Kluyveromyces Siamensis Sp. Nov., an Ascomycetous

RESEARCH ARTICLE Kluyveromyces siamensis sp. nov., an ascomycetous yeast isolated from water in a mangrove forest in Ranong Province,Thailand Somjit Am-In, Wichien Yongmanitchai & Savitree Limtong

Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand

Correspondence: Savitree Limtong, Abstract Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok Seven strains of a novel Kluyveromyces species were isolated from seven water 10900, Thailand. Tel.: 166 2 562 5444, samples collected from a mangrove forest. Analysis of the D1/D2 domain of the ext. 4017; fax: 166 2 579 2081; e-mail: large subunit (LSU) rRNA gene sequences revealed that the sequences of ﬁve [email protected] strains (RS8T, RS20, RS54, RV42 and RV89) were identical and differed from the other two strains (RS65 and RV153) by only one nucleotide substitution in Received 29 January 2008; revised 28 March 544 nucleotides (nt). The closest species in terms of pairwise sequences similarity 2008; accepted 26 April 2008. was Kluyveromyces aestuarii, but the level of nucleotide substitution (six to seven First published online 6 June 2008. nucleotide substitutions in 544 nt) was sufﬁcient to justify the description of a separate species. The phylogenetic tree based on the sequence of the D1/D2 DOI:10.1111/j.1567-1364.2008.00396.x domain of the LSU rRNA gene rather suggested that the new species is a sister

Editor: Cletus Kurtzman species of K. aestuarii and forms a clade with the other six recognized species of Kluyveromyces. Sequence analysis of the internal transcribed spacer (ITS1– Keywords 5.8S rRNA gene–ITS2) region supported their distinct status as a species. Kluyveromyces siamensis sp. nov.; novel The phenotypic characteristics of the seven strains were typical of the genus species; ascomycetous yeast; water; mangrove Kluyveromyces. On this basis, the seven strains were assigned to a single novel forest and Thailand. species of the genus Kluyveromyces,forwhichthenameKluyveromyces siamensis sp. nov. is proposed. The type strain is RS8T (BCC 25962T = NBRC 103859T = CBS 10860T).

Botanical Congress and K. maxianus was chosen as the new Introduction type species (Lachance, 2007). The ascomycetous yeast genus Kluyveromyces was ﬁrst In mangrove ecosystems, yeasts play an important role in proposed by van der Walt (1956) to accommodate Kluyver- the detrital food web in that they may be a food source for omyces polysporus. Van der Walt (1965) transferred a num- some marine invertebrates and zooplanktons (Nagahama, ber of Saccharomyces species to the genus Kluyveromyces.In 2006). Yeasts are abundant in the water adjacent to and the description of the genus Kluyveromyces in The Yeasts, A within mangrove swamps (Fell et al., 2004). Recently, Taxonomic Study, 4th edition, Kluyveromyces consisted of 15 Lachancea meyersii was described for 18 strains isolated species in which K. polysporus, the type species, was included from waters in mangrove habitats in the northern Bahamas (Lachance, 1998). Nagahama et al. (1999) described a novel (Fell et al., 2004). In Thailand, we reported that yeasts could species of the genus Kluyveromyces as Kluyveromyces non- be isolated from all 32 water samples collected from the fermentans. On the basis of multigene analyses of Kurtzman surface and below the surface in two mangrove forests in (2003), only six species, Kluyveromyces aestuarii, Kluyvero- Khao Lumpee-Haad Thaimueang National Park (81N, 981E) myces dobzhanskii, Kluyveromyces lactis, Kluyveromyces max- and Mu Ko Ra-Ko Prathong National Park (91N, 981E), ianus, Kluyveromyces wickerhamii and K. nonfermentans, Phang-Nga Province, and the two novel species, Candida continue to be the recognized species of the genus Kluyver- thaimueangensis and Candida phangngensis, were described omyces. Although the original type species K. polysporus was (Limtong et al., 2007b, 2008). transferred to the genus Vanderwaltozyma, Kurtzman et al. During the course of investigation of yeasts in waters in a (2001) proposed to conserve the genus Kluyveromyces. The mangrove forest in Laem Son National Park (91N, 981E), proposal has been ofﬁcially accepted by the International Ranong Province, Thailand, seven strains (RS8T, RS20,

