FEMS Microbiology Letters 168 (1998) 213^219

Comparison of Streptococcus thermophilus strains by pulse ¢eld

gel electrophoresis of genomic DNA
Tadhg F. O'Sullivan, Gerald F. Fitzgerald *
Department of Microbiology, University College, Cork, Ireland

Received 1 July 1998; received in revised form 14 September 1998; accepted 15 September 1998

Abstract

Pulse field gel electrophoresis (PFGE) was utilised to compare the genomes of 16 Streptococcus thermophilus cultures from
yoghurt, cheese, laban and dahi after digestion with the restriction endonucleases, SfiI, SmaI and BssHII. PFGE profiles could
be used for strain identification and were also useful in predicting relatedness of certain strains. Genetic variations between
specific morphotypes of a highly proteolytic culture were not detectable by PFGE in this study. Statistical analysis of SmaI
restriction patterns enabled the clustering of strains into two groups which corresponded with biochemical properties of the
strains examined and suggested that PFGE profiles could be useful in predicting biochemical characteristics. z 1998 Fed-
eration of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : Streptococcus thermophilus; Genome ; Pulse ¢eld gel electrophoresis

1. Introduction chemical characteristics. Perhaps the most commer-

Streptococcus thermophilus is an important compo-
nent of many dairy starter cultures, particularly in
those used for the manufacture of yoghurts, fer-
mented milks and Italian and Swiss style cheeses.
Taxonomically, the species is most closely related
to Streptococcus salivarius, of which it was for
some time considered a subspecies [1]. Despite the
fact that no natural habitat for this species outside
of the dairy environment has been identi¢ed, consid-
erable inter-strain diversity has been observed. Nu-
merous attempts have been made to categorise and
group strains of S. thermophilus on the basis of bio-
* Corresponding author. Tel.: +353 (21) 902730;
Fax: +353 (21) 903101; E-mail: g.fitzgerald@ucc.ie
cially useful of such studies have been those based on
milk acidi¢cation rates [2,3]. Other methods have
relied on serotyping [4], membrane protein analysis
[5] and enzymatic pro¢ling [6]. From a commercial
perspective, it is important that new strains of S.
thermophilus can be rapidly identi¢ed as such and
to this end several S. thermophilus speci¢c DNA
probes have been developed. The most useful of
these has been that based on the lacZ gene [7]. Other
probes are based on the 16S and 23S RNA sequences
[8^10] and these have also been useful in demonstrat-
ing both inter- and intra-strain polymorphism.
The technique of pulse ¢eld gel electrophoresis
(PFGE), whereby unsheared genomic DNA is re-
stricted with rare-cutting restriction endonucleases
and the resulting large fragments are separated on

