RESEARCH LETTER
A genus-speci¢c PCR method for di¡erentiation between
Leuconostoc and Weissella and its application in identi¢cation of
heterofermentative lactic acid bacteria from co¡ee fermentation
Ulrich Schillinger1, Benjamin Boehringer1, Sabrina Wallbaum1, Lily Caroline1, Almaz Gonfa2,
Melanie Huch (née Kostinek)1, Wilhelm H. Holzapfel1 & Charles M.A.P. Franz1
1
Federal Research Centre for Nutrition and Foods, Institute of Hygiene and Toxicology, Karlsruhe, Germany; and 2Ethiopian Health and Nutrition
Research Institute, Addis Ababa, Ethiopia
Correspondence: Ulrich Schillinger, Institut
für Mikrobiologie und Biotechnologie, Max
Rubner Institut, Bundesforschungsinstitut für
Ernährung und Lebensmittel, Haid- und
Neustr.9, 76131 Karlsruhe, Germany. Tel.:
149 721 6625 461; fax: 149 721 6625 453;
e-mail: [email protected]
Received 18 April 2008; accepted 19 June
2008.
First published online 24 July 2008.
DOI:10.1111/j.1574-6968.2008.01286.x
Editor: Wolfgang Kneifel
Keywords
Leuconostoc ; Weissella ; genus-specific PCR;
coffee fermentation.
Abstract
A genus-specific PCR analysis method was developed for a rapid and reliable
differentiation between the two heterofermentative lactic acid bacteria genera
Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA
genes were designed and the specificity of the PCR was evaluated using the type
strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the
newly developed genus-specific PCR analysis was applied to characterize 72 lactic
acid bacteria (LAB) strains isolated from coffee fermentation and which were
presumptively classified as Leuconostoc or Weissella species. Additionally, a total of
34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct
assignment to one of these two genera by the application of the genus-specific
primers was confirmed by further identifying the strains using repetitive extragenic
palindromic-PCR and 16S rRNA gene sequencing.
Introduction
Traditionally, the differentiation of the genera of lactic acid
bacteria (LAB) is based on phenotypic criteria such as cell
morphology, production of CO2 and the configuration of
the lactic acid produced from glucose fermentation. Among
the heterofermentative LAB, the genus Leuconostoc is generally classified on the basis of its ovoid cell appearance, the
absence of arginine deiminase and the production of predominantly D()-lactate. In addition, all leuconostocs have a
similar type of peptidoglycan, characterized by the
presence of alanine and/or serine in the interpeptide bridge,
which is not present in most other LAB genera. However, the
phylogenetically related genus Weissella contains species
sharing the same or similar properties (e.g. Weissella paramesenteroides, Weissella hellenica, Weissella thailandensis).
Therefore, the classical phenotypic criteria do not allow the
unequivocal allocation of a new isolate to either Leuconostoc
or Weissella. Several molecular techniques for identification
of Leuconostoc and Weissella species have been described
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c
such as multiplex PCR (Lee et al., 2000), ARDRA (Jang
et al., 2002, 2003), and restriction of internal spacer regionamplified fragments (Chenoll et al., 2003). Some of these
methods, however, are labour intensive and most studies did
not include all the known species of both genera. Therefore,
this investigation aimed to develop a rapid PCR method to
distinguish between Leuconostoc and Weissella. Furthermore, this genus-specific PCR analysis was applied and
verified using LAB field strains that were isolated mainly
from coffee fermentation from West Africa, as well as other
fermented foods.
