Letters in Applied Microbiology 2001, 32, 215219

Temporal temperature gradient gel electrophoresis (TTGE) as

a tool for identication of Lactobacillus casei, Lactobacillus
paracasei, Lactobacillus zeae and Lactobacillus rhamnosus

A. Vasquez1, S. Ahrne1, B. Pettersson2 and G. Molin1

1
Laboratory of Food Hygiene, Division of Food Technology, Lund University, Lund, and 2Department of
Biotechnology, Royal Institute of Technology, Stockholm, Sweden

291/00: received 16 August, revised 18 December and accepted 5 January 2001

A . V A S Q U E Z , S . A H R N E , B . P E T T E R S S O N A N D G . M O L I N . 2001.
Aims: To develop a tool for rapid and inexpensive identication of the Lactobacillus casei
complex.
Methods and Results: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and
Lactobacillus rhamnosus were identied by PCR-amplication of the segment between the U1
and U2 regions of 16S rDNA (position 8357, Escherichia coli numbering) and temporal
temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were
divided into three TTGE-subgroups.
Conclusions: TTGE successfully distinguished between the closely-related target species.
TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA
genes.
Signicance and Impact of the Study: Due to rapid and easy performance, TTGE of PCR-
amplied 16S rDNA fragments will be useful for the identication of extended numbers of
isolates.

Lact. casei, except Lact. casei senso stricto, should be

INTRODUCTION
Lactobacillus paracasei and Lactobacillus rhamnosus constitute
a substantial part of the Lactobacillus ora of the human
intestinal mucosa (Molin et al. 1993), and they can
occasionally be isolated from the blood of diseased individ-
uals (Sussman et al. 1986). Furthermore, they are found in
high numbers in dairy products such as cheese (Hull et al.
1992; Lindberg et al. 1996). Strains of the Lactobacillus casei
complex are often used as probiotics and are included in
yoghurt-like products (Saxelin et al. 1993; Ahrne et al.
1995) which are claimed to inuence the health of the host in
a benecial manner (Siitonen et al. 1990).
The systematics of the Lact. casei complex is under
discussion. There are differences in opinion as to whether
Lact. casei should remain as a heterogeneous species
divided into different subspecies (Dellaglio et al. 1991;
Dicks et al. 1996), or whether all the former subspecies of
Correspondence to: Dr Siv Ahrne, Laboratory of Food Hygiene, Food
Technology, PO Box 124, SE-22100 Lund, Sweden
(e-mail: siv.ahrne@livsteki.lth.se).
included in Lact. paracasei (Collins et al. 1989). Neverthe-
less, there is a need for rapid and inexpensive genomic
identication tools to distinguish between Lact. casei, Lact.
paracasei, Lact. zeae, and Lact. rhamnosus, especially when
dealing with large numbers of isolates. As phenotypic
characterization is problematic for the closely-related
species of the Lact. casei complex (Lindberg et al. 1996),
several workers have tried, without success, to develop
genetically-based methods to differentiate this group at the
species level (Tannock et al. 1999; Tynkkynen et al. 1999;
Walter et al. 2000). Ward and Timmins (1999) were able to
differentiate Lact. casei, Lact. paracasei and Lact. rhamnosus
by polymerase chain reaction, but the results were difcult
to interpret because the distance between the three bands
was hardly distinguishable. Furthermore, their approach
did not include Lact. zeae.
Variations in 16S ribosomal DNA (rDNA) sequences
among the type strains of Lact. casei, Lact. zeae, Lact.
paracasei and Lact. rhamnosus are situated between positions
69 and 100 (Escherichia coli numbering) (Mori et al. 1997),
which makes this region suitable for differentiation.
Electrophoretic methods, such as denaturing gradient gel