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 4 5 - 5

FEMSLE 8446 5-11-98

214 T.F. O'Sullivan, G.F. Fitzgerald / FEMS Microbiology Letters 168 (1998) 213^219
agarose gels using a constantly reorienting electric
¢eld [11] has been particularly useful in terms of:
(a) species and strain identi¢cation, characterisation
and comparison; (b) genome size estimation; and (c)
construction of physical and genetic maps. Boutrou
et al. [12] described the clustering of S. thermophilus
strains based on their PFGE pro¢les and considered
that the examination of a wider range of strains in
conjunction with detailed biochemical characteristics
could enable the prediction of new industrially useful
cultures based on their PFGE patterns. The size of
the S. thermophilus genome has been estimated as
1.82^1.85 Mb by this method [12,13]. PFGE has
also been instrumental in constructing and compar-
ing physical maps of three strains of this species
[13,14]. In addition, intra-strain polymorphism has
been demonstrated by comparing PFGE pro¢les of
di¡erent morphotypes.
In this study, we report the evaluation of PFGE to
di¡erentiate 16 strains of S. thermophilus from
cheese, yoghurt, laban and dahi using PFGE of
S¢I, SmaI and BssHII digests of genomic DNA. In
addition, the comparison of restriction pro¢les of
di¡erent morphotypes of the highly proteolytic strain
CNRZ385 and the statistical analysis of the resulting
data to construct phylogenetic clusters which corre-
sponded to phenotypic groupings is described.
2. Materials and methods
2.1. Bacterial strains, media and restriction enzymes
S. thermophilus cultures examined in this study,
their sources and relevant characteristics are sum-
marised in Table 1. Cultures were routinely trans-
ferred in Belliker broth [15] at 42³C. All strains
were characterised as S. thermophilus by their behav-
iour in API 50 CHS, API 20 Strep and API 20 ZYM
(Biomerieux, France). Restriction endonucleases and
bu¡ers were from New England Biolabs (Hitchin,
UK).
2.2. Preparation of genomic DNA
Unsheared genomic DNA of S. thermophilus cul-
tures was prepared essentially as described by
O'Riordan and Fitzgerald [16]. A 100 ml amount
FEMSLE 8446 5-11-98
of preincubated Belliker broth was inoculated at
2% (v/v) from an overnight broth culture and was
incubated at 42³C until an OD600 of 0.8^1.0 was
attained. Cells were harvested by centrifugation at
8000 rpm for 15 min, washed once in 100 ml of 50
mM EDTA, pH 8.5 and resuspended in 1.5 ml of the
same bu¡er. A 0.5 ml amount of cell suspension was
then warmed to 45³C and added to 1.5 ml of 1%
molten low melting point agarose (Bio-Rad, Rich-
mond, CA) in 50 mM EDTA, pH 8.5. Inserts were
prepared by dispensing 250 Wl aliquots of the molten
cell suspension into plug mould wells and allowing
them to solidify at 4³C for 15^30 min. Cells were
lysed in situ by immersing the inserts in 10 ml of
50 mM EDTA containing 2 mg/ml of lysozyme
and 0.05% (w/v) of N-lauryl sarcosine at 37³C for
4 h, followed by overnight incubation at 50³C in
1% (w/v) SDS, 10 mM Tris, 0.5 mM EDTA, pH
8.5, containing 2 mg/ml of proteinase K (Sigma,
Poole, Dorset, UK). Inserts were subsequently
washed three times for 30 min at 4³C in 10 ml 50
mM EDTA, pH 8.5, and were stored for up to 1 year
in the same bu¡er.
2.3. DNA restriction
Endonuclease restrictions of S. thermophilus ge-
nomic DNA were performed in 2 mm long sections
cut from agarose inserts. Sections were ¢rst washed
twice in 1.0 ml of ice cold, sterile distilled water for
15 min and then once in the appropriate restriction
bu¡er prior to the addition of 20 U of the relevant
restriction enzyme. Digestions were performed at the
optimum temperature for 8 h or overnight.
2.4. Pulse ¢eld gel electrophoresis
Endonuclease restricted genomic DNA in agarose
inserts was sealed into wells in 1.0^1.2% of PFGE-
grade agarose (Bio-Rad) gels in 0.5UTBE bu¡er (45
mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0)
and was separated at 180^200 V for 24^27 h at 8³C
in a CHEF DR-II PFG electrophoresis unit with a
model 1000 mini chiller (Bio-Rad). The gel running
conditions for each digest are summarised in Table
2. Gels were stained in 0.5 mg l31 of ethidium bro-
mide in 0.5UTBE for 2 h and photographed on a
UVP image store 5000 Gel Documentation System
 T.F. O'Sullivan, G.F. Fitzgerald / FEMS Microbiology Letters 168 (1998) 213^219 215

linked to a Sony video graphic printer. DNA size

standards consisted of yeast chromosome, V ladder
and low range PFG markers (New England Bio-
Labs).

2.5. Statistical analysis

Multivariate data analysis, speci¢cally cluster

analysis, using the unweighted average pair group
method, was performed using Minitab 10.5 Xtra
Power on a Macintosh computer.

