Annals of Microbiology, 50, 191-203 (2000)

Characterisation and typing of Saccharomyces strains

by DNAfingerprinting
C.VITI, D. FORNI, S. VENTURA, A. MESSINI, R. MATERASSI,
L. GIOVANNETTI*
Dipartimento di Biotecnologie Agrarie, Universit degli Studi di Firenze,
and Centro di Studio dei Microrganismi Autotrofi-CNR, Piazzale delle Cascine 27,
50144 Firenze, Italy.
Abstract - Three different molecular methods, total DNA restriction profile analysis,
restriction profile of mitochondrial DNA(mtDNA) and Southern hybridisation of mtDNA,
were used to characterise Italian wine yeast strains previously identified using convention-
al taxonomic techniques. Total DNA restriction profile analysis allowed the typing of all
strains and showed that they constitute two well separated genomic taxa, one including the
type strain of S. cerevisiae and the other the type strain of S. bayanus. The data obtained by
analysing mtDNA restriction profiles and mtDNA Southern hybridisation were consistent
with results of total DNArestriction profile analysis. This permitted the taxonomic assign-
ment of Italian wine yeast strains and the determination of the interspecific genomic relat-
edness.
Key words: yeast, Saccharomyces, DNAfingerprinting, identification, strain typing.
INTRODUCTION
The criteria traditionally employed for the classification of yeasts have been pre-
dominantly based on physiological and biochemical characteristics, and morpho-
logy of vegetative and sexual stages. Most of these properties tend to be influen-
ced by culture conditions and can give ambiguous results (Yamamoto et al., 1991;
Guillamn et al., 1996), therefore they often were inadequate for species delimi-
tation (Phaff, 1984). This was the case for species highly related to Saccharomy-
ces cerevisiae, which were defined mainly on the basis of their ability to use a
few different carbon sources (Barnett, 1992). For these reasons, and with the
knowledge that some different physiological features are due to single mutations
(Scheda and Yarrow, 1966; Scheda and Yarrow, 1968), several changes were
191
* Corresponding author. Phone +39-0553288307; Fax: +39-0553288393; e-mail:
luciana.giovannetti@unifi.it
made in the taxonomic status of the genus Saccharomyces in the past 30 years.
Yarrow (1984) combined the 21 species of Saccharomyces sensu stricto (Van der
Walt, 1970) into a single species: S. cerevisiae that also included S. bayanus, S.
pastorianus and S. paradoxus. Afterwards, studies on nDNA/nDNA (nuclear
DNA) reassociation within the species Saccharomyces cerevisiae sensu Yarrow
indicated the presence of four species, S. cerevisiae, S. bayanus, S. paradoxus and
S. pastorianus which formed the Saccharomyces sensu stricto complex (Vau-
ghan-Martini and Kurtzman, 1985; Vaughan-Martini and Martini, 1987; Vau-
ghan-Martini, 1989). These results were confirmed by other molecular approa-
ches such as electrophoretic karyotyping (Naumov et al., 1993; Cardinali and
Martini, 1994), random amplified polymorphic DNA analysis (Molnr et al.,
1995; Paffetti et al., 1995; Torriani et al., 1999), mitochondrial DNA restriction
analysis (Querol et al., 1992; Guillamn et al., 1994; Torriani et al., 1999), RFLP
(Restriction Fragment Length Polymorphism) and sequencing of nuclear rDNA
(ribosomal DNA) (Molina et al., 1992a; Molina et al., 1992b; Messner and Pril-
linger, 1995; Montrocher et al., 1998; Dlauchy et al., 1999). In recent years, stu-
dies have been carried out to clarify interspecific relationships at the phenotypic
level within the Saccharomyces sensu stricto complex (Vaughan-Martini and
Martini, 1993; Kishimoto and Goto, 1995; Rodrigues De Sousa et al., 1995; Tor-
nai-Lehoczki et al., 1996; Vaughan-Martini and Martini, 1998). In spite of a clea-
rer definition in the taxonomic status of the Saccharomyces sensu stricto com-
plex, a definitive identification of strains belonging to S. cerevisiae or some other
related species is often difficult although strain typing studies of this group con-
tinue to be relevant. The use of selected strains for commercial wine fermenta-
tion, beer brewing and backery products requires methods that are able to diffe-
rentiate highly related strains, to distinguish inoculated strains from indigenous
strains and to monitor the stability of used strains during fermentation process and
after storage. There is the need to control fermentation process and to ensure that
selected yeast strains conduct it to obtain a final product with specific characteri-
stics
In this study total DNAand mtDNArestriction profile analysis, and mtDNA
Southern hybridisation were applied in order to characterise and verify the taxo-
nomic assignment of Italian wine yeast strains belonging to the Saccharomyces
sensu stricto complex and originally identified as S. bayanus, S. cerevisiae, S.
ellipsoideus, S. oviformis or S. uvarum by using conventional taxonomic criteria.