2001 The Society for Applied Microbiology

216 A . V A S Q U E Z E T A L .

electrophoresis (DGGE) (Myers et al. 1987) and tempera-
ture gradient gel electrophoresis (TGGE) (Wartell et al.
1990), allow the separation of DNA fragments of the same
length but with different base-pair sequences. Temporal
temperature gradient gel electrophoresis (TTGE) was rst
introduced by Yoshino et al. (1991) as a modication of
TGGE. The temperature of a gel plate in TTGE increases
gradually and uniformly with time, which makes it easier to
modulate the temperature over time. This provides an
increased sensitivity as the separation range expands.
DGGE, TGGE and TTGE have been employed primarily
to screen for mutations in a variety of genes (Wartell et al.
1998), or to determine the genetic diversity of complex
microbial populations (Muyzer et al. 1993; Heuer et al.
1997; Muyzer 1999). Moreover, TGGE was applied as a
tool in bacterial taxonomy (Felske et al. 1999). This paper
demonstrates how TTGE of PCR-amplied 16S rDNA
fragments could be used to differentiate between Lact. casei,
Lact. paracasei, Lact. zeae and Lact. rhamnosus.
M A T E R I A LS A N D M E T H O D S
Bacterial strains
The test strains, including all representative type strains, are
listed in Table 1. The strains were grown overnight in
Lactobacillus Carrying Medium broth (Efthymiou and
Hansen 1962) at 37C.
The cells of the pure cultures were washed twice in 1 ml
sterile Milli-Q water, and the chromosomal DNA was
recovered by disrupting the cells in an Eppendorf tube with
glass beads (2 mm) in an Eppendorf Mixer 5432 (Eppen-
dorf, Hamburg, Germany). The disrupted cells were
pelleted by centrifugation at 20 817 g and 1 ll of the
supernate was used in the PCR.
Polymerase chain reaction
The segment between the U1 and U2 regions of the 16S
rRNA gene (position 8357 E. coli numbering) was PCR-
amplied (Saiki et al. 1985) with primers to conserved
regions: ENV1 (5-AGAGTTTGATIITGGCTCAG-3)
and TTGE7-gc adapted from Muyzer et al. (1993)
(5-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGG-
GCACGGGGGGCTGCTGCCTCCCGTAGG-3). PCR
amplication was performed in a DNA Thermal cycler
(Perkin-Elmer, Norwalk, CT, USA). The reaction mixture
(50 ll) consisted of 1 ll cell lysate, 25 pmol of each primer,
25 mmol l1 MgCl2, 02 mmol l1 PCR Nucleotide Mix
(Boehringer Mannheim), 5 ll 10 PCR buffer [100 mmol l1
TRIS-HCl (pH 83), 500 mmol l1 KCl], and 125 U of

Table 1 Lactobacillus (Lact.) strains

Species designation by TTGE
subjected to temporal temperature gradient
Lable and strain number Source 16S rDNA-sequencing group gel electrophoresis (TTGE); the source and
Lact. rhamnosus CCUG 21452T CCUG* Lact. rhamnosus 1 identity are according to partial 16S rDNA
Lact. rhamnosus CCUG 34682 CCUG* Lact. rhamnosus 1 sequencing
Lact. rhamnosus CCUG 27772 CCUG* Lact. rhamnosus 1
Lact. rhamnosus ATCC 53103 ATCC Lact. rhamnosus 1
Lact. rhamnosus 7D HIM Lact. rhamnosus 1
Lact. rhamnosus 49 A HIM Lact. rhamnosus 1
Lact. zeae ATCC 15820T ATCC Lact. zeae 2
Lact. casei ATCC 393T ATCC Lact. casei 3
Lact. casei NCFB 173 NCFB Lact. casei 3
Lact. paracasei subsp. paracasei NCFB 151T NCFB Lact. paracasei 4a
Lact. paracasei 240HI HIM Lact. paracasei 4a
Lact. casei ATCC 4646 ATCC Lact. paracasei 4b
Lact. paracasei subsp. tolerans DSM 20258T DSM** Lact. paracasei 4c
Lact.casei subsp. pseudoplantarum DSM 20008 DSM** Lact. paracasei 4c
Lact. casei ATCC 334 ATCC Lact. paracasei 4c
Lact. paracasei subsp. paracasei CCUG 31151 CCUG* Lact. paracasei 4c

* Culture Collection of the University of Gothenburg, Gothenburg, Sweden.

American Type Culture Collection, Rockville, MD, USA.
Human intestinal mucosa: strains 7D and 49 A (Ahrne et al. 1998); strain 240HI (Molin et al.
1993).
National Collection of Food Bacteria, Reading, UK.
The partial sequence of Lact. casei ATCC 4646 differed by one nucleotide compared with strains
NCFB 151T, 240HI, DSM 20258T, DSM 20008, ATCC 334, and CCUG 31151.
** Deutsche Sammlung von Mikroorganismen und Zellkulturen Braunschweig, Germany.