3. Results and discussion

3.1. Restriction pro¢les
The restriction endonucleases S¢I, BssHII, SmaI
and NotI were used to digest the genomic DNA of
16 strains of S. thermophilus. The DNA of only one
of the strains examined, strain NDI-6, was restricted
by the enzyme NotI and just one band of approxi-
mately 95 kb was evident after PFGE. The endonu-
clease S¢I cut the chromosomal DNA of most of the
S. thermophilus strains into 5 or 6 fragments, de-
Fig. 1. Pulse ¢eld gel electrophoresis of SmaI restricted genomic
DNA of Streptococcus thermophilus cultures. Lanes: 1 and 8,
V concatamers ; 2, 385; 3, 4ML; 4, 703 ; 5, 1020 ; 6, 985 ; and 7,
NDI-6
pending on the strain. Two atypical strains were
956 and 959, each of which gave rise to only two
bands after treatment with S¢I. Digests of S. ther-
mophilus DNA with BssHII yielded 8 or 9 fragments
ranging in size from 41 to 630 kb. SmaI digested

Table 1
Bacterial strains
Bacterial strain Characteristics Reference
Streptococcus thermophilus
013 Slowa (yoghurt) UCCb
019 Slow (yoghurt) UCC
026 Slow (yoghurt) UCC
030 Slow (yoghurt) UCC
054 Slow (yoghurt) UCC
NDI-6 Slow (dahi) Dr. S. Sannabhadtic
CNRZ 385 Fast (Eastern yoghurt) INRAd
CNRZ 703 Fast (Eastern yoghurt) INRA
4ML Fast (laban) UCC
956 Slow (Italian starter) UCC
957 Fast (Italian starter) UCC
958 Fast (Italian starter) UCC
959 Slow (Italian starter) UCC
985 Fast (Italian starter) UCC
1020 Fast (Italian starter) UCC
780 Slow (yoghurt) UCC
a
Refers to rates of milk coagulation.
b
University College, Cork, Ireland.
c
Dr. S. Sannabhadti, University of Anand, Province of Gujarat, India.
d
M. Desmazeaud, INRA, Jouy-en-Josas, France.

FEMSLE 8446 5-11-98

216 T.F. O'Sullivan, G.F. Fitzgerald / FEMS Microbiology Letters 168 (1998) 213^219

Fig. 2. PFGE genomic DNA of Streptococcus thermophilus CNRZ 385 morphotypes F, S1 and S2 digested with S¢1 (A), BssHII (B) and
SmaI (C). Lanes : 1, 385F; 2, 385S1; 3, 385S2; and 4, V concatamers.

genomic DNA into more than 13 fragments for
each of the strains tested, and some diversity was
observed between PFG pro¢les of strains after
digestion with this enzyme. Representative restric-
tion pro¢les after SmaI digestion are presented in
Fig. 1.
3.2. Estimation of genome size
The size of the S. thermophilus chromosome was
estimated by determining the sum of the individual
bands in each digest. Care was taken to eliminate
partial digest products and to include co-migrating
bands. Estimated sizes as determined with each en-
zyme are presented in Table 3. These data suggest
that the S. thermophilus genome is between 1.75 and
1.85 Mb in size, which is consistent with previous
estimations [12,13]. Some inconsistencies in sizing
with di¡erent enzymatic digests may be explained
by either co-migrating bands or bands which are
below the size range detectable on the gels.
3.3. Comparison of clonal variants of S. thermophilus
cultures
The S. thermophilus culture CNRZ385 is a highly
proteolytic, fast milk coagulating strain. Two spon-
taneous variants of this culture, S1 and S2, were
isolated which di¡ered in terms of their colony ap-
pearance on milk agar and rates of acid production
in pasteurised milk. Variant S1 was unable to coag-
ulate pasteurised milk but its milk-clotting ability
was restored when the culture was supplemented
with 0.1% (w/v) peptone. This variant was inactive
against casein in both qualitative and quantitative
assays, suggesting that it is lacking a proteinase ac-
tivity. Variant S2 had proteolytic activity equivalent
to that of the parent culture and its milk-clotting
ability was not restored upon supplementation with
peptone, indicating that its de¢ciency is in an area
other than proteinase activity (unpublished results).
When PF gels of their digested genomic DNA were
compared, no di¡erences in banding patterns were

