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Weissella Koreensis Sp. Nov., Isolated from Kimchi

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Weissella Koreensis Sp. Nov., Isolated from Kimchi International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1257–1261 DOI: 10.1099/ijs.0.02074-0 Weissella koreensis sp. nov., isolated from NOTE kimchi 1 Korea Research Institute Jung-Sook Lee,1,2 Keun Chul Lee,1 Jong-Seog Ahn,1 Tae-Ick Mheen,1 of Bioscience and 2 1 Biotechnology, PO Box Yu-Ryang Pyun and Yong-Ha Park 115, Yusong, Taejon 305-600, Korea 2 Author for correspondence: Yong-Ha Park. Tel: j82 42 860 4620. Fax: j82 42 862 1315. Department of e-mail: yhpark!mail.kribb.re.kr Biotechnology, Yonsei University, Seoul 120-749, Korea A taxonomic study was carried out on two strains that came from kimchi, a traditional Korean fermented-vegetable food. The DNA GMC content of these strains was 37 mol%. Both strains contained Lys–Ala–Ser in the cell walls. On the basis of morphological, physiological and chemotaxonomic characteristics, together with data from 16S rDNA sequence comparisons and DNA–DNA reassociation, it is proposed that these strains represent a novel species of the genus Weissella, Weissella koreensis sp. nov. The type strain is strain S-5623T (l KCTC 3621T l KCCM 41516T l JCM 11263T). Keywords: Weissella koreensis sp. nov., kimchi, taxonomy Lactic acid bacteria are widely distributed in Korean densis and Weissella viridescens. Members of the genus traditional foods such as kimchi. Kimchi is a generic Weissella are Gram-positive, non-spore-forming, term used to denote a group of fermented-vegetable heterofermentative and non-motile. The cells are foods produced in Korea. The flavour of kimchi is generally short rods with rounded to tapered ends or dependent on the ingredients, fermentation conditions coccoid in shape, occurring singly, in pairs or in short (e.g. temperature) and bacteria involved in the fer- chains. With the exception of W. paramesenteroides mentation process (Cheigh & Park, 1994; Lee et al., and W. hellenica, all species of the genus generally 1992; Mheen & Kwon, 1984). In particular, the genera produce -lactic acid from glucose. The peptido- Lactobacillus, Leuconostoc and Pediococcus are known glycan subunit contains lysine and the interpeptide to play an important role in kimchi fermentations bridge contains alanine or serine and alanine as typical (Cheigh & Park, 1994; Lee et al., 1992; Mheen & constituents (Collins et al., 1993). Kwon, 1984). Although many lactic acid bacteria have In this study, we report the morphological, biochemi- been isolated from Korean kimchi, studies of their cal and phylogenetic characteristics of novel strains systematic taxonomy have rarely been reported (Lee et isolated from kimchi. We also propose that two of the al., 1996a, b, 1997a, b). Most taxonomic studies on isolates be assigned to a novel species, Weissella isolates from kimchi have been based on phenotypic koreensis sp. nov. characteristics. A polyphasic approach, including phenotypic, chemotaxonomic and molecular methods, is needed to determine the taxonomic position of Micro-organisms and cultures kimchi isolates. Recently, novel strains from kimchi The bacterial strains used in this study were isolated have been reported, such as Lactobacillus kimchii from kimchi, a traditional Korean fermented- (Yoon et al., 2000) and Leuconostoc kimchii (Kim et vegetable food. Strains S-5623T and S-5673 were iso- al., 2000). lated using MRS agar medium (Difco). These strains and two reference strains, W. viridescens KCTC 3504T The genus Weissella was first proposed by Collins et al. T (1993) on the basis of the results of a 16S rRNA and W. kandleri KCTC 3610 , were cultivated at phylogenetic analysis. At the time of writing, it 30 mC. included Weissella confusa, Weissella halotolerans, Weissella hellenica, Weissella kandleri, Weissella Morphological and physiological characteristics minor, Weissella paramesenteroides, Weissella thailan- The morphology of the cells was examined by using ................................................................................................................................................. scanning electron microscopy. Growth at different The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences temperatures was observed in MRS broth at 10, 15, 20, of strains S-5623T and S-5673 are AY035891 and AY035892. 25, 30, 37 and 42 mC. The growth experiment was 02074 # 2002 IUMS Printed in Great Britain 1257 1258 J.