Materials and methods
Bacterial strains and growth conditions
The following type strains of Leuconostoc, Weissella and
Lactobacillus species were used in this study: Lactobacillus
brevis DSM 20054, Lactobacillus fermentum DSM 20052,
FEMS Microbiol Lett 286 (2008) 222–226
223
PCR identification of Leuconostoc and Weissella
Lactobacillus malefermentans DSM 20177, Lactobacillus
reuteri DSM 20016, Leuconostoc carnosum DSM 5576,
Leuconostoc citreum DSM 5577, Leuconostoc durionis LMG
22556, Leuconostoc fallax DSM 20189, Leuconostoc ficulneum
DSM 13613, Leuconostoc fructosum LTH 471, Leuconostoc
gasicomitatum LMG 18811, Leuconostoc gelidum DSM 5578,
Leuconostoc inhae DSM 15101, Leuconostoc lactis DSM
20202, Leuconostoc mesenteroides ssp. cremoris DSM 20346,
L. mesenteroides ssp. dextranicum DSM 20484, L. mesenteroides ssp. mesenteroides DSM 20343, Leuconostoc pseudomesenteroides DSM 20193, Leuconostoc pseudoficulneum DSM
15468, Weissella cibaria LMG 17699, Weissella confusa DSM
20196, Weissella halotolerans DSM 20190, W. hellenica LTH
7378, Weissella kandleri LTH 1396, Weissella koreensis DSM
15830, Weissella minor DSM 20014, W. paramesenteroides
DSM 20288, Weissella soli DSM 14420, W. thailandensis
DSM 19821 and Weissella viridescens DSM 20410. Weissella
kimchii is a synonym of W. cibaria (Ennahar & Cai, 2004)
and therefore was not included. The type strain of the novel
Leuconostoc species, Leuconostoc holzapfelii (De Bruyne et al.,
2007), was among the environmental isolates investigated.
The other type strains were received from DSMZ (Deutsche
Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany), LTH (Institute of Food Technology, University
of Hohenheim, Stuttgart, Germany) or LMG Bacteria Collection
(Laboratorium voor Microbiologie, Ghent University, Belgium).
The origin of the 106 environmental isolates investigated in this
study is listed in Tables 1 and 2. Among them, 10 presumptive
Weissella strains isolated from meat products were obtained
from our Kulmbach BfEL sister institute. All strains were
cultivated in MRS broth at 30 1C and stock cultures were
maintained at 80 1C with 15% (v/v) glycerol added.
DNA extraction
Genomic DNA was extracted by the guanidium thiocyanate
method of Pitcher et al. (1989) as modified by Björkroth &
Korkeala (1996).
Table 1. Identification of Leuconostoc and Weissella strains isolated
from coffee fermentation
Total number
of isolates
Genus-specific
PCR
Species
identificationw
72
Leuconostoc
(60)
L. citreum (19)
L. holzapfelii (1)
L. mesenteroides (23)
L. pseudomesenteroides (17)
W. cibaria (11)
W. soli (1)
Weissella
(12)
Strains producing a 1200-bp PCR product with Leuconostoc- or
Weissella-specific primers.
w
Strains were identified to species level by rep-PCR and 16S rRNA gene
sequencing.
FEMS Microbiol Lett 286 (2008) 222–226
Table 2. Leuconostoc and Weissella strains isolated from various fermented foods and examined using the Leuconostoc- and Weissellaspecific primer pairs Leucgrp and Weissgrp
Species
No. of
strains
L. carnosum
3
L. fallax
2
L. lactis
1
L. mesenteroides
2
L. pseudomesenteroides 6
W. confusa
W. halotolerans
W. hellenica
3
2
3
W. paramesenteroides
2
W. thailandensis
W. viridescens
1
7
Origin
Meat products (2), Korean cabbage
kimchi (1)
Fermented bamboo (1), fermented
cassava (1)
Fermented bamboo, Sikkim/India
Korean cabbage kimchi
Maasai fermented milk, Kenya (3),
fermented cassava, Benin (2),
cheese, Sikkim/India (1)
Ogi (fermented maize), Nigeria
Fermented meat product
Fermented bamboo (1), Korean
cabbage kimchi (2)
Fermented cassava (1), meat
product (1)
Cheese, Sikkim/India
Meat products
All strains except those belonging to Weissella halotolerans and two of
the seven Weissella viridescens strains generated a PCR amplification
product of the expected size with the respective primer.
The two Lactobacillus brevis strains from meat products are not included
in this table.
Genus-specific PCR
The specific primers for characterization of the Leuconostoc
and Weissella genera were designed from 16S rRNA gene
sequences. The 16S rRNA gene sequences of phylogenetically related species were retrieved from GenBank and were
aligned using DNASTAR’s LASERGENE MEGALIGN module (version 7.0). For Leuconostoc, the sequence of the forward
primer, named Leucgrp (5 0 -GCG GCT GCG GCG TCA
CCT AG-3 0 ) was used. For Weissella, a primer named
Weissgrp (5 0 -GAT GGT TCT GCT ACC ACT AAG-3 0 ) was
custom designed. The reverse primer for both genera was 5 0 GGN TAC CTT GTT ACG ACT TC-3 0 . Amplifications were
carried out in a total volume of 50 mL with 100 ng chromosomal DNA, 200 mM dNTPs, 0.5 mM of each primer, 1 U Taq
DNA polymerase (Amersham Pharmacia, Freiburg,
Germany) and 5 mL 10 PCR buffer (Amersham Pharmacia).