RS54, RS65, RV42, RV89 and RV153) were found to Examination of taxonomic characteristics represent a novel species of the genus Kluyveromyces; there- The strains were characterized morphologically, biochemi- fore, in this study we describe and propose Kluyveromyces cally and physiologically according to the standard methods siamensis sp. nov. described by Yarrow (1998). Assimilation of nitrogen compounds was investigated on solid media with starved inocula following the method of Nakase & Suzuki (1986). Vitamin Materials and methods requirement was determined according to the method of Komagata & Nakase (1967). Growth at various temperatures Yeast strains was determined by cultivation in YM broth. Seven yeast strains, RS8T, RS20, RS54, RS65, RV42, RV89 and RV153, were isolated from seven water samples col- Ubiquinone system lected from a mangrove forest in Laem Son National Park Ubiquinones were extracted from intact cells cultivated in (91N, 981E), Ranong Province, Thailand, by membrane yeast extract peptone dextrose (YPD) broth (1% yeast filtration following the method of Limtong et al. (2007b). extract, 2% peptone and 2% dextrose) on a rotary shaker at Purified yeast strains were suspended in yeast extract malt 28 1C for 24–48 h and purified according to the methods extract (YM) broth (0.3% yeast extract, 0.3% malt extract, described by Yamada & Kondo (1973) and Kuraishi et al. 0.5% peptone and 1% glucose) supplemented with 10% (1985). Isoprenologues were identified by HPLC as glycerol and maintained at 80 1C. described previously (Limtong et al., 2007a).

DNA sequencing and phylogenetic analysis Results and discussion The sequences of the D1/D2 domain of the large subunit Phylogenetic analysis (LSU) rRNA gene and the internal transcribed spacer (ITS) (ITS1–5.8S rRNA gene–ITS2) region were determined from The sequences of the D1/D2 domain of the LSU rRNA gene PCR products from genomic DNA extracted from yeast cells of the five strains (RS8T, RS20, RS54, RV42 and RV89) were using a slightly modified version of the method described by identical and differed from the other two strains (RS65 and Lachance et al. (1999). The D1/D2 domain of the LSU rRNA RV153) by only one nucleotide substitution in 544 nucleo- gene was amplified by a PCR with the forward primer NL1 tides (nt). The closest species in terms of pairwise sequence and the reverse primer NL4 (O’Donnell, 1993); amplifica- similarity was K. aestuarii but with 1.1–1.3% nucleotide tion of the ITS region including the 5.8S rRNA gene was substitutions (six to seven nucleotide substitutions in performed with the forward primer pITS-F and the reverse 544 nt). According to Kurtzman & Robnett (1998), yeast primer pITS-R (Sugita et al., 1999). The PCR product was strains showing nucleotide substitutions 41% in the D1/D2 checked by agarose gel electrophoresis, purified using the domain of the LSU rRNA gene are usually different species. QIAquick purification kit (Qiagen) and cycle-sequenced The phylogenetic tree based on the sequence of the D1/D2 using an ABI BigDye terminator cycle sequencing kit, domain of the LSU rRNA gene rather suggested that the new version 3.1 (Applied Biosystems), with the external primers, species is a sister species of K. aestuarii and forms a clade NL1 and NL4, for the D1/D2 domain (Kurtzman & Robnett, with the other six recognized species of the genus Kluyver- 1998) and the external primers, pITS-F and pITS-R, for the omyces, K. aestuarii, K. dobzhanskii, K. lactis, K. maxianus, ITS region (Sugita et al., 1999). The sequences were deter- K. wickerhamii and K. nonfermentans that constitute a mined using an ABI PRISM 3100 automated DNA sequen- monophyletic clade (Fig. 1). Therefore, the seven novel cer (Applied Biosystems) according to the manufacturer’s strains are considered to represent a single novel phylogen- instructions. The sequences were compared pairwise using etically distinct species. As the difference in the D1/D2 BLASTN homology search program (Altschul et al., 1997) domain of the LSU rRNA gene was relatively small, there- and were aligned with the sequences of related species fore, the ITS region was determined to confirm the distinct retrieved from GenBank using the multiple alignment status of the novel species. The sequences of the ITS region T program CLUSTAL_X version 1.81 (Thompson et al., 1997). of the five strains (RS8 , RS54, RV42, RV89 and RV153) A phylogenetic tree was constructed from the evolutionary were identical and differed from the other two strains (RS20 distance data with Kimura’s two-parameter correction and RS65) by only one nucleotide substitution in 636 (Kimura, 1980), using the neighbour-joining method nucleotides. The closest species to the seven strains in terms (Saitou & Nei, 1987). Confidence levels of the clades of pairwise ITS region sequence similarity was K. aestuarii were estimated from bootstrap analysis (1000 replicates) but with 1.0–1.1% nucleotide substitutions (six to seven (Felsenstein, 1985). nucleotide substitutions in 636 nt). Sugita et al. (1999)

c 2008 Federation of European Microbiological Societies FEMS Yeast Res 8 (2008) 823–828 Published by Blackwell Publishing Ltd. All rights reserved Kluyveromyces siamensis sp. nov. isolated in Thailand 825

