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1 Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Council of Scientific and Industrial Research (CSIR), Sector 39A, Chandigarh 160036, India
2 TERI School of Advanced Studies, The Energy and Resources Institute (TERI), Darbari Seth Block, India Habitat Centre, Lodhi Road, New Delhi 110003, India
3 TERI, Darbari Seth Block, India Habitat Centre, Lodhi Road, New Delhi 110003, India
Correspondence
G. S. Prasad
prasad{at}imtech.res.in
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ABSTRACT |
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Published online ahead of print on 29 October 2004 as DOI 10.1099/ijs.0.63313-0.
The GenBank/EMBL/DDBJ accession numbers for the 26S rRNA gene D1/D2 domain, ITS and 18S rRNA gene sequences of strains MTCC 4371T and MTCC 4372 are respectively AJ549212 and AJ549213, AJ697745 and AJ697746, and AJ697749 and AJ697750, and the accession numbers for the ITS sequences of C. blankii MTCC 1442T and MTCC 624 are AJ697747 and AJ697748.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Iizuka & Komagata, 1965). Recently, Candida bituminiphila from tar was reported (Robert et al., 2001). On the basis of conventional morphological and physiological tests, the strains were shown to be related to Candida blankii and Zygoascus hellenicus, though they differed from them in several physiological tests. No sexual reproduction was observed on corn meal agar (HiMedia), malt agar, Gorodkowa agar (Yarrow, 1998), YM agar (HiMedia) or V8 agar (Difco). Sequence analysis of rRNA genes also showed that the strains are closely related to C. blankii. All these data supported the assignment of strain TERI-6T to a novel species, for which we propose the name Candida digboiensis.
The yeast strains examined in this study are listed in Table 1 and are available from the MTCC, the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, and the Japan Collection of Microorganisms (JCM), Wako-shi, Japan. All the strains were grown on YM agar at 25 °C. Phenotypic characteristics were examined using standard methods for yeast taxonomy (Yarrow, 1998).
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Each PCR was performed in a final reaction mixture (50 µl) containing 50 ng genomic DNA, 25 pmol each primer, 200 mM each of dATP, dTTP, dGTP and dCTP (Promega), 2·5 mM MgCl2, 2·0 U Taq polymerase (Promega) and 5 µl 10x reaction buffer (Promega). Primers ITS1 and ITS4 were used to amplify the internal transcribed spacer (ITS) region, while NS1 and NS8 were used for amplifying the small-subunit (SSU) rRNA gene (White et al., 1990). The D1/D2 domain of the large-subunit rRNA gene was amplified with primers NL1 and NL4 (Kurtzman & Robnett, 1998). The primers were obtained from Integrated DNA Technologies. Amplification reactions were performed in a PTC 150 Mini Cycler (MJ Research) with the following cycling parameters: initial denaturation for 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C and 1·0 min at 72 °C (for ITS and D1/D2 regions) or 2·0 min (for SSU rRNA gene), with a final extension for 10 min at 72 °C, and cooled to 4 °C. The amplified products were separated on 1·2 % agarose (Sisco Research Laboratories) gel by electrophoresis and visualized by staining with ethidium bromide (0·5 µg ml–1).
The amplicons were purified using the Qiagen gel extraction kit. Direct sequencing of gel-purified PCR products was performed with the ABI BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems). Both strands of the PCR product were sequenced. The SSU rRNA gene was sequenced with primers NS1–NS8, the ITS region with primers ITS1 and ITS4 (White et al., 1990) and the D1/D2 domain with primers NL1 and NL4 (Kurtzman & Robnett, 1998). Sequencing reactions were purified by ethanol and sodium acetate precipitation. The pellet was washed twice with 70 % ethanol, which considerably improved the removal of dye terminators from the reaction. Processing of the samples for loading onto an ABI 310 model sequencer was performed according to the instructions of the manufacturer (Applied Biosystems).