At the same time, the applicability of our approaches to yeast strain typing was
tested.
MATERIALS AND METHODS
Yeast strains. On the basis of optimum growth temperature, the strains studied
were separated into two groups, denominated as cryotolerant and non-cryotole-
rant strains. A yeast strain was defined, in agreement with Zambonelli et al.
(1994) and Castellari et al. (1998), as cryotolerant when its optimal temperature
of growth was <30 C and as non-cryotolerant when its optimal temperature of
growth was > 30 C. Strains investigated together with corresponding collection
numbers, origin and optimal growth temperature are given in Table 1.
192
DNAextraction. Yeast strains were grown in YEPG (1% yeast extract, 1% pep-
tone and 2.5% glucose) at 25 C with continuous shaking. Cells were harvested in
exponential phase by centrifugation (5300 x g for 20 min at 4 C) and washed
twice with TEN buffer (10 mM TRIS, 1 mM EDTA, 100 mM NaCl, pH 8.0). Pel-
lets were kept frozen at 20 C until use. Spheroplasts were obtained by suspen-
ding frozen cells in TEN buffer supplemented with 20000 U mL
1
of lyticase
(Roche Diagnostics, Switzerland) and incubating the mixture at 37 C for about
3 hours. To lyse spheroplasts SDS was added to a final concentration of 1% and
the mixture was incubated at 65 C for 30 min. After the addition of 1/3 volume
of 5 M potassium acetate, the solution was chilled on ice for 1 hour and centrifu-
ged (5300 x g per 20 min). Two volumes of cold ethanol (-20 C) were added to
the recovered supernatant to precipitate nucleic acids. RNAse treatment and DNA
recovery were performed according to Giovannetti et al. (1990).
193
TABLE 1 - Strains used, their isolation source and optimal growth temperature
Ref.
a
Species
b
Species Strain Isolation source T
opt.
c
(original designations
epithet)
1 S. cerevisiae S. cerevisiae DBVPG
d
6173
T
Beer >30
g
2 S. cerevisiae B7
e
Fermenting grape juice >30
h
3 S. cerevisiae Cu11
e
Fermenting grape juice >30
h
4 S. cerevisiae G1
e
Fermenting grape juice >30
h
5 S. ellipsoideus PC80
e
Fermenting grape juice >30
i
6 S. cerevisiae S. oviformis DBVPG
d
6254
T
Fermenting grape juice <30
i
7 S. oviformis DBVPG
d
1589 Wine >30
i
8 S. bayanus 1024
f
Fermenting grape juice >30
j
9 S. bayanus 7541
f
Fermenting grape juice >30
j
10 S. uvarum 11052
f
Fermenting grape juice >30
k
11 S. bayanus S. bayanus DBVPG
d
6171
T
Beer <30
g
12 S. bayanus 12130
f
Fermenting grape juice <30
j
13 S. bayanus 12212
f
Fermenting grape juice <30
j
14 S. bayanus S. uvarum DBVPG
d
6179
T
Currant juice <30
i
15 S. uvarum 12138
f
Fermenting grape juice <30
k
16 S. bayanus 12000
f
Fermenting grape juice <30
k
a
Reference number in figures;
b
Vaughan-Martini and Martini, 1998;
c
optimal growth
temperature;
d
Collection of Dipartimento di Biologia Vegetale, Universit di Perugia,
Italy;
e
Collection of Dipartimento di Biotecnologie Agrarie, Universit di Firenze, Italy;
f
Collection of Ente per gli Studi e lAssistenza Viticola ed Enologica dellEmilia Roma-
gna, Italy;
g
Berardi and Fatichenti, 1989;
h
De Philippis et al., 1994;
i
our unpublished
results;
j
Castellari (Ente per gli Studi e lAssistenza Viticola ed Enologica dellEmilia
Romagna, Italy) personal communication;
k
Castellari et al., 1994;
T
type strain.