PCR was conducted using a Primus 96 Plus thermal cycler
(Peqlab Biotechnologie GmbH, Erlangen, Germany) with
the following steps: one cycle of denaturation for 2 min at
94 1C and 33 cycles of denaturation at 94 1C for 1 min,
primer annealing at 53 1C for the Leucgrp primer and 50 1C
for the Weissgrp primer for 1 min, and extension at 72 1C for
1.5 min. PCR products were subjected to electrophoresis on
1.2% (w/v) agarose gels in 1 TBE buffer solution at 100 V
for 2 h. Gels were stained in ethidium bromide and gel
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224
U. Schillinger et al.
images were captured using the Fluorchem Imager 5500
system (Alpha Innotech).
Repetitive extragenic palindromic-PCR (rep-PCR)
with GTG5 primer and sequencing of the 16S
rRNA gene
The method of Gevers et al. (2001) was used. Gel electrophoresis and evaluation of the fingerprints obtained was
performed as described by Kostinek et al. (2005). The almost
complete 16S rRNA gene of selected strains was sequenced
as described previously (Kostinek et al., 2005).
Results and discussion
A Weissella- and a Leuconostoc-specific genus primer set was
designed using the alignment of 16S rRNA gene sequences of
Weissella and Leuconostoc species, as well as those of related
heterofermentative lactobacilli. The specificity of the
primers was examined in silico using the National Center
for Biotechnology’s BLAST MEGABLAST search, as well as the
ribosomal database project II probe search tool. In addition,
the specificity was assessed by PCR reactions using DNA
from the type strains of all Weissella and Leuconostoc species.
Furthermore, a number of heterofermentative Lactobacillus
species were included for comparison.
With the Weissella-specific primer, a c. 1200-bp PCR
product was obtained with the type strains of 11 of the 12
Weissella species, but not with Leuconostoc strains (Fig. 1)
and other nontarget bacteria such as the heterofermentative
L. brevis, L. fermentum, L. malefermentans and L. reuteri
(results not shown). Thus, among the Weissella species, only
the type strain of W. halotolerans did not generate an
amplification product in this PCR reaction (see Fig. 1 lane
5). The PCR amplification reaction with the Leuconostocspecific primer generated a unique DNA fragment of about
1200 bp with the type strains of all L. mesenteroides ssp.,
L. lactis, L. carnosum, L. citreum, L. gelidum, L. gasicomitatum, L. pseudomesenteroides, L. fallax and L. inhae (Fig. 2).
Very weak bands of similar size were observed with
L. fructosum, L. ficulneum and L. durionis, whereas no amplification product was detected with L. pseudoficulneum and all
Weissella species, as well as the Lactobacillus strains tested.
Phylogenetic trees based on 16S rRNA gene sequences
and several protein-coding genes used as phylogenetic
markers showed that L. fructosum, together with
L. ficulneum, L. pseudoficulneum and L. durionis, form a
subcluster within the genus Leuconostoc, known as the
L. fructosum group (Chambel et al., 2006; Chelo et al.,
2007). Phenotypically, this group differs from other leuconostocs in cell morphology. They typically form slender rods
and do not have the coccoid appearance of most leuconostocs. Moreover, they are characterized by the production of
only small amounts or no gas from glucose (Leisner et al.,
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c
Fig. 1. PCR amplification products from genus-specific PCR assays with
Weissella strains. M, molecular marker. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17
Weissella-specific primer (Weissgrp); lanes 2, 4, 6, 8, 10, 12, 14, 16, 18
Leuconostoc-specific primer (Leucgrp). (a) Lanes 1,2: Weissella cibaria
LMG 17699T; 3,4: Weissella confusa DSM 20196T; 5,6: Weissella
halotolerans DSM 20196T; 7,8: Weissella hellenica LTH 7378T; 9,10:
Weissella kandleri LTH 1396T; 11,12: Weissella paramesenteroides DSM
20288T; 13,14: Weissella koreensis DSM 15830T; 15,16: Weissella minor
DSM 20014T; 17,18: Weissella soli DSM 14420T. (b) Lanes 1,2: Weissella
thailandensis DSM 19821T; 3,4: Weissella viridescens DSM 20410T; 5,6:
W. confusa BFE 7851; 7,8: W. hellenica BFE 2942; 9,10: W. cibaria BFE
6989; 11,12: W. paramesenteroides BFE 6649; 13,14: W. thailandensis
BFE 1637; 15,16: W. paramesenteroides Lb 637; 17,18: W. viridescens Lb
981.
