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Previous Article | Next Article 
Applied and Environmental Microbiology, December 2003, p. 7517-7522, Vol. 69, No. 12
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.12.7517-7522.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Comparative Sequence Analysis of the tuf and recA Genes and Restriction Fragment Length Polymorphism of the Internal Transcribed Spacer Region Sequences Supply Additional Tools for Discriminating Bifidobacterium lactis from Bifidobacterium animalis
Marco Ventura* and Ralf Zink
Nestlé Research Center, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland
Received 26 June 2003/
Accepted 9 September 2003
 |
ABSTRACT
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The
relationship between Bifidobacterium lactis and
Bifidobacterium animalis was examined by comparative analysis
of tuf and recA gene sequences and by restriction
fragment length polymorphism analysis of their internal 16S-23S
transcribed spacer region sequences. The bifidobacterial strains
investigated could be divided into two distinct groups within a single
species based on the tuf, recA, and 16S-23S spacer
region sequence analysis. Therefore, all strains of B. lactis
and B. animalis could be unified as the species B.
animalis and divided into two subspecies, Bifidobacterium
animalis subsp. lactis and Bifidobacterium
animalis subsp.
animalis.
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INTRODUCTION
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Bifidobacterium lactis and Bifidobacterium animalis
are closely related, representing a relatively small number of known
strains. B. lactis includes strains employed as probiotics for
dairy products and infant formula. Despite their high industrial
importance, the taxonomy of B. lactis and B. animalis
is still unclear. Historically, the species annotation of B.
animalis (12,
15) was introduced first,
followed by the isolation of a highly oxygen-tolerant
Bifidobacterium organism from yogurt, which was classified in
1997 as the new species B. lactis
(11). The definition
criteria for B. lactis and B. animalis were based on
phenotypic characteristics such as carbohydrate fermentation patterns
and high tolerance of oxygen stress. However, due to the high levels of
DNA relatedness and 16S ribosomal DNA (rDNA) sequence similarity
between B. lactis and B. animalis, Cai et al.
(1). proposed the
rejection of the name B. lactis and suggested that B.
lactis should be considered a junior subjective synonym of B.
animalis. The nomenclature of the species B. lactis and
B. animalis is still ambiguous, and a taxonomic decision by
the International Committee on Systematic Bacteriology to maintain them
as two separate species has been pending since 1999
(6).
Recently,
we have demonstrated that analysis of 16S rDNA sequences, analysis of
the 16S-23S spacer region, and enterobacterial repetitive intergenic
consensus-PCR have powerful potential for tracing B. animalis
or B. lactis
(20-23).
The tuf and recA genes have been proposed as useful
markers in inferring bacterial phylogeny
(2,
10,
18) and have recently
been successfully used to differentiate species and subspecies within
various bacterial genera
(3,
7,
8,
17). Our results show
that the sequence analysis of tuf and recA as well as
restriction fragment length polymorphism (RFLP) analysis of the
internal transcribed spacer (ITS) sequences are relatively simple and
rapid methods by which B. lactis and B. animalis can
be identified without resorting to the use of species-specific PCR
primer sets.
|
tuf and recA
sequence analysis.
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Bacterial
strains used in this study were either obtained from culture
collections or isolated from human or animal fecal samples (Table
1) and grown as described previously
(19). The DNA of all
bifidobacterial strains was prepared as described by Ventura and Zink
(19). The 940-bp
tuf fragment sequences and the 690-bp recA sequences
were amplified with the oligonucleotides tuf1
(5'-GAGTACGACTTCAACCAG-3')
(22) and tuf2
(5'-CAGGCGAGGATCTTGGT-3')
(22) and the
oligonucleotides rec1
(5'-TCGAGGTGATTCCCACC-3') and rec2
(5'-GAACCAAGAACCGGACTTC-3'),
respectively. Each PCR mixture (50 µl) contained a
reaction cocktail of 20 mM Tris-HCl at pH 8.0, 50 mM KCl, a 200
µM concentration of each deoxynucleoside triphosphate, 50 pmol
of each primer, 1.5 mM MgCl2, and 1 U of Taq DNA
polymerase (Gibco BRL, Paisley, United Kingdom). Each PCR cycling
profile consisted of an initial denaturation step of 3 min at
95°C, followed by amplification for 30 cycles as follows:
denaturation (30 s at 95°C), annealing (30 s at 52°C),
and extension (2 min at 72°C). The PCR was completed with an
elongation phase (10 min at 72°C). PCR fragments were purified
with a PCR purification kit (Qiagen, West Sussex, United Kingdom) and
were subsequently cloned in the pGEM-T Easy plasmid vector (Promega,
Southampton, United Kingdom) following the supplier's
instructions. Subsequently, the sequence of the inserted DNA fragment
was determined by sequencing three randomly selected clones on both
strands for each bacterial species to ensure that no sequencing errors
were attributable to misincorporation by the Taq
polymerase.
