International Journal of Systematic and Evolutionary Microbiology (2012), 62, 2383–2387
DOI 10.1099/ijs.0.035329-0
Reclassification of Lactobacillus kimchii and
Lactobacillus bobalius as later subjective synonyms
of Lactobacillus paralimentarius
Huili Pang,1 Maki Kitahara,2 Zhongfang Tan,1 Yanping Wang,1
Guangyong Qin,1 Moriya Ohkuma2 and Yimin Cai3
Correspondence
Yimin Cai
[email protected]
1
Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University, Zhengzhou,
Henan 450052, PR China
2
Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako,
Saitama 351-0198, Japan
3
Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan
Characterization and identification of strain CW 1 (5JCM 17161) isolated from corn silage were
performed. Strain CW 1 was a Gram-positive, catalase-negative and homofermentative rod that
produced the DL-form of lactic acid. This strain exhibited more than 99.6 % 16S rRNA gene
sequence similarity and greater than 82 % DNA–DNA reassociation with type strains of
Lactobacillus kimchii, L. bobalius and L. paralimentarius. To clarify the taxonomic positions of
these type strains, phenotypic characterization, 16S rRNA gene sequencing, ribotyping and
DNA–DNA relatedness were examined. The three type strains displayed different L-arabinose,
lactose, melibiose, melezitose, raffinose and N-acetyl-b-glucosaminidase fermentation patterns.
Phylogenetic analysis showed that L. paralimentarius is a closer neighbour of L. kimchii and L.
bobalius, sharing 99.5–99.9 % 16S rRNA gene sequence similarity, which was confirmed by the
high DNA–DNA relatedness (¢82 %) between L. paralimentarius JCM 10415T, L. bobalius JCM
16180T and L. kimchii JCM 10707T. Therefore, it is proposed that L. kimchii and L. bobalius
should be reclassified as later synonyms of L. paralimentarius.
Strain CW 1 was isolated from corn (Zea mays L.) silage
collected on Qinchuan Dairy Farm, Lanzhou, Gansu
Province, China (in 2006). Based on its chemotaxonomic
and morphological properties, this novel isolate was
presumed to be a member of the genus Lactobacillus. 16S
rRNA gene sequencing showed that strain CW 1 was
closely related to the type strains of Lactobacillus
paralimentarius JCM 10415T isolated from sourdough in
Japan by Cai et al. (1999), Lactobacillus bobalius JCM
16180T isolated from Bobal grape must in Spain by MañesLázaro et al. (2008) and Lactobacillus kimchii JCM 10707T
isolated from a type of kimchi in Korea by Yoon et al.
(2000). Therefore, strain CW 1 could not be identified at
the species level on the basis of its phenotypic and
phylogenetic characteristics.
The present study aimed to identify strain CW 1, and to
investigate whether L. paralimentarius, L. bobalius and L.
kimchii differ phenotypically and phylogenetically.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain CW 1 (5JCM 17161) is AB605665.
Two supplementary figures are available with the online version of this
paper.
035329 G 2012 IUMS
Comparative 16S rRNA gene sequencing, ribotyping and
DNA–DNA relatedness, as well as physiological analyses,
were carried out.
The type strains used in this study are shown in Table 1.
Strain CW 1 is a Gram-positive and catalase-negative rod,
and does not produce gas from glucose. CW 1 was cultured
on MRS agar (Difco Laboratories) at 30 uC for 24 h before
being transferred to nutrient broth (Difco) containing
10 % glycerol and stored as stock culture at 280 uC for
further analysis.
Strain CW 1 was compared with type strains of relevant
Lactobacillus species by carbohydrate fermentation using
analytical profile index (API 50 CH) strips (bioMérieux).