2005). They prefer fructose as carbon source and vigorous
gas production is observed when glucose is replaced by
fructose. Therefore, this group can be easily separated from
the ‘classical’ leuconostocs characterized by ovoid cell morphology, a strong gas production from glucose and also by
the absence of a PCR amplification product with both the
Weissgrp and Leucgrp primers in the genus-specific PCR
reported here. Indeed the 16S rRNA gene sequence to which
the Leucgrp primer was targeted was considerably more
diverse in this region for L. fructosum, L. durionis,
L. pseudoficulneum and L. ficulneum than the corresponding
sequences of all the other Leuconostoc species for whom
the Leucgrp primer was developed. This could explain the
absence of a PCR amplification product for the L. fructosum,
L. ficulneum, L. pseudoficulneum and L. durionis strains.
In the second part of the study, the applicability and
specificity of the primer pairs designed for discrimination
between Leuconostoc and Weissella were evaluated using 72
environmental isolates from coffee fermentation (Table 1),
and 34 additional strains isolated from various other
FEMS Microbiol Lett 286 (2008) 222–226
225
PCR identification of Leuconostoc and Weissella
Fig. 2. PCR amplification products from genus-specific PCR assays with
Leuconostoc strains. M, molecular marker. Lanes 1, 3, 5, 7, 9, 11, 13, 15,
17 Weissella-specific primer (Weissgrp); lanes 2, 4, 6, 8, 10, 12, 14, 16,
18 Leuconostoc-specific primer (Leucgrp); (a) Lanes 1,2: Leuconostoc
carnosum DSM 5576T; 3,4: Leuconostoc citreum DSM 5576T;5,6: Leuconostoc. fallax DSM 20189T; 7,8: Leuconostoc gasicomitatum LMG
18811T; 9,10: Leuconostoc gelidum DSM 5578T; 11,12: Leuconostoc
inhae DSM 15101T; 13,14: Leuconostoc lactis DSM 20202T; 15,16:
Leuconostoc mesenteroides ssp. cremoris DSM 20346T; 17,18: L. mesenteroides ssp. dextranicum DSM 20484T. (b) Lanes: 1,2: L. mesenteroides ssp. mesenteroides DSM 20343T; 3,4: Leuconostoc pseudomesenteroides DSM 20193T; 5,6: Leuconostoc durionis LMG 22556T;
7,8: L. ficulneum DSM 13613T; 9,10: Leuconostoc fructosum LTH 471T;
11,12: Leuconostoc pseudoficulneum DSM 15468T; 13,14: L. citreum
BFE 6854; 15,16: L. fallax BFE 6660; 17,18: L. pseudomesenteroides
BFE 5028.
fermented foods including kimchi, gari, ogi, fermented
bamboo and several meat products (Table 2). These were
presumptively classified by phenotypic properties as belonging to Leuconostoc or Weissella. This part of the study was
not intended as a complete diversity investigation of the
LAB involved in these fermentations, but rather in this study
the effectiveness of the genus-specific primers as identification tools was assessed, using isolates obtained from foods.
A total of 74 of 106 strains generated an amplification
product of about 1200 bp with the Leucgrp primer, but not
with the Weissgrp primer, indicating that these belong to the
genus Leuconostoc. Three of these (L. citreum BFE 6854,
L. fallax BFE 6660 and L. pseudomesenteroides BFE 5028)
are included in Fig. 2. The coffee isolates classified as
Leuconostoc by this PCR involved strain BFE 7000 (LMG
23990), which was characterized and described as a new
species, L. holzapfelii, during a previous study (De Bruyne
et al., 2007). On the other hand, 26 strains showed a 1200-bp
FEMS Microbiol Lett 286 (2008) 222–226
amplification product with the Weissella-specific primer,
and no PCR products were detected with Leucgrp primer.