A sequence-similarity search was done using GenBank BLASTN (Altschul et al., 1997). Sequences of closely related taxa were retrieved and aligned using the CLUSTAL X program (Thompson et al., 1997). For the neighbour-joining analysis (Saitou & Nei, 1987), distances between the sequences were calculated using Kimura's two-parameter model (Kimura, 1980). Bootstrap analysis was performed to assess the confidence limits of the branching (Felsenstein, 1985).
Latin diagnosis of Candida digboiensis G. S. Prasad, Mayilraj, Sood et Lal sp. nov.
Coloniae in agaro malti humiles convexae, integrae vel fimbriatae, albae vel cremeae, butyrosae. In medio liquido cum dextroso et peptono et extracto levedinis et extracto malti post 3 dies ad 25 °C cellulae sunt ellipsoideae ad cylindratae pro maxima partes irregulares (2·0–3·5x3·0–9·0 µm), singulae vel binae. Reproductio vegetativa per holoblastice gemmationem. In lamina Dalmau post 7 dies pseudohyphae formantur dense ramosae. Hyphae verae nonnumquam praesentes. Fermentatio nulla. Sucrosum, galactosum, L-sorbosum, D-ribosum, D-xylosum, L-arabinsum, D-arabinosum, L-rhamnosum, maltosum, trehalosum, methyl 
-D-glucosidum, cellobiosum, salicinum, melibiosum, lactosum, raffinosum (exigue), melezitosum, inulinum, erythritolum, ribitolum, xylitolum (lente), D-arabinitolum, L-arabinitolum, glucitolum, mannitolum, galactitol, myo-inositolum, glucono-
-lactonum (lente), acidum gluconicum (exigue), ethanolum, arbutinum, amylum solubile et glycerolum (lente) assimilantur, neque glucosaminum, acidum glucuronicum, acidum succinicum, acidum citricum, aut methanolum. Ethylaminum, lysinum et cadaverinum velut substrata nigrogeni utuntur, neque sodii nitratum aut nitritum. Vitaminis vel acidis aminosis absentibus haud crescit. 42 °C crescit. In agaro calcii carbonato addito acidum non formatur. In liquido 50 % glucosiii addito non crescit. 0·01 % cycloheximidum non crescit. Amylum non formatur, urea non finditur, diazolium coeruleo B probatio negativa. Holotypus TERI-6T (=MTCC 4371T) lyophilus, isolatus e terra inquinata ‘acid tar sludge’, circa Digboi oil refinery, Digboi, Assam, India.
Description of Candida digboiensis G. S. Prasad, Mayilraj, Sood & Lal sp. nov.
Candida digboiensis [dig.boi.en'sis. N.L. nom. fem. adj. digboiensis referring to Digboi (27·33° N 95·40° E), a town in Assam State, north-eastern India, where the type strain was isolated].
Colonies on malt agar are low-convex, entire or fringed, white to cream and butyrous. After 3 days in YM broth at 25 °C, cells are ellipsoidal to short cylindrical (2·0–3·5x3·0–9·0 µm) and occur singly, in pairs or in short chains (Fig. 1). Some irregular shapes and a few elongated (up to 15 µm) cells are also present. Sympodial holoblastic conidiogenesis results in ovoid to obclavate conidia (2–4 µm) that arise from pronounced protuberances. After 1 week in Dalmau plate culture, pseudohyphae consisting of branched chains of elongated cells are visible. True hyphae may be present. Fermentation absent. Sucrose, galactose, sorbose, ribose, xylose, D-arabinose, L-arabinose, L-rhamnose, maltose, trehalose, methyl -D-glucoside, cellobiose, salicin, melibiose, lactose, raffinose, melezitose, inulin, erythritol, ribitol, xylitol, D-arabitol, L-arabitol, D-glucitol, D-mannitol, galactitol (weak), myo-inositol, D-glucono-1,5-lactone (weak), D-glucuronate, ethanol, arbutin, starch (slow) and glycerol (weak) are assimilated. D-Gluconate, D-glucosamine, succinate, citrate and methanol are not assimilated. Ethylamine, lysine and cadaverine are utilized as sole nitrogen sources; sodium nitrate and nitrite are not. Does not grow in the absence of vitamins. Grows in the absence of amino acids. Grows at 42 °C. Acid is not produced on chalk agar. Does not grow in the presence of 50 % (w/w) glucose, 0·01 % (w/v) cycloheximide or 1 % (v/v) acetic acid. Does not produce starch-like compounds. Urease-negative. Diazonium blue B reaction is negative.