-S. Lee and others Table 1. Differential characteristics of species of the genus Weissella ...................................................................................................................................................................................................................................................................................................................................................................................................................... Taxa are listed as: 1, W. kandleri;2,W. viridescens;3,W. minor;4,W. halotolerans;5,W. confusa;6,W. paramesenteroides;7,W. hellenica;8,W. thailandensis; 9, W. koreensis sp. nov. (strains S-5623T and S-5673 gave identical results). Data were taken from this study and from Collins et al. (1993) and Tanasupawat et al. (2000). Characters are scored as: j, " 90% of strains positive; k, " 90% of strains negative; d, 11–89% of strains positive; ( ), delayed reaction; , not tested. Characteristic 1 2 3 4 5 6 7 8 9 Acid produced from: -Arabinose kk k k k d jjj Cellobiose kk j k j (d) kkk Galactose jk k k j j kjk Maltose kj j j j j jjk International Journal of Systematic and Evolutionary Microbiology Melibiose kk k k k j kjk Raffinose kk k k k d kjk Ribose jk j j j d kjj Sucrose k d jkjjjjk Trehalose k d jkkjjjk Xylose kk k k j d kkj Hydrolysis of aesculin kk j k j (j) jk NH$ from arginine jk j j j k kkj Dextran formation j k jkkkj Lactic acid configuration* Murein type Lys–Ala–Gly–Ala# Lys–Ala–Ser Lys–Ser–Ala# Lys–Ala–Ser Lys–Ala Lys–Ala; Lys–Ser–Ala# Lys–Ala–Ser Lys–Ala# Lys–Ala–Ser Cell morphology Irregular rods Small, irregular Irregular, short, Irregular, short Short rods, thickened Spherical or lenticular Large, spherical Cocci in pairs Irregular, short rods coccoid rods with or coccoid rods at one end cells or lenticular cells or in chains or coccoid rods rounded to tapered ends DNA GjC content 39 41–44 44 45 45–47 37–38 39–40 38–41 37 (mol%) * Scored as: , " 90% of the lactic acid is (k); , " 25% of the total lactic acid is (j). 52 Weissella koreensis sp. nov. Table 2. DNA–DNA reassociation between strains S-5623T, S-5673, W. kandleri KCTC 3610T and W. viridescens KCTC 3504T Strain Reassociation (%) with labelled DNA from: 1234 1. Strain S-5673 100 103 25 16 2. Strain S-5623T 90 100 24 16 3. W. kandleri KCTC 3610T 21 20 100 14 4. W. viridescens KCTC 3504T 13 16 9 100 performed using a cap tube containing 5 ml MRS instructions as described previously (Lee et al., broth at a pH of 1n0–10n0 and a temperature of 25 mC. 1996a, b; Yang et al., 1993). Growth was estimated by monitoring the OD . '!! Both strains contained Lys–Ala–Ser in the cell walls. MRS broth containing 200 mM KCl\HCl buffer at The major whole-cell fatty acids in the test strains pH 1 0–2 0, 100 mM citric acid\200 mM Na HPO n n # % were octadecenoic acid (18:1) and hexadecanoic acid at pH 3 0–5 0, 100 mM Na HPO \NaH PO buffer at n n # % # % (16:0). pH 6n0–8n0 and 100 mM NaHCO$\Na#CO$ buffer at pH 9n0–10n0 was used. This experiment was done on the basis of the methods of Yumoto et al. (1998). API DNA base composition 50CHL strips (bioMe! rieux) were used to determine the sugar-fermentation patterns of the organisms. API DNA was extracted and purified by a modification of 20E strips were used for other physiological and the method of Marmur (1961). The GjC content of biochemical characteristics. All API tests were per- the DNA was determined by using the reverse-phase formed in accordance with the manufacturer’s direc- HPLC method described by Tamaoka & Komagata tions. Catalase activity was determined by bubble (1984). The DNA base composition of the isolates was production in 3% (v\v) H#O# and oxidase activity was 37 mol%. determined using 1% (w\v) tetramethyl p-phenylene- diamine. We tested the configuration of lactate by DNA–DNA hybridization using the -lactate enzymic kit (Boehringer Mann- heim). Production of dextran (slime) from sucrose was DNA–DNA hybridization was carried out by fluoro- observed on MRS agar in which glucose had been metric hybridization in microdilution wells, using replaced by 5% sucrose (Hitchener et al., 1982). Gas biotinylated DNA (Ezaki et al., 1989). As shown in (CO#) production from glucose was determined using Table 2, DNA–DNA reassociation values between the two isolates (S-5623T and S-5673) and two reference Durham tubes. Tolerance of NaCl was examined on T MRS agar with 8 and 10% NaCl. strains (W. kandleri KCTC 3610 and W. viridescens KCTC 3504T) were less than 25%. Isolates S-5623T Cells of both strains were irregular, short or coccoid and S-5673 exhibited high levels of homology (90– rods. They were Gram-positive, catalase-negative, 103%) to each other. facultative anaerobes. Both strains grew at 10 and 37 mC but not at 42 mC; the optimum temperature for growth was 25 mC. The strains grew at pH 4n0–8n0; the Phylogenetic analysis optimum pH was pH 6n0. They did not grow in 8 or Two universal primers described by Stackebrandt & 10% NaCl. As shown in Table 1, both strains gave Liesack (1993), 9F (5h-GAGTTTGATCCTGGCTC- positive results for arginine hydrolysis, dextran for- AG-3h; positions 9–27, Escherichia coli 16S rRNA mation from sucrose and acid production from - numbering) and 1542R (5h-AGAAAGGAGGTGAT- arabinose, ribose and xylose and negative results for CCAGCC-3h; positions 1542–1525), were used for aesculin hydrolysis and acid production from cellob- PCR amplification of the 16S rDNA. The amplified iose, galactose, maltose, melibiose, raffinose, sucrose PCR product was purified using the QIAquick PCR and trehalose.
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