Seven of these are included in Fig. 1 (W. confusa BFE 7851,
W. hellenica BFE 2942, W. cibaria BFE 6989, W. paramesenteroides BFE 6649, Lb 637, W. thailandensis BFE 1637 and
W. viridescens Lb 981). To confirm the genus-specific PCR
method’s allocation of the 74 strains to Leuconostoc and of
the 26 strains to Weissella, all of them were further identified
to species level by rep-PCR genotyping with (GTG)5 primer,
previously shown to be a powerful tool for identification of
Leuconostoc and other LAB (Gevers et al., 2001; Kostinek
et al., 2005; Tamang et al., 2005; Franz et al., 2006; Nielsen
et al., 2007). The dendrograms obtained as a result of the
comparison of the fingerprints with reference strains (not
shown) allowed the allocation of the environmental isolates
to Leuconostoc or Weissella species (Tables 1 and 2). In
addition, representatives of the clusters obtained by repPCR were analysed using 16S rRNA gene sequencing to
confirm the identification. According to the results of these
two molecular methods, 71 strains isolated from different
stages of coffee fermentation from Ethiopia and Tanzania
belonged to the species L. citreum, L. mesenteroides,
L. pseudomesenteroides, W. cibaria and W. soli, and one strain
consisted of the novel species L. holzapfelii (Table 1). This
confirmed the accuracy of the genus-specific PCR to correctly allocate these strains to the genus Leuconostoc or
Weissella. Moreover this investigation, which served to
demonstrate the effectiveness of the genus-specific PCR,
also yielded further information on the LAB involved in
coffee fermentation. Thus, L. mesenteroides was already
reported to be a part of the LAB population of coffee, as well
as Lactobacillus plantarum and Lactobacillus brevis (Avallone
et al., 2001). However, the predominant occurrence of other
Leuconostoc species and of W. cibaria and W. soli in the coffee
fermentation is a new finding and has not yet been reported.
Further identification results of the strains from other fermented products are given in Table 2.
With only six out of the 106 investigated strains, no
amplification products were observed in the PCR reaction
with the two primer sets. Two of these six strains had been
misclassified as Leuconostoc/Weissella as rep-PCR and 16S
rRNA gene sequencing resulted in their identification as
L. brevis. This actually again confirms the specificity of the
primers for Leuconostoc and Weissella 16S rRNA gene
sequences. Two strains isolated from meat products shared
a highly similar rep-PCR fingerprint and grouped closely
with W. halotolerans type strain in the resulting dendrogram
(results not shown). The 16S rRNA gene sequencing of one
of the two strains clearly showed that it belonged to
W. halotolerans, and this confirms our observation that with
the reference strain of this species, a PCR product with the
Weissgrp primer could not be generated. Nevertheless, the
absence of a genus-specific PCR amplicon in this case also
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c
226
has value in characterization of Weissella strains when
considered together with the phenotypic data and Leuconostoc-specific PCR data, which indicate that the strain belongs
to Weissella. The remaining two strains were identified as
W. viridescens by rep-PCR and 16S rRNA gene sequencing.
However, attempts to demonstrate a PCR product with the
Weissella-specific primer were not successful, indicating that
for some very few strains possible differences in 16S rRNA
gene nucleotide sequence may lead to no amplification
signals, thus requiring further investigation.
In conclusion, the specificity of both primer pairs was
verified using 106 environmental isolates. All (100%) of
the Leuconostoc isolates could be correctly classified as
Leuconostoc with the specific primer designed for this genus.
The PCR reactions performed with the primer designed for
Weissella enabled the correct allocation of most, but not all
Weissella strains to this genus. Nevertheless, the applicability of
the two primer sets designed for Leuconostoc and Weissella for a
rapid discrimination of the two genera was demonstrated and
could be useful in biodiversity investigations for a relatively
rapid and accurate initial identification of these strains.
Acknowledgements
The authors would like to thank Mrs Simone Britsch for
excellent technical assistance. Thanks are also due to
Dr Lothar Kröckel for providing presumptive Weissella
strains isolated from meat products. The study was partly
accomplished within the framework of the INCO RTD
programme of the EU (Project ICA4-CT-2001-10060: An
integrated approach to prevent ochratoxin A contamination
in postharvest processing of coffee in East Africa).
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