Strain CW 1 exhibited an almost identical carbohydrate
fermentation pattern to L. paralimentarius JCM 10415T
except for melezitose, to L. kimchii JCM 10707T except for
L-arabinose, D-xylose and gluconate, and to L. bobalius
JCM 16180T except for lactose, melibiose and gluconate,
but a very different pattern from Lactobacillus alimentarius
JCM 1095T and Lactobacillus farciminis JCM 1097T in
terms of fermentation of ribose, D-xylose, galactose, amethyl-D-glucoside, amygdalin, b-gentiobiose, lactose,
melibiose, melezitose, raffinose, D-tagatose and gluconate
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:22:17
Printed in Great Britain
2383
H. Pang and others
Table 1. Phenotypic properties of strain CW 1 and closely
related Lactobacillus species
cysteine arylamidase, phosphoamidase, b-glucosidase and
N-acetyl-b-glucosaminidase.
Strains: 1, L. paralimentarius JCM 10415T; 2, L. kimchii JCM 10707T;
3, L. bobalius JCM 16180T; 4, L. alimentarius JCM 1095T; 5, L.
farciminis JCM 1097T. JCM, Japan Collection of Microorganisms. All
strains were Gram-positive, catalase-negative and homofermentative
rods that produce the DL form of lactic acid and could grow at 40 uC,
in 3.0 % NaCl and at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, but
not at 5 uC. These strains produced acid from galactose, D-glucose, Dfructose, D-mannose, N-acetylglucosamine, aesculin, salicin, cellobiose, maltose, sucrose and trehalose, but failed to produce acid from
glycerol, erythritol, D-arabinose, L-xylose, b-methyl-xyloside, Lsorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, a-methylD-mannoside, inulin, glycogen, xylitol, turanose, D-lyxose, D-fucose,
L-fucose, D-arabitol, L-arabitol and 5-keto-gluconate. All strains have
enzymic activity with leucine arylamidase, valine arylamidase, cysteine
arylamidase, phosphoamidase and b-glucosidase. No strains had
enzymic activity with alkaline phosphatase, esterase (C4), esterase
lipase (C8), lipase (C14), trypsin and b-glucuronidase. +, Growth; –,
no growth; w, weak growth.
The optical activity and quantity of lactic acid were
determined by using an enzymic bioanalysis and food
analysis kit, in accordance with the manufacturer’s
instructions (R-Biopharm). Strain CW1 produced both
the D and L forms of lactic acid (75.6 and 24.4 %,
respectively), as did L. paralimentarius (19.7 and 80.3 %),
L. kimchii JCM 10707T (20.0 and 80.0 %) and L. bobalius
JCM 16180T (78.2 and 21.8 %). Cai et al. (1999) and
Mañes-Lázaro et al. (2008) also reported that L.
paralimentarius and L. bobalius produced the D and L
forms of lactic acid.
Characteristic
Growth at:
10 uC
45 uC
Growth in NaCl:
6.5 %
Growth at pH:
3.0
3.5
Acid from:
L-Arabinose
Ribose
D-Xylose
Galactose
a-Methyl-D-glucoside
Amygdalin
Arbutin
Lactose
Melibiose
Melezitose
Raffinose
Starch
b-Gentiobiose
D-Tagatose
Gluconate
2-keto-gluconate
Enzymic activity:
Cystine arylamidase
N-acetyl-b-glucosaminidase
1
2
3
4
5
6
w
–
–
w
–
–
–
w
–
w
–
–
–
+
–
+
+ +
–
+
w
+
w
+
–
w
–
w
–
w
–
+
–
–
–
+
+
–
–
+
–
–
+
–
–
–
–
+
–
–
–
+
+
–
–
–
–
–
+
–
–
–
+
+
+
w
–
+
+
–
–
+
–
w
+
–
+
–
–
+
+
+
–
+
+
+
+
+
+
–
+
–
+
w
–
+
+
+
–
+
+
+
+
+
+
–
+
–
+
w
–
–
–
+
+
–
w
+
–
–
–
–
–
+
–
–
+
+
–
+
+
+
w
–
w
–
w
–
(Table 1). The enzymic activities of API ZYM were
investigated by using the bioMérieux system, according
to the manufacturer’s instructions. CW 1, L. paralimentarius JCM 10415T and L. kimchii JCM 10707T were each
positive for leucine arylamidase, valine arylamidase,
2384
The automated RiboPrinter microbial characterization
system (Qualicon) was used for ribotyping, in accordance
with the manufacturer’s instructions, with EcoRI as a
restriction enzyme. Ribopatterns were analysed by using
BioNumerics version 2.5 software (Applied Maths) and
compared by Pearson similarity coefficient analysis and
the unweighted pair group method, using the arithmetic
average (UPGMA) algorithm (Cai et al., 2011). Ribotyping
was applied to investigate the relationships among CW
1, L. paralimentarius JCM 10415T, L. kimchii JCM 10707T,
L. alimentarius JCM 1095T and L. farciminis JCM 1097T
(Fig. 1). Strain CW 1 formed a distinct cluster with L.