Table 2
Agarose gel electrophoresis conditions
Enzyme % Agarose Voltage Pulse (s) Time (h)
SmaI 1.0 200 5^15 24
S¢I 1.2 200 60 24
BssHII 1.1 200 5^30 24
Not1 1.1 200 5^30 24

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 T.F. O'Sullivan, G.F. Fitzgerald / FEMS Microbiology Letters 168 (1998) 213^219
Fig. 3. Dendrogram of SmaI digests of genomic DNA of Streptococcus thermophilus cultures.
observed with any of the enzymes utilised in this
study (Fig. 2).
3.4. Statistical analysis
The statistical analysis of the genomic restriction
pro¢les of S. thermophilus cultures allowed the clus-
tering of these strains into groups from which a phy-
logenetic dendrogram could be constructed. The
most clear-cut dendrogram resulted from the analy-
sis of SmaI digests and suggested that the S. thermo-
philus strains fell into two distinct groups which cor-
responded broadly with acidi¢cation rates and
ultimately with their proteolytic capacities (Fig. 3).
The ¢rst grouping included all of the European yo-
ghurt cultures, the dahi strain, NDI-6 and the slow
acidifying cheese cultures. The second grouping con-
sisted of the proteolytic, fast acid producers and the
bacteriocinogenic strain 780. Within the second
grouping there were two clusters, one of which con-
tained the laban strain 4ML and the cheese cultures
1020 (the pro¢le of which is virtually identical to
4ML), 957 and 958. The second Group II cluster
contains the eastern yoghurt cultures 385, and 703,
along with the cheese cultures 985 and 780. Strain
985 is in turn closely related to strain 385 on the
basis of restriction pro¢les. The dendrograms result-
ing from the analysis of S¢I and BssHII restriction
pro¢les were largely in agreement with that derived
from SmaI pro¢les except that the atypical S¢I pro-
FEMSLE 8446 5-11-98
217
¢les of strains 956 and 959 tended to distort the S¢I
dendrogram and the BssHII restriction pro¢les were
somewhat more conserved.
The ability to rapidly identify, compare and char-
acterise new cultures for application in the food in-
dustry is an important facet of a starter culture de-
velopment programme. It is also of use in
monitoring the survival and passage of particular
cultures in food, clinical and environmental systems.
Molecular methodologies currently favoured for
such purposes include, PAGE protein analysis, sero-
typing, ribotyping, the use of species-speci¢c DNA
probes and RAPD-PCR. Of the above techniques,
RAPD-PCR is the only one which has not yet
been applied to S. thermophilus to any signi¢cant
extent. In terms of rapid speciation, the use of
DNA probes appears to o¡er the best option; how-
ever, this method does not detect polymorphism and
therefore does not permit strain identi¢cation or
comparison. From this perspective, PFGE, although
more time-consuming, is a far more useful tool. As
indicated by this study it may have an application in
determining to which group a particular strain be-
longs and thus to predict biochemical traits of indus-
trial importance and potential end-product applica-
tion as suggested by Boutrou et al. [12]. The only
exception observed during this study was the bacter-
iocinogenic strain 780, which, despite being a slow
acidifying culture, is clustered along with the fast
acidifying strains. This may re£ect an evolutionary
218 T.F. O'Sullivan, G.F. Fitzgerald / FEMS Microbiology Letters 168 (1998) 213^219

