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Int J Syst Evol Microbiol 53 (2003), 1123-1126; DOI  10.1099/ijs.0.02463-0
© 2003 International Union of Microbiological Societies

Leuconostoc inhae sp. nov., a lactic acid bacterium isolated from kimchi

Bongjoon Kim, Jongho Lee, Jichan Jang, Jeongho Kim and Hongui Han

Research Laboratory for Microbiology, Department of Biological Sciences, Inha University, Incheon 402-751, Republic of Korea

Correspondence
Hongui Han
biohan{at}inha.ac.kr


   ABSTRACT
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Six strains of a hitherto unknown bacterium isolated from kimchi, a fermented vegetable food produced in Korea, were characterized by using phenotypic methods, phylogenetic analysis and DNA–DNA hybridization. The novel strains were Gram-positive, non-spore-forming, heterofermentative and spherical or lenticular lactic acid bacteria. Comparative 16S rRNA gene sequencing and DNA relatedness demonstrated that the unknown strains represented a novel clade within the genus Leuconostoc and were close to, but distinct from, Leuconostoc gelidum. The unknown strains were clearly distinguished from all described members of the genus Leuconostoc by using RFLP patterns of genus-specific 16S rRNA gene PCR products with a single endonuclease, BsmAI. Based on the polyphasic evidence, the unknown isolates are classified as Leuconostoc inhae sp. nov. The type strain is strain IH003T (=KCTC 3774T =DSM 15101T).

Published online ahead of print on 13 December 2002 as DOI 10.1099/ijs.0.02463-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains IH003T and IH611 are AF439560 and AY117686.


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Kimchi is a traditional Korean food prepared from various vegetables and consumed as a main side dish. It is kept in a cryogenic storage system for between 2 weeks and 4 months, thereby allowing leuconostocs to predominate in the whole fermentation. We have been focusing on the diversity of species and, at present, five Leuconostoc species have been isolated from kimchi: Leuconostoc gelidum, Leuconostoc mesenteroides, Leuconostoc citreum (Kim et al., 2000aGo), Leuconostoc kimchii (Kim et al., 2000bGo) and dextran-producing Leuconostoc lactis (Kim et al., 2001Go). As a part of a study of the microflora of kimchi, we have isolated six strains that fall into a group distinct from all known members of the genus Leuconostoc (Garvie, 1986Go; Shaw & Harding, 1989Go; Farrow et al., 1989Go; Martinez-Murcia & Collins, 1991Go; Dicks et al., 1993Go; Björkroth et al., 2000Go; Kim et al., 2000bGo; Antunes et al., 2002Go), but are closely related to L. gelidum. In this article, we report the characterization of these unknown lactic acid bacteria and the results of a polyphasic taxonomy study. Based on the results, a novel species is described, Leuconostoc inhae sp. nov.

Six strains, IH003T, IH101, IH201, IH316, IH515 and IH611, were isolated from kimchi fermented for 18 days at 5–8 °C. These unknown strains were cultured with 14 Leuconostoc and one Weissella species as reference type strains at 25 °C on MRS agar (Difco) or in broth. The morphology of isolates was examined by using scanning electron microscopy (S-4200; Hitachi). Additional biochemical and physiological tests were performed according to the methods of Smibert & Krieg (1994)Go and Garvie (1984)Go. Growth at different temperatures and pH and tolerance of NaCl were tested in MRS broth. Carbohydrate fermentation patterns were determined at 25 °C by using API CH50 strips and the API CHL medium system according to the manufacturer's instruction (bioMérieux). The optical isomer of lactic acid was determined by using a D-/L-lactate dehydrogenase kit (tc D-/L-lactic acid; Boehringer Mannheim).

Sequencing of 16S rDNA was carried out as described by Kim et al. (2000b)Go. The 16S rDNA sequences of isolates were aligned by using CLUSTAL X software (version 1.8). Similarity values were calculated using SIMILARITY MATRIX (version 1.1) in RDP-II (Maidak et al., 2001Go). Calculation of an evolutionary distance matrix (Kimura two-parameter model), construction of a neighbour-joining phylogenetic tree and bootstrap analysis (1000 replicates) were carried out by using PHYLIP (Felsenstein, 1993Go). DNA–DNA hybridization was performed as described by Kim et al. (2000b)Go, using the protocol described in the ECL direct nucleic acid labelling and detection system (RPN 3000; Amersham Pharmacia Biotech). G+C contents were determined by HPLC as described by Tamaoka & Komagata (1984)Go.

Differentiation of Leuconostoc species was carried out by the method described by Jang (2002)Go. For the detection of Leuconostoc species, the following genus-specific primers were used to amplify 16S rRNA genes: forward primer 5'-CGAAAGGTGCTTGCACCTTTCAAG-3' (Escherichia coli numbering system, positions 74–98); reverse primer 5'-TTTGTCTCCGAAGAGAACA-3' (positions 1023–1040). These primers produced a 976 bp PCR fragment. PCR amplification was carried out with the method described by Kim et al. (2000b)Go. For species identification, restriction fragment length polymorphism (RFLP) analysis of PCR products (976 bp) was performed with four endonucleases, MseI, HaeIII, Tsp509I and BsmAI (New England Biolabs). Digestions were performed at 37 (MseI, HaeIII), 55 (BsmAI) or 65 (Tsp509I) °C according to the manufacturer's instructions.