Nucleotide sequencing of both strands
from cloned DNA was performed with a fluorescence-labeled primer cycle
sequencing kit (Amersham Buchler, Braunschweig, Germany) following
supplier's instructions. The primers used were tuf1, tuf2, rec1,
and rec2 labeled with IRD800 (MWG Biotech, Germany). The tuf
and recA sequences of all Bifidobacterium strains
determined here as well as those available in the GenBank database were
used for comparison. The partial nucleotide sequences of the
tuf and recA genes from 11 Bifidobacterium
strains belonging to B. lactis and B. animalis were
determined, and phylogenetic trees based on these data as well as those
retrieved from GenBank databases were constructed. Phylogenetic trees
were constructed with the programs Clustal X, DNAML (maximum
likelihood), and DNAPARS (parsimony) (PHYLIP [Phylogeny Inference
Package], version 3.5c; J. Felsenstein, University of Washington,
Seattle, Wash.).
The topologies of the
recA- and tuf-based trees were comparable (Fig.
1). In these trees, B. lactis and B. animalis strains
were grouped into two clusters; the first one contained nine strains,
including all B. lactis strains as well as B.
animalis ATCC 27673, ATCC 27674, and ATCC 27536, while the second
one contained only the type strain of B. animalis and B.
animalis ATCC 27672. The phylogenetic distances among strains of
the B. lactis are virtually zero (Fig.
1). The individuality of
each of these strains was supported by various molecular typing tools,
such as pulsed-field gel electrophoresis, random amplified polymorphic
DNA PCR, and triplicate arbitrarily primed PCR
(19; data not
shown).
Twenty-seven nucleotide substitutions were observed
between the tuf gene sequences of B. lactis DSM 10140
and B. animalis ATCC 25527, but only four contributed to an
amino acid substitution. In parallel, only 13 synonymous nucleotide
substitutions were noticed among the recA gene sequences in
the same set of strains. Interestingly, many of the base differences
observed between the tuf and recA sequences of the
two taxa are guanine or cytosine in B. lactis and adenine and
thymine in B. animalis. The spontaneous deamination of
cytosine leading to a thymine is a frequent event, and it has already
been described in relation to the species identification of
Lactobacillus delbrueckii
(4). Because of the
similar G+C content of the organisms under consideration,
sequence differences are likely due to real evolutionary divergence.
Therefore, we focused our interest on the tuf and
recA nucleotide sequences. When we aligned the tuf
and the recA sequences of all B. lactis and B.
animalis strains investigated, we noticed that these 27- and
13-nucleotide differences could be directly used to distinguish all
B. lactis from B. animalis strains (Fig.
2). Some of these sequence signatures are typical of either B.
lactis strains or B. animalis strains but are not
exhibited in all other microorganism investigated here (Fig.
1b and d). These sequence
signatures could be directly used for designing PCR-specific primers,
or they could be a target for specific restriction enzymes, providing
species-specific RFLP patterns. In fact, theoretical restriction
profiles with different restriction enzymes were obtained from the
tuf and recA sequences. The restriction enzymes
HindII, BamHI, and ClaI were found to give
the clearest and most reliable distinction in theoretical RFLP patterns
in order to differentiate B. lactis and B. animalis
strains (Fig. 2c and
d).