Kluyveromyces siamensis RV42 (AB330825) Kluyveromyces siamensis RS54 (AB330829) Kluyveromyces siamensis RS65 (AB330830) Kluyveromyces siamensis RS8T (AB330824) 96 Kluyveromyces siamensis RV89 (AB330826) Fig. 1. Phylogenetic tree based on the Kluyveromyces siamensis RS20 (AB330828) sequences of the D1/D2 domain of the LSU rRNA 90 Kluyveromyces siamensis RV153 (AB330827) gene, showing positions of the seven strains of 57 T the novel species, Kluyveromyces siamensis sp. Kluyveromyces aestuarii CBS 4438 (U69579) T nov., with respect to closely related species. The Kluyveromyces nonfermentans CBS 8778 (AF398490) T phylogenetic tree was constructed from 95 Kluyveromyces marxianus CBS 712 (U94924) 89 T evolutionary distance data corrected by Kluyveromyces lactis CBS 683 (U94922) 91 two-parameter transformation of Kimura Kluyveromyces wickerhamii CBS 2745T (U69577) (1980), using the neighbour-joining method. Kluyveromyces dobzhanskii CBS 2104T (U69575) Numbers indicate percentages of bootstrap Saccharomyces cerevisiae CBS 1171T (U44806) 0.01Knuc sampling, derived from 1000 samples. indicated that conspeciﬁc yeast strains usually have a Table 1. Characteristics that distinguish Kluyveromyces siamensis sp. nucleotide difference of less than 1% in the ITS (ITS1–5.8S nov. from Kluyveromyces aestuarii rRNA gene–ITS2) region. These results lend further support Characteristic K. siamensis K. aestuariiÃ to the conclusion that the seven strains represent a single Fermentation novel species of the genus Kluyveromyces closely related to Galactose v K. aestuarii. Rafﬁnose w 1 Assimilation Phenotypic characteristics D-Xylose 1 Ribitol 1 Cells of K. siamensis were spherical to ellipsoidal, prolifer- Growth ated by multilateral budding, and formed one to four At 37 1Cw spherical ascospores in a conjugated and evanescent ascus At 42 1Cw With 50% glucose 1 that may be produced parthenogenetically or by conjugation With 60% glucose 1 between a cell and its bud or between independent cells. Vitamin-free medium 1 Pseudohyphae were formed but true hyphae were not 1, positive; , negative; l, latent; w, weak; v, variable. formed. Kluyveromyces siamensis fermented glucose, did not Ã assimilate nitrate, gave negative results for the diazonium Data from Lachance (1998). blue B and urease reactions and had Q-6 as the major sum nec melibiosum. D-Glucosum, D-galactosum, ubiquinone as do other members of the genus Kluyveromyces. L-sorbosum, sucrosum, cellobiosum, lactosum, raffinosum, The novel species, K. siamensis, differed from K. aestuarii, its closest phylogenetic relative by some phenotypic char- D-xylosum, ethanolum, glycerolum, ribitolum (lente), D D D d acteristics as shown in Table 1. -mannitolum, -glucitolum, salicinum, -glucono- -lacto- On the basis of the data reported above, it is therefore num, acidum DL-lacticum, acidum succinicum assimilantur concluded that the seven strains represent a single novel at non maltosum, trehalosum, melibiosum, melizitosum, species of the genus Kluyveromyces. The name K. siamensis inulinum, amylum solubile, L-arabinosum, D-arabinosum, sp. nov. is proposed for these strains. D-ribosum, L-rhamnosum, N-acetyl-D-gluosaminum, metha- nolum, erytritolum, galactitolum, a-methyl-D-glucosidum, acidum D-gluconicum, acidum D-glucuronicum, acidum Latin diagnosis of K. siamensis Am-In, D-galacturonicum, acidum 2-keto-D-gluconicum, acidum Yongmanitchai et Limtong sp. nov. 5-keto-D-gluconicum, acidum citricum nec inositolum. In medio liquido: ‘YM’,post dies 3 ad 28 1C cellulae globosae Ethylaminum, L-lysinum et cadaverinum assimilantur at non aut ellipsoideae (2.3–5.4 mm 2.3–6.9 mm), singulae aut nitrosum nec nitricum. Vitamina externa ad crescentiam binae, per germinationem multipolarem reproducentes. In necessaria non sunt. Crescit in 10% NaCl/5% glucosum agaro ‘YM’, post dies 3 ad 28 1C, cultura butyrosa, cremea, et non crescit in 15% NaCl/5% glucosum. Non crescit in sublatum, glabra et margine glabra. Pseudohyphae forman- 0.01% cycloheximido et 0.1% cycloheximido. Crescit in 50% tur nec hyphae non formantur. Ascus formatur per parthe- glucosum et 60% glucosum. Crescere potest in temperatura nogenesis vel conjunctio. Ascosporae globosae, 1–4 in asco. 37 (infirme) et 42 1C (infirme). Diazonium caeruleum B D-Glucosum, sucrosum et raffinosum (exigue) fermen- non respondens. Ureum non hydrolysatur. Ubiquinonum tantur at non D-galactosum, maltosum, lactosum, trehalo- majus: Q-6.