DNA digestion. Each g of total DNA was digested with 3 units of restriction
endonuclease. The mixture was incubated according to the supplier (Roche Dia-
gnostics, Switzerland).
Total DNA restriction profile analysis. Restriction endonucleases SfuI, EcoRI,
ClaI, ScaI, BglII, XbaI, PvuII, BamHI, HindIII, EcoRV, BglI, SacI, BclI, DraI,
NaeI, Alw44I, StuI and Asp718 were tested with DNAs extracted from the non-
cryotolerant yeast strain G1 and the cryotolerant yeast strain 12138 in order to
select enzymes giving electrophoretic patterns with several well separated low
molecular weight bands suitable for computer analysis. SacI and StuI were selec-
ted to perform SDS-PAGE of DNA of all yeast strains studied. Ten g of total
DNA of each sample were digested with the chosen endonucleases. SDS-PAGE
of total DNA digests was performed as described by Giovannetti and Ventura
(1995) and Ventura et al. (1993). Band patterns were recorded using a LKB
Ultroscan laser densitometer, using the procedure described by Giovannetti and
Ventura (1995) and Ventura et al. (1993). The similarity between all pairs of pat-
terns was determined using the Dice similarity coefficient (S
D
) (Sneat and Sokal,
1973). Strains were clustered by analysing the matrix of S
D
values with UPGMA
(Sneat and Sokal, 1973) using the cluster and tree procedures of the SAS packa-
ge (SAS Institute Inc., 1987).
It has been previously reported that total DNA restriction profile analysis
performed on polyacrilamide gel and stained with silver nitrate is a highly repro-
ducible molecular technique (Degli-Innocenti et al., 1990; Viti et al., 1996).
However in this study to ensure that total DNArestriction profile remained stable
over time, DNA of the strains G1 and 12138, obtained from two cultures, were
digested with StuI and submitted to electrophoretic run. DNA samples from a
same strain gave identical electrophoretic profiles (data not shown).
mtDNA restriction profile and mtDNA Southern hybridisation. Restriction
endonucleases such as AluI, DdeI, HaeIII, HinfI, MaeI, MaeII, Sau3A1, RsaI
recognise a high number of sites in yeast nDNA, but few sites in mtDNA. As a
result, restriction fragments of high molecular weight can be obtained from
mtDNA sequences that are easily distinguishable by gel electrophoresis from
fragments of low molecular weight obtained from nDNA (Querol et al., 1992).
This makes it possible to obtain mtDNA restriction profiles without previously
isolating mitochondria and/or purifying mtDNA.
Five g of total DNA were digested with AluI or RsaI following the manu-
facturers instruction (Roche Diagnostics, Switzerland). DNA fragments were
separated by 0.8% (wt/vol) agarose gel electrophoresis in Tris-borate buffer (90
mM Tris-borate, 2 mM EDTA, pH 8.3) at 4 V/cm for about 6 h. After depurina-
tion (0.25 M HCl, for 30 min), denaturation (0.5 M NaOH, 1.5 M NaCl for 30
min) and neutralisation (0.5 M Tris-HCl, 1.5 M NaCl, pH 7.5 for 40 min), DNA
fragments were transferred under low vacuum (5 inches of Hg) for 90 min in 10
x SSC (1.5 m NaCl, 0.15 M Na
3
citrate) to a nylon membrane (Hybond-N, Amer-
sham International).
The hybridisation probe was purified mtDNA of the strain S. cerevisiae C1
(dr. Casalone) labelled with digoxigenin using the Nonradiactive DIG DNA
Labelling and Detection Kit (Roche Diagnostics, Switzerland).
Hybridisation and the immunoenzymatic detection of hybridised fragments
194
were performed, in accord to the Nonradiactive DIG DNALabelling and Detec-
tion Kit handbook (Roche Diagnostics Switzerland), inside the glass tube of a
hybridisation oven. Hybridisation was performed at 42 C for 16 h.
RESULTS
Total DNArestriction profile analysis
The restriction endonucleases StuI or SacI gave electrophoretic profiles with well
separated bands in the range between 298 and 1230 bp and between 394 and
1033 bp, respectively. As an example the profiles obtained with StuI are reported
in figure 1. The sections of band patterns subjected to computer analysis contai-
ned from 54 to 85 bands when using StuI and from 53 to 64 bands with SacI.