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FIG. 2. (a
and b) Pairwise alignment of the tuf sequences (a) and
recA sequences (b) of B. lactis DSM 10140T
and B. animalis ATCC 25527T. Nucleotides which are
different between B. lactis strains and B. animalis
ATCC 25527 are shaded in black. (c and d) Theoretical RFLP profiles
from B. lactis-B. animalis tuf sequences (c) and from
B. lactis-B. animalis recA sequences
(d).
|
|
The synonymous distances calculated by the method
of Nei et al. (13) from
the nucleotide substitution ratios at synonymous positions in
tuf and recA were examined for all possible
combinations of these 22 Bifidobacterium gene sequences. A
significant correlation between the synonymous distances in the
tuf genes and those in the recA genes were observed,
with a correlation coefficient of 0.99. This result was not unexpected,
because it has been demonstrated before that a synonymous substitution
rate is constant for many chromosomal genes in various organisms and
can thus serve indeed as a molecular clock of their evolution
(9). The evolutionary
distances calculated for tuf, recA, ITS, and 16S rDNA
sequences for strains of B. lactis and B. animalis
(Table
2) clearly underline the inadequacy of the 16S rDNA sequences for
distinguishing these closely related taxa. From these data, it appears
that the Knuc values (average extent of sequence
change at any position in two homologous sequences
[5]) calculated
for 16S rDNA sequences are at least threefold lower than those
calculated for the other taxonomic molecular markers (tuf and
recA gene sequences) and sevenfold lower than those of the
ITS. Hence, tuf, recA, and ITS sequences provide
different complementary phylogenetic information. While tuf
and recA sequences are excellent tools for inferring
interspecific relationships, 16S-23S rDNA spacer sequence comparisons
provide information concerning intraspecific links (e.g.,
characterization of strains).
View this table:
[in this window]
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|
TABLE 2. Knuc
values for tuf, recA, ITS, and 16S rDNA sequences of
B. lactis DSM 10140 with respect to B. lactis
and B. animalis strains
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PCR-RFLP
analysis of the 16S-23S ITS region.
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Theoretical restriction profiles for
the ITS region of B. lactis and B. animalis with
different enzymes were obtained by using the Webcutter online analysis
tool
(http://users.unimi.it/
camelot/tools/cut2.html).
The enzyme Sau3AI gave the clearest and most reliable
theoretical PCR-RFLP pattern, in order to differentiate between the
B. animalis and B. lactis type strains. PCR was used
to amplify the 16S-23S ITS region of all B. lactis and B.
animalis strains with the primers 16S-for
(5'-GCTAGTAATCGCGGATCAG-3')
(19) and 23Si
(5'-CATTCGGACACCCTGGGATC-3')
(19). Before a
restriction digestion of all PCR products was done, amplicons were
purified with the QIAquick PCR purification kit (Qiagen, Valencia,
Calif.). In order to achieve complete digestion, reactions were carried
out for 3 h at 37°C in a 10-µl volume of
incubation buffer containing 2 U of Sau3AI
(Boehringer-Mannheim, Mannheim, Germany) and 1 µg of the
purified PCR product. The restriction products were loaded on 3%
(wt/vol) agarose gel (NuSieve; FMC Bioproducts, Rockland, Maine) and
separated at 7 V/cm, followed by ethidium bromide staining (UV at 260
nm). PCR-amplified 16S-23S rDNA ITS sequences of all B. lactis
and B. animalis strains were digested with Sau3AI,
leading to polymorphic patterns and hence allowing direct species-level
identification (Fig.
3). The clustering from these patterns consistently grouped all restriction
fragment patterns into two distinct subsets. One shared the restriction
pattern of the type strain of B. lactis and includes B.
lactis DSM 10140, B. animalis ATCC 27536, B.
animalis ATCC 27673, B. animalis ATCC 27674, and all
other B. lactis isolates, and the other includes the type
strain of B. animalis, ATCC 25527, and B. animalis
ATCC 27672. In all experiments, we obtained clearly distinguishable and
always reproducible RFLP patterns of the 16S-23S ITS region, confirming
all theoretically expected restriction
profiles.