Fig. 2. Kluyveromyces siamensis sp. nov. RS8T. (a) Vegetative cells and ascospores on YM agar after 4 days at 28 1C. (b) Pseudohyphae formed on corn meal agar after 7 days at 28 1C. Scale bar = 10 mm.

Holotypus: Stirps RS8 isolatus aqua, Ranong Provincia, Lactose Thailandia. Cultura et conservatus in Collectionie Culturar- Trehalose um in BIOTEC Culture Collection (BCC), National Center Rafﬁnose Weak for Genetic Engineering and Biotechnology (BIOTEC), Melibiose Pathumthani, Thailandia ut BCC 25962T; NITE Biological Assimilation of carbon compounds Resources Center (NBRC), Department of Biotechnology, D-Glucose 1 National Institute of Technology and Evaluation, Chiba, D-Galactose 1 Japonia conservatus ut NBRC 103859T et Centraalbureau L-Sorbose 1 voor Schimmelcultures (CBS), Utrecht, the Netherlands, ut Sucrose 1 CBS 10860T. Maltose Cellobiose 1 Description of K. siamensis Am-In, Trehalose Yongmanitchai & Limtong sp. nov. Lactose 1 Melibiose 1 Growth in YM broth: After 3 days at 28 C, cells are spherical Rafﬁnose 1 to ellipsoidal (2.3–5.4 mm 2.3–6.9 mm) and occur singly or Melizitose in pairs (Fig. 2). Budding is multilateral. Growth on YM Inulin 1 agar: After 3 days at 28 C, the streak culture is butyrous, Soluble starch cream-coloured, raised, with a smooth surface and has an D-Xylose 1 entire margin. Formation of hyphae: Slide culture on corn L-Arabinose meal agar after 7 days at 28 1C, pseudohyphae are formed D-Arabinose but true hyphae are not formed (Fig. 2). D-Ribose Formation of ascospores: Acospores are produced on YM L-Rhamnose agar, 5% malt extract agar, corn meal agar, Gorodkowa agar N-Acetyl-D-glucosamine 1 and Fowell’s acetate agar after 3 days at 28 C. The ascus is Methanol evanescent and formed parthenogenetically or by conjuga- Ethanol 1 tion between a cell and its bud or between independent cells. Glycerol 1 Ascospores are spherical and one to four ascospores are Erythritol formed in one ascus. No pellicle is present on the surface of Adonitol (ribitol) Delayed assimilation medium. Galactitol D-Mannitol 1 Fermentation D-Glucitol 1 D-Glucose 1 a-Methyl-D-glucoside D-Galactose Salicin 1 Sucrose 1 D-Gluconic acid Maltose D-Glucuronic acid