paralimentarius JCM 10415T and L. kimchii JCM 10707T,
supported by similar ribotyping patterns, which were wellseparated from L. alimentarius JCM 1095T and L. farciminis
JCM 1097T. In addition, strain CW 1, L. paralimentarius
and L. kimchii fell into a distinct cluster, as they did in the
phylogenetic tree (Fig. 2) based on 16S rRNA gene
sequences, and so these data are consistent with the
sequence similarity analysis.
Amplification, purification and sequencing of the 16S
rRNA gene of strain CW 1 was performed as described by
Cai (1999). High 16S rRNA gene sequence similarities to L.
paralimentarius JCM 10415T (99.5 %), L. bobalius JCM
16180T (99.6 %) and L. kimchii JCM 10707T (99.7 %) were
detected. In addition, the overall level of sequence
similarity of L. paralimentarius JCM 10415T to L. kimchii
JCM 10707T and L. bobalius JCM 16180T was 99.9 % (1517
of 1519 bases) and 99.5 % (1488 of 1519 bases),
respectively. On the basis of the 16S rRNA gene sequences,
a phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). This analysis
placed strain CW 1 in the same cluster as L. kimchii, L.
bobalius and L. paralimentarius, and confirmed its equally
close relationship to each of these species.
As shown in Figs. 2, S1 and S2 (available in IJSEM Online),
the cluster including Lactobacillus crustorum, Lactobacillus
mindensis, Lactobacillus nantensis and Lactobacillus
versmoldensis showed 16S rRNA gene sequence similarities
with L. paralimentarius JCM 10415T of ,98.3 %. As for the
further separated species L. bobalius, it showed a high 16S
rRNA gene sequence similarity with L. paralimentarius
JCM 10415T (99.5 %) and L. kimchii JCM 10707T (99.1 %).
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:22:17
L. kimchii and L. bobalius are synonyms of L. paralimentarius
Fig. 1. A dendrogram illustrating the relatedness of the ribotyping patterns of strain CW 1, L. paralimentarius JCM 10415T, L.
kimchii JCM 10707T, L. alimentarius JCM 1095T, L. farciminis JCM 1097T and L. manihotivorans JCM 12514T. The
dendrogram was analysed by using Pearson’s similarity coefficient analysis and UPGMA algorithm.
Mañes-Lázaro et al. (2008) reported that the type strain of
L. bobalius differed from L. paralimentarius JCM 10415T in
terms of its ability to ferment lactose, melibiose and
gluconate, but showed no N-acetyl-b-glucosaminidase
activity, and, unlike L. kimchii JCM 10707T, L. bobalius
JCM 16180T was able to ferment lactose and melibiose, but
not L-arabinose and D-xylose, and showed no cysteine
arylamidase activity. Moreover, DNA–DNA hybridization
Fig. 2. Phylogenetic tree showing the relative positions of strain CW 1 and related Lactobacillus species as inferred by using
the neighbour-joining method of complete 16S rRNA gene sequences. Bootstrap values for a total of 1000 replicates are
shown at the nodes of the tree. Bacillus subtilis is used as an outgroup. The bar indicates 1 % sequence divergence. Knuc,
Nucleotide substitution rates.