Table 3 possible to show such genetic polymorphism when

Estimated genome sizes of Streptococcus thermophilus cultures
examining di¡erent morphotypes of strain
based on the sum of their restriction fragment sizes after diges-
tion with the enzymes S¢I, BssHII and SmaI. All sizes represent
CNRZ385. It is probable that small DNA rearrange-
the mean of at least three determinations ments or deletions would not be detectable within
Culture Estimated genome size (kb)
the limits of the macrorestriction pro¢les examined.
PFGE data are readily amenable to statistical
Enzyme
analysis. While results from the S¢I, BssHII and
S¢I BssHII SmaI SmaI digests yielded broadly similar dendrograms,
013 1830 1818 1830 the most useful data in terms of strain comparison,
019 1838 1805 1833 clustering and correspondence to phenotypic charac-
026 1840 1825 1859
teristics, were those derived from SmaI pro¢les. Re-
030 1833 1822 1768
054 1839 1818 1716 striction fragment length polymorphism (RFLP) as
NDI-6 1840 1840 1713 demonstrated by PFGE has also been shown to be a
385 1860 1845 1819 reliable method of strain identi¢cation and di¡eren-
4ML 1851 1835 1876 tiation, particularly within a species in which plas-
703 1860 1829 1803
mids are rarely found. RFLP using SmaI PFGE pro-
780 1885 1897 1823
956 1870 1832 1804 ¢les has previously been utilised in strain comparison
957 1815 1823 1815 of Lactococcus lactis and S. thermophilus cultures
958 1830 1837 1832 [19,20]. In this study, certain strains had virtually
959 1870 1832 1804 identical restriction pro¢les. In particular, those of
985 1840 1812 1833
4ML and 1020 were similar for all three enzymes.
1020 1851 1846 1777
Both of these strains are highly proteolytic and the
two cultures are lysed by 4ML-speci¢c bacterio-
phage, while the apparently closely related culture,
development of this strain from a fast acidifying an- 958, was resistant to this phage. Similarly, the pro-
cestor due to loss of a proteolytic or peptide uptake ¢les of strains 956 and 959, which were atypical on
system. S¢I, were also identical on SmaI and BssHII.
The number and sizes of the genomic restriction The usefulness of PFGE in genome size determi-
fragments generated by S¢I, SmaI and BssHII dur- nation, speciation and strain identi¢cation within S.
ing this study are generally in agreement with those thermophilus has been demonstrated. In particular,
reported by other researchers [12,13]. NotI cut the the PFGE patterns of SmaI digests are of value in
genome of only one of the strains (NDI-6) examined strain identi¢cation and in determining strain relat-
in this study. This is consistent with the report of edness. There is also some evidence, on the basis of
Simonet et al. [17]. The size of the S. thermophilus statistical analysis, that such pro¢les can be utilised
genome has previously been determined as being to predict phenotypic and ultimately industrially rel-
from 1.82 Mb in the case of strain A054 (which evant characteristics. It would be of interest to ex-
was utilised as a control strain in this study) to tend this study to a wider range of strains, including
1.86 Mb for strain NST 2280 [14] and these values further highly proteolytic cultures along with texture
are consistent with the data emanating from this and £avour producing isolates with a view to build-
study. ing up databases of such strains concerning their
Intra-strain polymorphism has previously been at- relevant characteristics and their PFGE pro¢les.
tributed to DNA rearrangements, deletions due to These data could then be utilised to predict the use-
homologous recombination events between closely fulness of new isolates.
linked rRNA loci and to insertions or duplications,
some of which in£uence colony morphology [18].
Roussel et al. [13] utilised PFGE of genomic double Acknowledgments
digests to demonstrate genetic alterations in speci¢c
morphotypes of strain A054. In this study, it was not We acknowledge the assistance of Therese Uni-

FEMSLE 8446 5-11-98

 T.F. O'Sullivan, G.F. Fitzgerald / FEMS Microbiology Letters 168 (1998) 213^219 219

acke who helped with statistical analysis. This work [10] Ehrmann, M., Wolfgang, L. and Schleifer, K.H. (1992) Spe-
cies speci¢c oligonucleotide probe for the identi¢cation of
was ¢nancially supported by the Centre Internation-
Streptococcus thermophilus. Syst. Appl. Microbiol. 15, 453^
al de Recherche, Daniel Carasso of the Danone 455.
Group. [11] Lai, E., Birren, B.W., Clarke, S.M., Simon, M.I. and Hood,
M. (1989) Pulsed ¢eld gel electrophoresis. Biotechniques 7,
34^42.
[12] Boutrou, R., Thuault, D. and Bourgeois, C.M. (1995) Identi-
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