1 Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
2 Graduate school of the Chinese Academy of Sciences, Beijing 100039, China
Correspondence
Feng-Yan Bai
baify{at}sun.im.ac.cn
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ABSTRACT |
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Published online ahead of print on 13 May 2005 as DOI 10.1099/ijs.0.63675-0.
The GenBank/EMBL/DDBJ accession numbers for the 18S rRNA, ITS and 26S rRNA gene sequences of Kazachstania aquatica sp. nov. and K. solicola sp. nov. are AY881650–AY881652.
A more detailed phylogenetic tree based on 18S rRNA gene sequence analysis is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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, has recently been shown to represent a separate species by Lu et al. (2004). The novel species represented by strains AS 2.0706T and CBS 6904T are described in the present study. Strain AS 2.0706T was isolated from the wastewater of a paper mill in north-east China. The type strain of Kazachstania aerobia (AS 2.2384T) was obtained from the CGMCC. Strain CBS 6904T and the type strains of Kazachstania unispora (CBS 398T) and Kazachstania servazzii (CBS 4311T) were obtained from the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands.
Most of the morphological, physiological and biochemical characteristics were examined according to standard methods (Yarrow, 1998). Assimilation of nitrogen compounds was investigated on solid media with starved inocula (Nakase & Suzuki, 1986). Extraction, purification and identification of ubiquinones were carried out according to Yamada & Kondo (1973).
Nuclear DNA was extracted by the method of Makimura et al. (1994). The DNA fragment covering the ITS region (including 5·8S rDNA) and the large-subunit rDNA D1/D2 domain was amplified and sequenced according to Lu et al., 2004. The small-subunit rRNA gene was amplified and sequenced according to Sugita & Nakase (1999). Molecular phylogenetic analysis was performed by the methods described by Bai et al. (2002). Reference sequences were retrieved from GenBank under the accession numbers indicated in the trees.
Intact yeast chromosomal DNA was prepared by the method of Bai et al. (2000). Pulsed-field gel electrophoresis (PFGE) was performed according to Lu et al. (2004).
Sequence analyses
The close phylogenetic relationship of strain CBS 6904T with K. aerobia, K. servazzii and K. unispora, based on D1/D2 and ITS sequence analysis, has been shown by Lu et al. (2004). The position of strain AS 2.0706T was not clearly resolved in the tree drawn from the sequences of D1/D2 (data not shown). In trees drawn from the combined ITS (including 5·8S rDNA) and D1/D2 regions (Fig. 1) and from the small-subunit rDNA (see Supplementary Fig. S1 in IJSEM Online), the strain was located in a basal position relative to a small clade containing strain CBS 6904T and three previously described Kazachstania species.
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).
Electrophoretic karyotyping
The electrophoretic karyotypes of strains AS 2.0706T and CBS 6904T bore a superficial resemblance to those of the phylogenetically closely related species K. aerobia, K. servazzii and K. unispora (Fig. 2). Approximately 9 (600 to 2200 kb) and 11 (585 to 2200 kb) bands were resolved for AS 2.0706T and CBS 6904T, respectively. As wider bands or bands with higher intensities may correspond to doublets or triplets, the actual number of chromosomes may be higher. The patterns were nonetheless sufficiently distinct to differentiate the species from one another. Although strain CBS 6904T was more closely related to K. aerobia than to K. servazzii and K. unispora based on the sequence data, its chromosomal banding profile had more pronounced differences from that of K. aerobia compared with K. servazzii and K. unispora (Fig. 2).
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).
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). Kurtzman & Robnett (2003) examined two strains of K. unispora and found them to have identical ITS sequences. The ITS sequences of two additional strains of K. unispora (GenBank accession nos AF321542 and AF455430) differ from that of the type strain by only one indel. Nevertheless, since the D1/D2 sequences of K. solicola sp. nov. and K. aerobia are identical and only one strain is available for the former at present, DNA–DNA hybridization would certainly be helpful to justify the establishment of this novel species.