2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 215219
 IDENTIFICATION OF THE LACT. CASEI-COMPLEX BY TTGE 217

AmpliTaq gold DNA polymerase (Perkin Elmer). The
amplication programme was 94C for 10 min; 30 cycles of
94C for 30 s, 56C for 30 s, 72C for 60 s and nally,
72C for 10 min.
Temporal temperature gradient gel
electrophoresis
TTGE was done with a DCode universal mutation detection
system (Bio-Rad, Hercules, CA, USA) in a 075 mm
polyacrylamide gel [7% (w/v) Acrylamide/Bis (375:1),
7 mol l1 urea] with 125 Tris-acetate-EDTA buffer (Bio-
Rad) at a constant voltage of 90 V. The temperature was
raised continuously at a rate of 15C h1 from 61 to 70C
during the run. The gel was stained with ethidium bromide
solution (3 lg ml1 for 8 min), washed for 30 min in water,
visualized at 302 nm in a u.v. transilluminator (UVP Inc.,
San Gabriel, CA, USA) and photographed. The method
exhibited good reproducibility when comparing the TTGE
signals in different runs.
All strains were identied by partial sequencing of the 16S
rRNA gene (Tables 1 and 2). The target was the rst 350
nucleotides of the 5-end of the gene; primers and experi-
mental conditions used for amplication by PCR and
subsequent sequence determination have been described
previously (Pettersson et al. 1996).
RESULTS AND DISCUSSION
The test strains were divided into four major groups as
judged from the TTGE proles (groups 14, Table 1). Each
group demonstrated bands in different positions, with one
main TTGE band supplemented with a side-product
pattern type (Fig. 1). The rst group consisted of the Lact.
rhamnosus strains, with one uniform band pattern (Fig. 1,
lanes 13 and 56) with a slight deviation of ATCC 53103
(Fig. 1, lane 4). Sequence data conrmed that the 16S
rDNA sequences in the actual region were identical for the
six isolates (Table 2). Lactobacillus zeae ATCC 15820T
made up a one-member group with a unique band pattern
(Fig. 1, lane 7) and 16S rDNA sequence (Table 2). A third
group consisted of Lact. casei ATCC 393T and Lact. casei
NCFB 173, with identical band patterns (Fig. 1, lanes 8
and 9) and 16S rDNA sequences (Table 2). The different
TTGE proles show the divergence between Lact. zeae and
Lact. casei, in disagreement with Dicks et al. (1996) who
proposed that Lact. casei ATCC 393 T should be reclassied
as Lact. zeae.
The fourth group included, among others, the type strains
of Lact. paracasei subsp. paracasei and Lact. paracasei subsp.
tolerans, together with the type strain of the former Lact.
casei subsp. pseudoplantarum (Table 1). Group 4 was divided
into three subgroups exhibiting three different band patterns
but with the major TTGE band in common (Fig. 1, lanes
1016). Three strains, Lact. paracasei subsp. paracasei
NCFB 151T, 240HI and ATCC 4646 displayed two parallel
bands. The gene sequences of strains 240HI and NCFB
151T were identical (Table 2), as were the positions of the
double bands in the gel (Fig. 1, lanes 11 and 12). Strain
ATCC 4646, however, differed both in the 16S rDNA
sequence by one nucleotide (Table 2) and in the position of
its double bands (Fig. 1, lane 13). The remaining strains
(subcluster 4c, Fig. 1, lanes 10 and 1416) exhibited a single
clear band and a uniform gene sequence (Table 2).
Pure cultures should theoretically produce a single band
in TTGE analysis. However, all the groups formed a
specic side-product pattern type that was the same for all
the strains of a specic group. Felske et al. (1999) previously
observed the occurrence of multi-band gels. The extra bands
did not disappear by dilution, or with DNA puried by
alcohol extraction, and could be due either to sequence-
specic DNA polymerization artefacts, or to mutations in
one of the rrn operons. Whether the species in the Lact. casei
complex possess different rrn operons remains to be
determined.
The distinct double band pattern derived from Lact.
paracasei strains NCFB 151T and 240HI, and Lact. casei
strain ATCC 4646, may indicate heterogeneity of 16S rRNA
genes in the genome. Sequence heterogeneities of genes
encoding 16S rRNAs have previously been detected by

Table 2 Sequence differences between TTGE groups revealed by partial sequencing of the 16S rRNA gene

Nucleotide at position*
TTGE
group 73 75 76 78 79 80 84 85 101 102 103 106 114 115 117 118 120 121 208 246 300 314

1 C G A T A T A A T C T T T A T T T G A T C A
2 T G G C G A A C Y Y G T C A C T A A A T C C
3 T G G C G A A C C T G A C G C T A A A C C G
4a C C G T G A T C C C G A C A C A G G G T T G
4b C C G T G A T C C C G A C A C A G G G T C G
4c C C G T G A T C C C G A C A C A G G G T T G

* Position from the 5-end of the 16S rRNA sequence of Lact. casei.

2001 The Society for Applied Microbiology, Letters in Applied Microbiology, 32, 215219
218 A . V A S Q U E Z E T A L .
TGGE (Nubel et al. 1996) but never before in Lactobacillus.
The extent of sequence polymorphisms within a species, or
even within the same strain, must receive attention as the
heterogeneities may inuence the interpretation of the
sequence data and thereby interfere with classication.
The present TTGE method successfully distinguished
between the closely-related target species. Furthermore, due
to its rapid and easy performance, the method is useful for
identication of extended numbers of isolates. TTGE is also
a powerful method for revealing sequence heterogeneities in
the 16S rRNA genes.
ACKNOWLEDGEMENTS
B.P. is indebted to the Swedish Foundation for strategic
research.
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Fig. 1 TTGE of Lactobacillus strains.
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