Cellular morphology of the six isolates was spherical or lenticular, showing the shape typical of members of the genus Leuconostoc (Garvie, 1986Go). Cells of a representative strain IH003T are shown in Fig. 1Go. They were Gram-positive, catalase-negative, arginine dihydrolase-negative and produced gas and over 92 % D-lactic acid from glucose. They grew at pH 4·8 and in 3 % NaCl and at 1, 5 and 30 °C but not at 37 °C. Dextran production from 5 % sucrose agar medium was variable (~50 %). Cellular morphology and general biochemical characteristics of the isolates were consistent with their assignment to the genus Leuconostoc (Garvie, 1986Go). The details are presented in Table 1Go and in the species description below.



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  		Fig. 1. Scanning electron micrograph of Leuconostoc inhae sp. nov. IH003T. Cells were grown in MRS medium at 25 °C for 24 h. Bar, 2 µm.

 

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  		Table 1. Differential characteristics of Leuconostoc inhae sp. nov. and related leuconostocs

Species/strains: 1, L. inhae sp. nov. (n=6; data from this study); 2, L. gelidum DSM 5578T (data from Shaw & Harding, 1989Go); 3, L. carnosum DSM 5576T (Shaw & Harding, 1989Go); 4, L. gasicomitatum LMG 18811T (Björkroth et al., 2000Go); 5, L. kimchii KCTC 2386T (Kim et al., 2000bGo). Results are scored as: +, >=90 % strains positive; -, >=90 % strains negative; d, 11–89 % strains positive (number of strains positive is shown). All strains/species showed the following results for acid production: positive for D-fructose, D-glucose, D-mannose, methyl {alpha}-D-glucoside, ribose, sucrose and trehalose; negative for D-arabinose, dulcitol, erythritol, glycerol, melezitose, rhamnose, sorbitol and L-sorbose. Other characteristics are mentioned in the species description.

 
The 16S rRNA genes of IH003T and IH611 were sequenced and subjected to a comparative analysis to establish the phylogenetic relationship of the isolates. The almost-complete gene sequences (1474 and 1505 nt) were determined and found to be identical. Sequence database searches showed that the sequence similarity of IH003T and IH611 was 99·1 and 98·9 % to L. gelidum DSM 5578T. The phylogenetic position of these isolates within the genus Leuconostoc is presented in Fig. 2Go. The isolates were most closely related to, but different from, L. gelidum DSM 5578T, and constituted a ‘cold cluster’ with L. kimchii KCTC 2386T, Leuconostoc carnosum DSM 5576T, Leuconostoc gasicomitatum LMG 18811T and L. gelidum DSM 5578T, frequently isolated at low temperatures (Shaw & Harding, 1989Go; Kim et al., 2000bGo; Björkroth et al., 2000Go). The mean G+C content of the six isolates was 39·9±0·5 mol%. This value is within a range for members of the genus Leuconostoc (38–44 mol%; Garvie, 1986Go). The isolates showed more than 73 % DNA relatedness to each other but less than 26 % to all reference strains used (Table 2Go). Thus, the isolates were considered to represent a novel species belonging to Leuconostoc.



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  		Fig. 2. Unrooted phylogenetic tree based on 16S rDNA comparisons showing the relationships of L. inhae sp. nov. IH003T and IH611 to other Leuconostoc strains. Bootstrap percentages obtained with 1000 resamplings are given at branch points. Accession numbers are given in parentheses. Bar, genetic distance of 0·01.

 

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  		Table 2. Levels of DNA relatedness among L. inhae sp. nov. and Leuconostoc species within the same cluster

Percentage reassociation is shown. Reassociation values are means of duplicate determinations.

 
However, the separation of the isolates from L. gelidum was not definite, since they formed a phylogenetically close branch and had high sequence similarity, as mentioned above. We applied a PCR-RFLP method (Jang, 2002Go) by using a set of Leuconostoc genus-specific primers present in the 16S rRNA gene. The resultant PCR products were subjected to cascade digestion with three restriction enzymes, MseI, HaeIII and Tsp509I, yielding different combined PCR-RFLP patterns and enabling not only their detection but also identification at the genus and species level. Jang (2002)Go demonstrated that all described members of the genus Leuconostoc were successfully distinguished by RFLP patterns, excluding phylogenetically quite distant species such as Leuconostoc fallax, Leuconostoc ficulneum and Leuconostoc fructosum (Antunes et al., 2002Go; Martinez-Murcia & Collins, 1991Go). Nevertheless, our isolates could not be differentiated with these enzymes, since they showed identical RFLP patterns to L. gelidum DSM 5578T (data not shown). BsmAI was chosen theoretically from a highly variable region of the 16S rRNA gene (positions 183–207) to allow separation between the novel isolates and L. gelidum DSM 5578T by using WWWtacg version 2.38 (http://genzi.virus.kyoto-u.ac.jp/tacg2/tacg2.form.html). Experimental RFLP patterns are shown in Fig. 3Go. L. kimchii KCTC 2386T, L. carnosum DSM 5576T, L. gasicomitatum LMG 18811T and L. gelidum DSM 5578T (a ‘cold cluster’) revealed identical RFLP patterns (fragment sizes 232 and 711 bp), but the pattern differed from that of the six novel isolates (fragment sizes 107, 125 and 711 bp). Interestingly, RFLP patterns with BsmAI appeared to be identical in all Leuconostoc species with the exception of the novel isolates (data not shown). This indicates that the isolates can be differentiated independently with a single restriction enzyme, BsmAI, without using three enzymes as suggested above. PCR-RFLP analyses confirmed the phylogenetic homogeneity of the six isolates and their separation from other species of the genus Leuconostoc. These results are also supported by RFLP as a good distinct method for strain characterization (Heyndrickx et al., 1996Go). Thus, based on evidence from cellular morphology, phylogenetic analysis, DNA relatedness and PCR-RFLP patterns and their distinctive biochemical characteristics, the six isolates from kimchi are assigned to the genus Leuconostoc as Leuconostoc inhae sp. nov.