Each strain showed a unique electropherogram, differing in at least some bands
from all of the others. After the determination of the S
D
coefficient between each
pair of profiles, the genomic relationship among strains was evaluated with
195
strains
b
p
FIG. 1 Total DNA restriction profiles of Saccharomyces strains performed by SDS-
PAGE and silver staining. DNA was digested with StuI. Reference numbers
listed in Table 1 designate strains. Lanes M contain DNA molecular weight
marker VI (Roche Diagnostics, Switzerland).
UPGMA. The dendrograms obtained from UPGMAanalysis indicated a striking
correspondence of the data independently from the endonuclease used (Fig. 2, 3).
All strains were clearly differentiated and constituted two well defined and geno-
mically separate groups: one cluster (A) contained all non-cryotolerant strains
including the type strain of S. cerevisiae (DBVPG 6173) and the cryotolerant
196
FIG. 2 Dendrogram based on UPGMAclustering of total DNArestriction profile data
obtained with StuI. Reference numbers listed in Table 1 designate strains.
FIG. 3 Dendrogram based on UPGMAclustering of total DNArestriction profile data
obtained with SacI. Reference numbers listed in Table 1 designate strains.
strain DBVPG 6254; the other (B) comprised cryotolerant strains including the
type strain of S. bayanus (DBVPG 6171). The two clusters joined, with both
enzymes, at S
D
value around 0.60. Inside each group the strains showed S
D
levels
higher than 0.70. Differences in the degree of genomic relationship among strains
in the two dendrograms were modest.
mtDNArestriction profile and mtDNASouthern hybridisation
The mtDNArestriction profiles, obtained with enzymes RsaI or AluI, are reported
in figures 4 and 5. Inside the group of cryotolerant strains, except for the strain
DBVPG 6254, a high level of similarity of mtDNA profiles was evident. The
strains DBVPG 6171 (type strains of S. bayanus), DBVPG 6179 and 12212, and
the strains 12138 and 12000 (Fig. 4a, 5a) showed identical profiles. On the other
hand the non-cryotolerant strains showed more mtDNA divergence. Most of
strains exhibited a specific profile with only the pair DBVPG 6173 (type strain of
S. cerevisiae) and DBVPG 1589 showing the same profile. The mtDNAprofiles
of non-cryotolerant strains, independently of the enzyme used, were characterised
by the presence of some larger bands than those found in non-cryotolerant strains.
To further test the effectiveness of mtDNA profiles to distinguish cryotolerant
from non-cryotolerant strains, mtDNA profiles obtained with RsaI or AluI, were
hybridised with purified mtDNAof S. cerevisiae. While, independently from the
enzyme used, the polymorphism of non-cryotolerant strains was confirmed, all
cryotolerant strains, except DBVPG 6254, did not hybridise with the S. cerevisiae
mtDNAprobe (Fig. 4b, 5b).
DISCUSSION
The application of total DNArestriction profile analysis, independently from the
restriction endonuclease used, permitted a clear typing of strains tested and
showed that these constitute two well separated genomic taxa. One taxon inclu-
197
FIG. 4 mtDNA restriction profiles (a) and mtDNA Southern hybridisation (b) of Sac-
charomyces strains obtained with RsaI. Reference numbers listed in Table 1
designate strains. Lanes M contain digoxigenin-labelled DNAmolecular weight
marker II (Roche Diagnostics, Switzerland).
strains
b
p
b
p
ded the type strain of S. cerevisiae, all the other non-cryotolerant strains plus the
cryotolerant strain DBVPG 6254. The other taxon comprised the type strain of S.
bayanus and all remaining cryotolerant strains. The degree of genotypic diversity
found inside each cluster and between the two clusters (Fig. 2, 3) was much lower
than that found by Barberio et al., (1994) who applied the same molecular
approach to strains identified, by classical methods, as S. cerevisiae or S. baya-
nus. These authors reported that strains attributed to S. cerevisiae or S. bayanus
showed a value S
D
of 0 (no DNA restriction fragment in common). This is very
puzzling since it would be expected that strains belonging to the same or highly
correlated species should have at least some DNA bands in common. It is likely
that the apparent absence of similarity found by these authors was due to the use
of restriction endonucleases that yield a very small number of fragments (not
more than sixteen with HpaI and eight with KspI). On the contrary the advantage
of total DNA restriction profile obtained using polyacrylamide gel and stained
with silver nitrate is that of giving finely resolved restriction profiles with a large
number of well separated low molecular weight bands (Giovannetti and Ventura,
1995). This is advantageous because the analysis of a large number of restriction
sites allows the genome structure to be randomly assayed at many loci.