http://ijs.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:22:17
2385
H. Pang and others
Table 2. DNA base composition and DNA relatedness among strain CW 1 and phylogenetically closely related Lactobacillus species
Values are means of three tests. Strains: 1, L. paralimentarius JCM 10415T; 2, L. kimchii JCM 10707T; 3, L. bobalius JCM 16180T; 4, CW 1; L.
alimentarius JCM 1095T; 6, L. farciminis JCM 1097T.
Strain
1
2
3
4
5
6
DNA G+C content (mol%)
37.3
37.4
36.8
37.7
37.0
36.4
using L. bobalius JCM 16180T and the type strains of L.
paralimentarius and L. kimchii showed the highest DNA–
DNA relatedness (64.2 %) (Mañes-Lázaro et al., 2008).
To clarify the taxonomic status of strain CW 1, a DNA–
DNA relatedness assay was performed using CW 1, L.
paralimentarius JCM 10415T, L. kimchii JCM 10707T, L.
bobalius JCM 16180T, L. alimentarius JCM 1095T and L.
farciminis JCM 1097T. Furthermore, their DNA G+C
content was again determined. For the DNA base
composition and DNA–DNA relatedness analyses, the
DNA was extracted from cells harvested from MRS broth
culture after incubation for 8 h at 30 uC. DNA was then
purified by following the procedure of Saito & Miura
(1963), and base composition was determined by using the
method of Tamaoka & Komagata (1984) by HPLC
following enzymic digestion of DNA into deoxyribonucleotides. An equimolar mixture of four deoxyribonucleotides in a GC kit (Yamasa Shoyu) was used as the
quantitative standard. DNA–DNA relatedness was determined by the method of Ezaki et al. (1989), using
photobiotin and microplates. The DNA base composition
and DNA–DNA relatedness data are shown in Table 2.
Strains CW 1, L. paralimentarius JCM 10415T, L. bobalius
JCM 16180T and L. kimchii JCM 10707T had G+C
contents of 37.7, 37.3, 36.8 and 37.4 mol%, respectively.
The data are within the range of 35.0–38.0 mol% G+C
content for L. paralimentarius. This is in agreement with
the results of our previous investigation (Cai et al., 1999),
as well as those of Yoon et al. (2000) and Mañes-Lázaro
et al. (2008).
Yoon et al. (2000) also reported that the closest
phylogenetic relatives of L. kimchii are L. alimentarius
and L. farciminis, with 16S rRNA gene sequence similarities
of 98.4 and 98.2 %, respectively. In addition, their levels of
DNA–DNA relatedness were less than 11 %, indicating that
L. kimchii is a different species from L. alimentarius and L.
farciminis. However, comparative analyses of chemotaxonomic and morphological properties, 16S rRNA gene
similarity, and DNA–DNA relatedness between L.
paralimentarius JCM 10415T and L. kimchii JCM 10707T
2386
DNA–DNA reassociation (%) with:
1
2
3
4
100
82
82
96
17
16
87
100
85
95
19
16
86
84
100
91
15
10
97
83
90
100
16
17
were not carried out when the new species L. kimchii JCM
10707T was proposed by Yoon et al. (2000). In the present
study, strain CW 1, L. paralimentarius JCM 10415T and L.
kimchii JCM 10707T had a high DNA–DNA relatedness of
82.0–97.0 % and constituted a single taxonomic unit, as
currently delineated (Rosselló-Mora & Amann, 2001;
Stackebrandt & Goebel, 1994); they also had low DNA–
DNA relatedness (¡19 %) against other relevant type
strains of previously described species. In addition, we
confirmed the high DNA–DNA relatedness (¢82 %)
between L. paralimentarius JCM 10415T, L. bobalius JCM
16180T and L. kimchii JCM 10707T.
