International Journal of Systematic and Evolutionary Microbiology (2002), 52, 507–511 DOI: 10.1099/ijs.0.01957-0 Weissella kimchii sp. nov., a novel lactic acid NOTE bacterium from kimchi 1 Department of Hak-Jong Choi,1 Chan-Ick Cheigh,1 Seong-Bo Kim,1 Jung-Choul Lee,1 Biotechnology and 1 2 3 1 Bioproducts Research Dong-Woo Lee, Sung-Won Choi, Jung-Min Park and Yu-Ryang Pyun Center, Yonsei University, 134 Shinchon-Dong, Sodaemun-Gu, Seoul Author for correspondence: Yu-Ryang Pyun. Tel: j82 2 2123 2883. Fax: j82 2 312 6821. 120-749, South Korea e-mail: yrpyun!yonsei.ac.kr 2 Greenbiotech Co. Ltd, 687-2 Sangisug-Ri, Kyoha- Myun, Paju City, Kyungki- A Gram-positive, catalase-negative, non-sporulating, facultatively anaerobic, Do 413-830, South Korea short rod-shaped bacterium, with cells measuring 03–05i1–2 µm and T 3 Korean Culture Center of designated strain CHJ3 , was isolated from partially fermented kimchi, a Microorganisms, 361-221 traditional Korean fermented vegetable food. The strain produced CO2 gas, Hongje 1-Dong, D-lactate from glucose and dextran from sucrose and hydrolysed aesculin Sodaemun-Gu, Seoul 120-091, South Korea and arginine. It also fermented N-acetylglucosamine, amygdalin, arbutin, cellobiose, D-fructose, galactose, β-gentiobiose, gluconate, D-glucose, maltose, D-mannose, salicin, sucrose and D-xylose. The GMC content of the DNA was 482 mol%. Phylogenetic analysis of 16S rRNA showed that strain CHJ3T is a member of the genus Weissella. The nearest phylogenetic relative of strain CHJ3T was Weissella confusa, with 16S rRNA similarity of 983%. However, strain CHJ3T could be differentiated from W. confusa on the basis of some phenotypic characteristics, analysis of whole-cell protein patterns and DNA–DNA hybridization data. These data suggest that strain CHJ3T be classified in the genus Weissella as a novel species, Weissella kimchii sp. nov. The type strain is CHJ3T (l KCCM 41287T l DSM 14295T l KCTC 3746T). Keywords: Weissella kimchii, kimchi, lactic acid bacteria Lactic acid bacteria have been used as starter cultures Martinez-Murcia & Collins, 1990, 1991). However, the for dairy, meat and vegetable fermentations in many morphological and physiological features of Weissella countries. They contribute to flavour development as species do not support this grouping directly, which well as preservation of foods. They also produce a now incorporates species that produce (k)- as well as variety of antimicrobial compounds such as organic -lactate (Stiles & Holzapfel, 1997). Recently, Weis- acids, hydrogen peroxide and bacteriocins. The genus sella thailandensis was isolated and identified from Weissella encompasses a phylogenetically coherent fermented fish products in Thailand (Tanasupawat et group of lactic acid bacteria and includes eight al., 2000). Leuconostoc-like species, including Weissella confusa (formerly Lactobacillus confusus), Weissella minor Kimchi is a traditional Korean vegetable food, fer- (formerly Lactobacillus minor), Weissella kandleri (for- mented by a variety of lactic acid bacteria. The main merly Lactobacillus kandleri), Weissella halotolerans micro-organisms responsible for kimchi fermentation (formerly Lactobacillus halotolerans), Weissella viri- are known to be Leuconostoc mesenteroides and descens (formerly Lactobacillus viridescens), Weissella Lactobacillus plantarum (Mheen & Kwon, 1984). In paramesenteroides (formerly Leuconostoc paramesen- particular, Leuconostoc mesenteroides is known to teroides) and Weissella hellenica (Collins et al., 1993). predominate in the early stage of kimchi fermentation Based on 16S rRNA sequencing studies of the above and provides anaerobic conditions due to the pro- Weissella species, they constitute a natural phylo- duction of CO# gas. As the pH of kimchi is decreased genetic group separated from the genus Leuconostoc gradually to pH 4n0byLeuconostoc mesenteroides, within the lactic acid bacteria (Yang & Woese, 1989; Lactobacillus plantarum then dominates in the later stage of fermentation. It is generally recognized that ................................................................................................................................................. Lactobacillus plantarum is a major cause of excessive The GenBank accession number for the 16S rRNA gene sequence of strain acidification of kimchi in the later stages of fermen- CHJ3T is AF312874. tation (Mheen & Kwon, 1984). To date, various kinds 01957 # 2002 IUMS Printed in Great Britain 507 H.