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Physiological and phylogenetic relationships
C. digboiensis is physiologically similar to C. blankii and less so to Z. hellenicus. However, compared to these two species, it shows differences in eight physiological tests. It differs from C. blankii in its ability to assimilate melibiose and inulin, its inability to assimilate 2-keto-D-gluconate and D-gluconate and its inability to grow at 45 °C (two strains each of C. digboiensis and C. blankii were compared) and in the presence of 0·01 % and 0·1 % (w/v) cycloheximide. C. digboiensis differs from Candida auringiensis and Candida salmanticensis in its ability utilize melibiose and inulin and its inability to grow in the presence of 0·01 % and 0·1 % (w/v) cycloheximide. It differs from Z. hellenicus in its ability to assimilate melibiose, inulin, erythritol, arabitol and its inability to assimilate D-glucosamine. Z. hellenicus cannot grow at 42 °C, whereas strains TERI-6T and TERI-7 show growth at this temperature. Selected phenotypic differences between C. digboiensis and related species are shown in Table 2.
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). These studies have shown that strains belonging to separate species generally exhibit greater than 1 % sequence divergence. Strains TERI-6T and TERI-7 show 2·8 % divergence (16 base substitutions out of 556 nt) from C. blankii, indicating that C. digboiensis could be a novel species. However, in a recent study with the yeast species Clavispora lusitaniae, it was found that the sequence variation in the D1/D2 region among mating strains of that species could exceed 6·0 % (Lachance et al., 2003). At present, it is not clear whether this constitutes a rare example of polymorphism in the D1/D2 region or whether some other species are also polymorphic. For further confirmation of the novelty of the strains under study, we also sequenced the SSU rRNA gene and the ITS region (comprising ITS1, 5·8S rRNA gene and ITS2 regions) of strains TERI-6T and TERI-7.
A BLAST search (Altschul et al., 1997) using the C. digboiensis SSU rRNA gene sequence showed that C. blankii is the closest relative, although most other sequences retrieved in this search were those of filamentous fungal species. A discontiguous Mega BLAST (http://www.ncbi.nlm.nih.gov/blast/) search retrieved the yeast sequences. In the SSU rRNA gene sequence, C. digboiensis differs from C. blankii by 11 base substitutions and two deletions. C. salmanticensis and C. auringiensis are also related but show sequence divergence of 2·3 and 2·7 %, respectively, from C. digboiensis. Most species of the Stephanoascus clade (Stephanoascus smithiae, Stephanoascus farinosus, Arxula adeninovorans, Z. hellenicus, C. bituminiphila etc.) show more than 5 % divergence, in the SSU rRNA gene sequence, from C. digboiensis, which suggests that they may be distantly related to C. digboiensis. Further confirmation of the novelty of C. digboiensis came from sequencing of the ITS region. As the ITS region of C. blankii was not available in the nucleic acid databases, we sequenced the ITS regions of two strains of C. blankii (MTCC 1442T and MTCC 624). In this region, C. digboiensis differs from C. blankii by 58 base substitutions; in addition, there are 33 base deletions in C. blankii and nine deletions in C. digboiensis. This strongly suggests the separation of C. digboiensis from C. blankii. A BLAST search with the ITS sequence of C. digboiensis retrieved the partial ITS sequence of C. blankii (deposited as part of this study) and only the 5·8S rRNA gene sequences of other species, the first being C. bituminiphila, followed by Z. hellenicus and different varieties of Zygoascus species. This again shows that the strains isolated from acidic tar sludge-contaminated soil represent a novel species.