On the basis of the evidence presented here, although strain
CW 1 and the type species of L. paralimentarius, L. bobalius
and L. kimchii were isolated from quite different environments (silage, grape must, sourdough and kimchii) and
from geographically separate locations (China, Japan,
Spain and Korea), we propose that the three species L.
paralimentarius, L. bobalius and L. kimchii should be united
under the same name. According to the rules of priority
(rules 38 and 42 of the Bacteriological Code; Lapage et al.,
1992), the name L. paralimentarius should be retained,
together with its type strain TB 1T (5CCUG 43349T5CIP
106794T5DSM 13238T5JCM 10415T5LMG 19152T).
Consequently, strains of L. kimchii and L. bobalius should
be reclassified as belonging to L. paralimentarius, and strain
CW 1 should be identified as L. paralimentarius. Due to the
very similar traits reported in the descriptions of the two
species, an amended description of L. paralimentarius is
considered unnecessary.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (grant no. 41101244), by the Application of Nuclear
Techniques in Agriculture (grant 200803034), by the Chinese Ministry
of Agriculture, the Super Wheat Breeding and Demonstration (grant
081100110500), by the Henan Province and the State Scholarship Fund
(2009704013), by the China Scholarship Council, and the Project of
Exploration, and the Introduction of Microbial Genetic Resources
in Foreign Countries from National Institute of Agro biological Science
of Japan.
Downloaded from www.microbiologyresearch.org by
International Journal of Systematic and Evolutionary Microbiology 62
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:22:17
L. kimchii and L. bobalius are synonyms of L. paralimentarius
References
Mañes-Lázaro, R., Ferrer, S., Rodas, A. M., Urdiain, M. & Pardo, I.
(2008). Lactobacillus bobalius sp. nov., a lactic acid bacterium isolated
Cai, Y. (1999). Identification and characterization of Enterococcus
from Spanish Bobal grape must. Int J Syst Evol Microbiol 58, 2699–2703.
species isolated from forage crops and their influence on silage
fermentation. J Dairy Sci 82, 2466–2471.
Rosselló-Mora, R. & Amann, R. (2001). The species concept for
Cai, Y., Okada, H., Mori, H., Benno, Y. & Nakase, T. (1999).
Saito, H. & Miura, K. I. (1963). Preparation of transforming
Lactobacillus paralimentarius sp. nov., isolated from sourdough. Int
J Syst Bacteriol 49, 1451–1455.
deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta
72, 619–629.
Cai, Y., Yang, J., Pang, H. & Kitahara, M. (2011). Lactococcus fujiensis
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
sp. nov., a lactic acid bacterium isolated from vegetable matter. Int J
Syst Evol Microbiol 61, 1590–1594.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
prokaryotes. FEMS Microbiol Rev 25, 39–67.
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
DNA–DNA reassociation and 16S rRNA sequence analysis in the present
species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in
which radioisotopes are used to determine genetic relatedness among
bacterial strains. Int J Syst Bacteriol 39, 224–229.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
Lapage, S. P., Sneath, P. H. A., Lessel, E. F., Skerman, V. B. D.,
Seeliger, H. P. R. & Clark, W. A. (1992). International Code of
Yoon, J. H., Kang, S. S., Mheen, T. I., Ahn, J. S., Lee, H. J., Kim, T. K.,
Park, C. S., Kho, Y. H., Kang, K. H. & Park, Y. H. (2000). Lactobacillus
Nomenclature of Bacteria (1990 Revision): Bacteriological Code.
Washington, DC: American Society for Microbiology.
kimchii sp. nov., a new species from kimchi. Int J Syst Evol Microbiol
50, 1789–1795.
http://ijs.sgmjournals.org
composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 14:22:17
2387