-J. Choi and others Strain CHJ3T was isolated from 4-d-old kimchi using MRS agar (Difco). Reference strains were obtained from the Korean Culture Center of Microorganisms (KCCM), the Korean Collection for Type Cultures (KCTC), the ATCC and the DSMZ. Morphological, cultural and biochemical tests were performed at 30 mC unless stated otherwise. Colony morphology, Gram staining and catalase production were determined on cells grown anaerobically on MRS agar and MRS broth incubated for 20 h. Cell morphology was ex- amined by scanning electron microscopy. Cells were tested for the ability to grow on acetate agar adjusted to pH 5n4 (Whittenbury, 1965). Gas (CO#) production from glucose was determined in MRS broth without ................................................................................................................................................. citrate using Durham tubes (Holzapfel & Gerber, T Fig. 1. Scanning electron micrograph of strain CHJ3 . Bar, 2 µm. 1983). Production of dextran from sucrose was ob- served on MRS agar in which glucose was replaced by 5% sucrose (Holzapfel & Schillinger, 1991). Hydroly- sis of arginine was tested in Moeller decarboxylase medium with 1% arginine (Phillips & Nash, 1985). Carbohydrate fermentation tests were performed ac- cording to Orberg & Sandine (1984) and the API Rapid CH system (bioMe! rieux) using the recom- mended CHL medium. The isomers of lactic acid formed from glucose were determined enzymically by using -lactate and -lactate dehydrogenase (Roche). SDS-PAGE analysis of whole-cell proteins was carried out as described by Elliott et al. (1991) and Pot et al. (1993). The GjC content of the DNA and DNA– DNA hybridization were determined by the DSMZ. DNA was isolated and purified by chromatography on a hydroxyapatite column. The GjC content was determined by using HPLC as described by Mesbah et ................................................................................................................................................. al. (1989); non-methylated λ DNA (Sigma) was used T Fig. 2. Whole-cell protein patterns of strain CHJ3 isolated from as the standard. Spectroscopic DNA–DNA hybridiza- kimchi and reference Weissella type strains. Lanes: 1 and 10, mole- cular mass standards [from top to bottom; myosin (200 kDa), tion was carried out as described by De Ley et al. β-galactosidase (116 kDa), phosphorylase B (97n4 kDa), BSA (1970), with the modifications described by Huß et al. (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (31 kDa)]; (1983) and Escara & Hutton (1980). Extraction of 2, strain CHJ3T ;3,W. confusa DSM 20196T ;4,W. minor genomic DNA suitable for amplification of the 16S DSM 20014T ;5,W. viridescens DSM 20410T ;6,W. halotolerans DSM 20190T ;7,W. paramesenteroides DSM 20288T ;8,W. rRNA gene was performed by the method of Choi et hellenica NCFB 2973T ;9,W. kandleri DSM 20593T. al. (2000). A large fragment of the 16S rRNA gene of strain CHJ3T was amplified by PCR using universal 16S rRNA primers. The amplified PCR products were cloned into pGEM-T easy vector (Promega) of lactic acid bacteria from genera such as Lactococcus, according to the manufacturer’s recommendations Lactobacillus, Pediococcus and Leuconostoc have been and sequenced using a Tag Dye-Deoxy terminator isolated from kimchi (Lim et al., 1989; Cheigh & Park, cycle sequencing kit (Perkin-Elmer) and ABI PRISM 1994; So & Kim, 1995; Lee et al., 1997). Moreover, 373 automatic DNA sequencer (Perkin-Elmer). For newly isolated and identified strains have been re- the comparison of 16S rDNA sequences, 16S rRNA ported such as Lactobacillus kimchii (Yoon et al., gene sequences published previously were obtained 2000) and Leuconostoc kimchii (Kim et al., 2000). from the Ribosomal Database Project (Maidak et al., Taxonomic studies on micro-organisms from kimchi 1996), EMBL and GenBank databases. Multiple are not yet complete. Thus, it is important to classify alignment of sequences, calculation of the nucleotide and identify micro-organisms from kimchi on the basis substitution rate (Knuc; Kimura, 1980), construction not only of phenotypic characteristics but also phylo- of a neighbour-joining phylogenetic tree (Saitou & genetic analysis by 16S rRNA sequencing and other Nei, 1987) and 1000-replicate bootstrap analysis molecular tools. In this study, we describe the pheno- for evaluation of the phylogenetic tree topology typic, phylogenetic and genetic characteristics of a (Felsenstein, 1985) were carried out with novel isolate. These polyphasic taxonomic studies version 1.6 (Thompson et al., 1994). Alignment gaps suggest that strain CHJ3T represents a novel species, and unidentified base positions were not taken into Weissella kimchii sp. nov. account. 508 International Journal of Systematic and Evolutionary Microbiology 52 Weissella kimchii sp. nov. from kimchi ..................................................................................................... Fig. 3. Phylogenetic tree showing the position of strain CHJ3T, Weissella species and related bacteria