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  		Fig. 3. PCR-RFLP patterns of L. inhae sp. nov. and closely related species derived from digestion of genus-specific PCR products with BsmAI. Lanes: 1, L. carnosum DSM 5576T; 2, L. gasicomitatum LMG 18811T; 3, L. gelidum DSM 5578T; 4, L. kimchii KCTC 2386T; 5–10, L. inhae sp. nov. IH003T, IH101, IH201, IH316, IH515 and IH611; M, 100 bp DNA ladder. RFLP patterns with BsmAI were identical in all strains of Leuconostoc examined except L. inhae strains and the phylogenetically distant strains L. fallax DSM 20189T, L. ficulneum DSM 13613T and L. fructosum DSM 20349T (not shown). Only four species in the same cluster as L. inhae sp. nov. are shown.

 
Description of Leuconostoc inhae sp. nov.
Leuconostoc inhae (in'ha.e. N.L. gen. n. inhae of Inha, from the Inha University, Republic of Korea).

Cells are Gram-positive, spherical or lenticular, occurring singly or in pairs. Cells are 0·8–1·0x0·8–1·8 µm. Colonies on MRS agar after 48 h at 25 °C are small (1·0–1·5 mm), round, smooth, convex, opaque and greyish-white. Cells are non-motile and non-spore-forming. Facultatively anaerobic. Catalase-negative. Obligately heterofermentative. Arginine is not hydrolysed. Some strains produce slime materials. Growth occurs in 3 % NaCl but not in 7 % NaCl and at pH 4·8 but not at pH 3·8. All strains grow at 30 °C but not at 37 °C. Good growth is obtained at 1–5 °C. Over 92 % D-lactic acid is produced from glucose. Acid is produced from L-arabinose, cellobiose, aesculin, D-fructose, {beta}-gentiobiose, D-glucose, D-mannose, mannitol, maltose, methyl {alpha}-D-glucoside, N-acetylglucosamine, ribose, sucrose and trehalose. Acid is not produced from adonitol, D-arabinose, D-arabitol, arbutin, 2-ketogluconate, 5-ketogluconate, dulcitol, erythritol, D-fucose, L-fucose, glycerol, glycogen, inositol, inulin, lactose, D-lyxose, melezitose, melibiose, methyl {alpha}-D-mannoside, methyl {beta}-xyloside, D-raffinose, rhamnose, sorbitol, L-sorbose, starch, D-tagatose, xylitol, D-xylose or L-xylose. Some strains produce acid from amygdalin, galactose, gluconate, salicin and D-turanose. The G+C content of the type strain is 39·9±0·5 mol% (as determined by HPLC).

The type strain, strain IH003T (=KCTC 3774T =DSM 15101T), was isolated from kimchi. The description of the type strain corresponds to that of five other isolates, except that no growth occurs at pH 3·8 and slime material is not produced from sucrose. Amygdalin, galactose, gluconate, salicin and D-turanose are not fermented.


   ACKNOWLEDGEMENTS
 
Microbia Co. Ltd supported this study. The authors are indebted to Dr J. P. Euzéby and Professor J. Chun for their kind help in the denomination of the novel species. We also thank Dr J.-H. Yoon (Korea Research Institute of Bioscience and Biotechnology) for helping with DNA base composition analysis.


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Farrow, J. A. E., Facklam, R. R. & Collins, M. D. (1989). Nucleic acid homologies of some vancomycin-resistant leuconostocs and description of Leuconostoc citreum sp. nov. and Leuconostoc pseudomesenteroides sp. nov. Int J Syst Bacteriol 39, 279–283.

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Garvie, E. I. (1986). Genus Leuconostoc van Tieghem 1878. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1071–1075. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.

Heyndrickx, M., Vauterin, L., Vandamme, P., Kersters, K. & De Vos, P. (1996). Applicability of combined amplified ribosomal DNA restriction analysis (ARDRA) patterns in bacterial phylogeny and taxonomy. J Microbiol Methods 26, 247–259.

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