The genus Kazachstania was redefined recently based on multigene sequencing. A relatively large number of species in an only moderately supported clade resolved from the combined sequences of six regions or genes were assigned into the genus (Kurtzman, 2003; Kurtzman & Robnett, 2003). This was apparently a provisional treatment. The phylogenies of these yeasts resolved from the sequences of the small-subunit rRNA gene and other single genes were not congruent with that resolved from the combined gene analysis (James et al., 1997; Kurtzman & Robnett, 2003; Mikata et al., 2001; 
pírek et al., 2003). The addition of novel species may help to reclassify the species in this genus into more homogeneous groups. The two novel species described in the present study and the three already described species, K. aerobia, K. servazzii and K. unispora, formed a strongly supported clade in the trees based on the small-subunit rRNA and combined ITS and D1/D2 sequences (see Supplementary Fig. S1 in IJSEM Online and Fig. 1
). Interestingly, the species in this group have similar karyotypes (Fig. 2), as do those of Saccharomyces sensu stricto (Fischer et al., 2000; Vaughan-Martini et al., 1993).
Latin diagnosis of Kazachstania aquatica Bai & Wu sp. nov.
In medio liquido YM post dies 3 ad 25 °C, cellulae globosae vel subglobosae, 3·7–6·2x5·0–7·5 µm, singulae, binae et adhaerentes. Per gemmationem multipolarem reproducentes. Post 1 mensem sedimentum formatur. In agaro YM post 1 mensem ad 25 °C, butyrosa, cremea, initida, verrucosa, glabra cum radiatis, margo undulato. In agaro farinae Zea mays post dies 7, pseudohyphae nullae. Asci inconjugatio fiunt. Ascosporae globosae, 1 in quoque asco.
Glucosum et galactosum fermentantur at non sucrosum, maltosum, lactosum nec raffinosum. Glucosum, galactosum, trehalosum (infirme, lente), ethanolum (infirme, lente) assimilantur at non L-sorbosum, sucrosum, maltosum, cellobiosum, lactosum, melibiosum, raffinosum, melezitosum, inulinum, amylum solubile, D-xylosum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, D-glucosaminum, methanolum, glycerolum, erythritolum, ribitolum, galactitolum, D-mannitolum, D-glucitolum, methyl 
-D-glucosidum, salicinum, acidum DL-lacticum, acidum succinicum, acidum citricum, inositolum nec hexadecanum. Ammonium sulfatum, ethylaminum, L-lysinum et cadaverinum assimilantur at non natrum nitrosum nec kalium nitricum. Ad crescentiam vitaminae externae necessariae sunt. Maxima temperatura crescentiae: 37 °C. Materia amyloidea iodophila non formantur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur. Systema coenzymatis Q-6 adest. Typus depositus in collectione China General Microbiological Culture Collection Center, Academia Sinica (AS 2.0706T).
Description of Kazachstania aquatica Bai & Wu sp. nov.
Kazachstania aquatica (L. fem. adj. aquatica aquatic, referring to the source of the type strain).
In YM broth (Yarrow, 1998), after 3 days at 25 °C, the cells are globose to subglobose, 3·7–6·2x5·0–7·5 µm and occur singly, in pairs or in groups (Fig. 3). Budding is multilateral. After 1 month at 25 °C, sediment is present. On YM agar (Yarrow, 1998), after 1 month at 25 °C, the streak culture is butyrous, cream-coloured, raised, glossy, verruciform and smooth with faint striations; the margin is undulating. In Dalmau plate culture on cornmeal agar, pseudohyphae are not formed. Sporulation was observed on acetate agar (Fowell, 1952) after 5 days at 25 °C; vegetative cells transform directly into persistent asci each containing one globose ascospore (Fig. 3).
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-D-glucoside, salicin, DL-lactic acid, succinic acid, citric acid, inositol and hexadecane are not assimilated. Ammonium sulfate, ethylamine hydrochloride, L-lysine and cadaverine dihydrochloride are assimilated; sodium nitrite and potassium nitrate are not assimilated. Growth in vitamin-free medium is negative. Maximum growth temperature is 37 °C. Starch-like compounds are not produced. Diazonium blue B reaction is negative. Urease activity is negative. Ubiquinone type is Q-6. The type strain, AS 2.0706T (=CBS 10102T), was isolated from wastewater collected in a paper mill in north-east China. This strain has been deposited in the CGMCC, Academia Sinica, Beijing, China.
Latin diagnosis of Kazachstania solicola Bai & Wu sp. nov.
