Kluyveromyces Siamensis Sp. Nov., an Ascomycetous
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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, FEMS Yeast Res 8 (2008) 823–828 c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 824 S. Am-In et al. 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 com- pounds 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 À 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