The analysis of mtDNArestriction profiles and mtDNASouthern hybridisa-
tions provided further evidence that the strains studied constitute two distinct
genomic groups, one including all cryotolerant strains (except DBVPG 6254),
and the other with non-cryotolerant strains. Restriction fragments of mtDNA
higher than 6557 bp were only found in non-cryotolerant strains and DBVPG
6254. These bands could be typical of the species S. cerevisiae (Guillamn et al.,
1994; Guillamn et al., 1996). Moreover no hybridisation signal was obtained in
cryotolerant strains when the mtDNA of a S. cerevisiae strain was used as a
probe. Nevertheless the analysis of mtDNA, with the used restriction endonu-
cleases, did not allow for the distinction of all strains as was seen for total DNA
restriction profile analysis.
198
FIG. 5 mtDNA restriction profiles (a) and mtDNA Southern hybridisation (b) of Sac-
charomyces strains obtained with AluI. Reference numbers listed in Table 1
designate strains. Lanes M contain digoxigenin-labelled DNAmolecular weight
marker II (Roche Diagnostics, Switzerland).
strains
b
p
b
p
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The high genetic similarity found, with molecular approaches applied in this
study, between the type strain of S. cerevisiae (DBVPG 6173) and the non-cryo-
tolerant strains originally identified as S. uvarum (11052) and S. bayanus (1024
and 7541) suggests that these should be included in the species S. cerevisiae
(Table 2). This is not surprising, since phenotypic criteria like the ability to fer-
ment or to assimilate sugars, originally employed for identification of these
strains, have been demonstrated to be inadequate to clearly distinguish strains
belonging to S. cerevisiae from those belonging to S. bayanus. In regard to the
other strains the original identification was confirmed, since S. ellipsoideus and S.
oviformis are synonyms of S. cerevisiae, and the S. uvarum is a synonym of S.
bayanus (Vaughan-Martini and Martini, 1998).
The presence of the cryotolerant strain DBVPG 6254 in the non-cryotolerant
genomic group (S. cerevisiae cluster) is consistent with its attribution to the spe-
cies S. cerevisiae on the basis of nDNA/nDNArelatedness (Vaughan-Martini and
Martini, 1987). The behaviour of this strain in respect to growth temperature is
atypical for strains belonging to S. cerevisiae, it did not grow above 32 C (Vau-
ghan-Martini and Martini, 1993). Rodrigues De Sousa et al. (1995) suggested that
DBVPG 6254 could be a thermosensitive mutant, since Madeira-Lopes and Van
Uden (1979) reported a shift of maximum growth temperature to a lower tempe-
rature in a thermosensitive mutant of S. cerevisiae.
In conclusion, the data obtained in this study showed that total DNArestric-
tion profile analysis represents a useful tool for typing, and studying the intraspe-
cific genetic relatedness of yeast isolates and their attribution to the species S.
cerevisiae or S. bayanus. Indeed this technique, since it is relatively elaborate,
could not be considered as the first method of choice when many strains are
analysed. However, recently the use of AFLP (Amplified Fragment Length Poly-
morphism), a molecular method comparable for time requirement, specialised
equipment and software to total DNA restriction profile performed on polyacry-
lamide gel and stained with silver nitrate, has been proposed for the taxonomy
and the genome fingerprinting of bacteria and yeasts (Janssen et al., 1996; de
Barros Lopes et al., 1999; Duim et al., 1999). On the other hand RAPD finger-
print, suggested as a fast molecular technique for the typing and the differentia-
tion of microorganisms, sometimes does not yield reproducible bands and often
requires the use of several primers to generate profiles which discriminate yeast
strains at the interspecific level (Fernndez-Espinar et al., 1998; Xufre et al.,
1998). Moreover, it has been shown that co-migration of RAPD-PCR bands of
almost identical size but different sequences occurs (Oakey et al., 1998). This
suggests that caution must be used when very similar RAPD profiles are
employed for strain typing. Therefore, total DNA restriction profile, for its high
reproducibility, resolution and ability to point out subtle variations in genome
structure, can be useful for typing yeast strains which appear indistinguishable by
other molecular methods.
Acknowledgements
The authors tank A. Vaughan-Martini for a critical review of the manuscript. We
are also grateful to E. Casalone (Dipartimento di Biologia Animale e Genetica,
Firenze, Italy) for providing the purified mtDNAof S. cerevisiae.
200
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