In medio liquido YM post dies 3 ad 25 °C, cellulae ovoideae vel subglobosae, 2·5–5·0x3·7–7·5 µm, singulae, binae et adhaerentes. Per gemmationem multipolarem reproducentes. Post 1 mensem sedimentum formatur. In agaro YM post 1 mensem ad 25 °C, butyrosa, cremea, seminitida, glabra cum radiatis, margo glabro vel undulato. In agaro farinae Zea mays post dies 7, pseudohyphae nullae. Asci inconjugatio et conjugatio fiunt. Ascoaporae subglobosae, 1 in quoque asco.
Glucosum et galactosum fermentantur at non sucrosum, maltosum, lactosum nec raffinosum. Glucosum, galactosum, trehalosum (lente), D-ribosum (lente), D-glucosaminum (lente) et glycerolum (lente) assimilantur at non L-sorbosum, sucrosum, maltosum, cellobiosum, lactosum, melibiosum, raffinosum, melezitosum, inulinum, amylum solubile, D-xylosum, L-arabinosum, D-arabinosum, L-rhamnosum, methanolum, ethanolum, erythritolum, ribitolum, galactitolum, D-mannitolum, D-glucitolum, methyl -D-glucosidum, salicinum, acidum DL-lacticum, acidum succinicum, acidum citricum, inositolum nec hexadecanum. Ammonium sulfatum assimilantur at non natrum nitrosum, kalium nitricum, ethylaminum, L-lysinum nec cadaverinum. Ad crescentiam vitaminae externae necessariae sunt. Maxima temperatura crescentiae: 37 °C. Materia amyloidea iodophila non formantur. Diazonium caeruleum B non respondens. Ureum non hydrolysatur. Systema coenzymatis Q-6 adest. Typus depositus in collectione Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS 6904T).
Description of Kazachstania solicola Bai & Wu sp. nov.
Kazachstania solicola (N.L. n. solicola inhabitant of the soil, referring to the source of the type strain).
In YM broth (Yarrow, 1998), after 3 days at 25 °C, the cells are ovoid to subglobose, 2·5–5·0x3·7–7·5 µm and occur singly, in pairs or in groups (Fig. 4). Budding is multilateral. After 1 month at 25 °C, sediment is present. On YM agar (Yarrow, 1998), after 1 month at 25 °C, the streak culture is butyrous, cream-coloured, raised, semi-glossy, smooth with faint striations; the margin is entire to slightly undulating. In Dalmau plate culture on corn meal agar, pseudohyphae are not formed. Sporulation is observed on acetate agar (Fowell, 1952) after 7 days at 25 °C, vegetative cells transform directly into persistent asci each containing one subglobose ascospore (Fig. 4).
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-D-glucoside, salicin, DL-lactic acid, succinic acid, citric acid, inositol and hexadecane are not assimilated. Ammonium sulfate is assimilated; sodium nitrite, potassium nitrate, ethylamine hydrochloride, L-lysine and cadaverine dihydrochloride are not assimilated. Growth in vitamin-free medium is negative. Maximum growth temperature is 37 °C. Starch-like compounds are not produced. Diazonium blue B reaction is negative. Urease activity is negative. Ubiquinone type is Q-6. The type strain, CBS 6904T (=AS 2.2406T), was isolated from soil collected from the Elburz Mountains, Iran. This strain has been deposited in the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bai, F.-Y., Zhao, J.-H., Takashima, M., Jia, J.-H., Boekhout, T. & Nakase, T. (2002). Reclassification of the Sporobolomyces roseus and Sporidiobolus pararoseus complexes, with the description of Sporobolomyces phaffii sp. nov. Int J Syst Evol Microbiol 52, 2309–2314.[Abstract]
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James, S. A., Cai, J., Roberts, I. N. & Collins, M. D. (1997). A phylogenetic analysis of the genus Saccharomyces based on 18S rRNA gene sequences: description of Saccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. nov. Int J Syst Bacteriol 47, 453–460.[Abstract]
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Yamada, Y. & Kondo, K. (1973). Coenzyme Q system in the classification of the yeast genera Rhodotorula and Cryptococcus and the yeast like genera Sporobolomyces and Rhodosporidium. J Gen Appl Microbiol 19, 59–77.
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