D-Galacturonic acid BRTT_151006. We thank Dr Sasitorn Jindamorakot and D-Glucono-d-lactone 1 Ms Suthida Tuntigumton for laboratory assistance. 2-Keto-D-gluconate 5-Keto-D-gluconate DL-Lactic acid 1 Succinic acid 1 References Citric acid Altschul SF, Madden TL, Schaffer¨ JZ, Zhang J, Zhang Z, Miller W Inositol & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new Assimilation of nitrogen compounds generation of protein database search programs. Nucleic Acids Nitrate Res 25: 3389–3402. Nitrite Fell JW, Statzell-Tallman A & Kurtzman CP (2004) Lachancea Ethylamine 1 meyersii sp. nov., an ascosporogenous yeast from mangrove L-Lysine 1 regions in the Bahama Islands. Mycologia 50: 359–363. Cadaverine 1 Felsenstein J (1985) Confidence limits on phylogenies: an Growth in vitamin-free medium 1 approach using the bootstrap. Evolution 39: 783–791. Growth on NaCl 10% and Glucose 5% 1 Kimura M (1980) A simple method for estimating evolutionary Growth on NaCl 15% and Glucose 5% rate of base substitutions through comparative studies of Growth on medium containing 0.01% nucleotide sequences. J Mol Evol 16: 111–120. cycloheximide Komagata K & Nakase T (1967) Reitoshokuin no biseibutsu Growth on medium containing 0.1% nikannsuru kenkyu V. Shihan reituoshokushin yori bunri shita cycloheximide kobo no seijo (Microbiological study in foods. V. General Growth on medium with 50% glucose 1 properties of yeasts isolated from frozen foods) (in Japanese). Shokuhin Eiseigaku Zasshi 8: 53–57. Growth on medium with 60% glucose 1 Kuraishi H, Katayama-Fujimura Y, Sugiyama J & Yokoyama T Growth at 20 1C 1 (1985) Ubiquinone systems in fungi I. Distribution of Growth at 25 1C 1 ubiquinones in the major families of ascomycetes, Growth at 37 1C Weak basidimycetes and deuteromycetes, and their taxonomic 1 Growth at 42 C Weak implications. Trans Mycol Soc Jpn 26: 383–395. Diazonium blue B color reaction Kurtzman CP (2003) Phylogenetic circumscription of Urease Saccharomyces, Kluyveromyces and other members of the Acid formation from glucose 1 Saccharomycetaceae, and the proposal of the new genera Amyloid production Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Major ubiquinone Q-6 Zygotorulaspora. FEMS Yeast Res 4: 233–245. Kurtzman CP & Robnett CJ (1998) Identification and phylogeny Holotype: RS8 is the holotype of K. siamensis. The strain of ascomycetous yeasts from analysis of nuclear large subunit was isolated from estuarine water collected from a mangrove (26S) ribosomal DNA partial sequences. Antonie van forest in Laem Son National Park, Ranong Province, Thai- Leewenhoek 73: 331–371. land. The living culture from type was deposited at the Kurtzman CP, Lachance MA, Nguyen HV & Prillinger H (2001) BIOTEC Culture Collection (BCC), National Center for Proposal to conserve the name Kluyveromyces with a conserved Genetic Engineering and Biotechnology (BIOTEC), type (Ascomycota: Hemiascomycetes, Saccharomycetaceae). Taxonomy 50: 907–908. Pathumthani, Thailand, as BCC 25962T; NITE Biological Lachance MA (1998) Kluyveromyces. van der Walt emend.van der Resources Center (NBRC), Department of Biotechnology, Walt. The Yeasts, A Taxonomic Study, 4th edn (Kurtzman CP National Institute of Technology and Evaluation, Chiba, & Fell JW, eds), pp. 227–247. Elsevier, Amsterdam, the Japan, as NBRC 103859T and Centraalbureau voor Schim- T Netherlands. melcultures (CBS), Utrecht, the Netherlands, as CBS 10860 . Lachance MA (2007) Current status of Kluyveromyces 0 Etymology: The species epithet siamensis (si.a.men sis systematics. FEMS Yeast Res 7: 642–645. N.L. fem. adj. siamensis) referring to Siam, the old name of Lachance MA, Bowles JM, Starmer WT & Barker SF (1999) Thailand, where the strains were isolated. Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Can J Microbiol 45: 172–177. Acknowledgements Limtong S, Yongmanitchai W, Tun MM, Kawasaki H & Seki T (2007a) Kazachstania siamensis sp. nov., an ascomycetous yeast This work was supported by the TRF/BIOTEC Special species from forest soil in Thailand. Int J Syst Evol Microbiol 57: Program for ‘Biodiversity Research and Training’, grant 419–422.

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