In the phylogenetic tree constructed using the D1/D2 variable domain of the large-subunit rRNA gene (Fig. 2), C. digboiensis along with C. blankii were placed within a broad cluster comprising Stephanoascus/Arxula/Blastobotrys/Zygoascus (the Stephanoascus clade) and some species of Candida supported by high bootstrap values (90 %). However, C. digboiensis and C. blankii appear to be more closely related to C. auringiensis, C. salmanticensis and Candida tartarivorans than to the other species of the Stephanoascus clade. Kurtzman & Robnett (1998) showed that C. blankii is phylogenetically distantly related to the Stephanoascus clade. Similarly, C. auringiensis, C. salmanticensis and C. tartarivorans appear to be only distantly related to the Stephanoascus clade (Fonseca et al., 2000). Middelhoven & Kurtzman (2003) examined the ability of several yeast species to assimilate glycine, uric acid, n-hexadecane, putrescine and branched-chain aliphatic compounds such as isobutanol, leucine and isoleucine. Among the Saccharomycetales, most of the species belonging to the Stephanoascus clade utilized most, or all, of these compounds. C. blankii, which was considered as distantly related to the above clade, utilized n-hexadecane and five other compounds, indicating that it is physiologically more similar to members of the Stephanoascus clade. It would be interesting to examine the ability of C. digboiensis strains to utilize the above compounds.
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commented that ‘unfortunately too many times in the past, workers engaged in yeast isolation have not gone beyond the mere nomenclatural description of new species, failing to typify also their community, habitat, and possible interactions’. As C. digboiensis strains were isolated from an acid tar sludge-contaminated soil, we have investigated which other species have been isolated from hydrocarbon habitats. Although very few reports are available on the isolation of yeast species from oil-contaminated soil, yeast strains have been isolated from oilfields and oil brines in Japan (Iizuka & Goto, 1965; Iizuka & Komagata, 1965), and some yeast species have been reported to utilize petroleum hydrocarbons (Ismailov, 1985a, b; Palittapongarnpim et al., 1998; Radwan et al., 2001). We have examined the habitats of the closest relatives of C. digboiensis. Candida hydrocarbofumarica MTCC 624 (=CBS 6734), a synonym of C. blankii, was isolated from soil; it has been reduced to synonymy with C. blankii because of the high degree (>90 %) of nuclear DNA reassociation between the type strains of two species (Meyer et al., 1998). We have determined the sequence of the D1/D2 variable domain of the type strain of C. hydrocarbofumarica and found it to be identical to the sequence of the type strain of C. blankii, confirming their conspecificity. The SSU rRNA gene and ITS sequences of C. blankii and C. hydrocarbofumarica were also identical. C. blankii strains utilize hydrocarbons (Furukawa et al., 1970, 1978; Yamada et al., 1970). Strains of C. digboiensis could utilize alkane as well as aromatic fractions of acidic tar sludge (data not shown). On the basis of D1/D2 sequence analysis, C. auringiensis, C. salmanticensis and C. tartarivorans are the other relatives of C. digboiensis. Both C. auringiensis and C. salmanticensis were isolated from ‘alpechin’, the waste produced by olive oil extraction (Meyer et al., 1998). C. bituminiphila and Z. hellenicus are the other relatives of this cluster. Interestingly, C. bituminiphila was isolated from tar, which is a by-product of the crude oil industry (Robert et al., 2001). Three strains of Z. hellenicus (CBS 4028, CBS 4075 and CBS 5839) have the ability to split fat (CBS web site: http://www.cbs.knaw.nl/databases/index.htm). Yeasts are relatively rarely isolated from crude oil or hydrocarbon sources. However, phylogenetic analysis of C. digboiensis and related species suggests that several closely related yeast species may exist in nature. Physiological and biochemical aspects of acidic tar sludge degradation by C. digboiensis strains are under investigation and will be reported separately.
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ACKNOWLEDGEMENTS |
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