View larger version (87K):
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|
FIG. 3. Restriction
patterns of PCR-amplified fragments of 16S-23S rDNA digested with
Sau3AI. Lane 1, B. animalis ATCC 25527; lane 2,
B. animalis ATCC 27672; lane 3, B. lactis DSM 10140;
lane 4, B. animalis ATCC 27674; lane 5, B. animalis
ATCC 27673; lane 6, B. animalis ATCC 27536; lane 7, B.
lactis NCC 363; lane 8, B. lactis NCC 311; lane 9, B.
lactis NCC 402; lane 10, B. lactis NCC 311; lane 11,
B. lactis NCC 383; lane 12, B. lactis NCC 239; lanes
M, 50-bp DNA ladder (Gibco
BRL).
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Conclusions.
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Our study shows that comparison of
tuf and recA sequences as well as RFLP analysis of
the ITS sequences of B. lactis and B. animalis
strains provides additional and much further detailed practical means
to discriminate these closely related taxa. The analysis of
tuf and recA gene sequences and classical molecular
species identification tools (rDNA sequencing and species-specific
oligonucleotide probes) data should therefore be incorporated in a
modern polyphasic approach for bifidobacterial taxonomy. Our results
suggest that tuf and recA gene sequencing and RFLP
analysis of the ITS sequences have specific advantages over other
molecular tools for tracing B. lactis-B. animalis
strains (19). In fact,
tuf and recA are relatively short sequences that can
easily be sequenced on both polynucleotide strands. In addition, it has
been shown that bifidobacterial genomes carry only one tuf and
one recA gene
(16), which might be
advantageous because the interpretation and power of rRNA-based data
(e.g., the use of a single gene or operon) in molecular taxonomy appear
sometimes to be questionable
(24). Finally, the
application of tuf and recA gene sequencing and RFLP
analysis of the 16S-23S spacer sequences for our species-specific
identification allows simultaneous handling and comparison of many
isolates, in contrast to a required repeated PCR amplification with
species-specific primers. Establishing PCR primers based on
tuf and recA sequences for quantitative detection of
B. lactis and B. animalis is a task for the future,
and research into real-time quantitative detection avoiding the use of
multicopy genes (e.g., ribosomal genes) is rapidly proceeding
(14).
In the
present study, genotypic analysis carried out on B. lactis and
B. animalis strains targeting the tuf and
recA genes showed a consistent similarity between these two
taxa. However, taxonomic trees based on tuf and recA
indicate a separate branching of B. lactis and B.
animalis strains. Recently, a reorganization of the B.
animalis species with the rejection of few strains (ATCC 27673,
ATCC 27674, and ATCC 27536) from this species and a reclassification of
them as B. lactis have been proposed
(19). This hypothesis is
also validated by the tuf and recA gene analyses,
which suggested a revision of various strains, actually designed to the
B. animalis species. This study provides a clear image of the
genetic variability within the B. lactis-B. animalis taxa. In
fact, the use of genes encoding proteins (e.g., tuf and
recA) instead of ribosomal genes
(19) shows that the taxon
B. lactis is in fact highly homogeneous. In contrast, the
analysis of the tuf gene in all investigated B.
animalis strains depicted a significant variability. This might be
due to the fact that the taxon B. animalis-B. lactis
was originally a single group that has diverged only recently as a
result of different environmental conditions (e.g., growth in different
ecological niches). Therefore, based on the distinct phenotypic
characteristics (12,
19) of all B.
lactis strains and further detailed molecular evidence
(tuf and recA sequences as well as their ITS
restriction patterns), we proposed that B. lactis and B.
animalis be unified into one species and that this species should
be divided into two subspecies, Bifidobacterium animalis
subsp. animalis and Bifidobacterium animalis subsp.
lactis.
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Nucleotide sequence
accession numbers.
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The
accession numbers for the tuf and recA sequences are
AY370912 to AY370929 and AY372028 to
AY372031.
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FOOTNOTES
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* Corresponding author. Present address: Bioscience Institute, Department of Microbiology, University College Cork, Western Road, Cork, Ireland. Phone: 41-21-7858021. Fax: 41-21-7858925. E-mail: m.ventura{at}ucc.ie. 
Present address: COGNIS Germany, 40551 Düsseldorf, Germany.
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Applied and Environmental Microbiology, December 2003, p. 7517-7522, Vol. 69, No. 12
0099-2240/03/$08.00+0 DOI: 10.1128/AEM.69.12.7517-7522.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
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Top
Abstract
Introduction
75% identity, while residues
in dark grey have 
76%
identity.