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

Characterisation and typing of Saccharomyces strainsby DNA fingerprinting

C.VITI, D. FORNI, S. VENTURA, A. MESSINI, R. MATERASSI,L. GIOVANNETTI*

Dipartimento di Biotecnologie Agrarie, Università 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 allstrains and showed that they constitute two well separated genomic taxa, one including thetype strain of S. cerevisiae and the other the type strain of S. bayanus. The data obtained byanalysing mtDNA restriction profiles and mtDNA Southern hybridisation were consistentwith results of total DNA restriction 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, DNA fingerprinting, 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;Guillamón 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 afew different carbon sources (Barnett, 1992). For these reasons, and with theknowledge 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 derWalt, 1970) into a single species: S. cerevisiae that also included S. bayanus, S.pastorianus and S. paradoxus. Afterwards, studies on nDNA/nDNA (nuclearDNA) reassociation within the species Saccharomyces cerevisiae sensu Yarrowindicated the presence of four species, S. cerevisiae, S. bayanus, S. paradoxus andS. 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 andMartini, 1994), random amplified polymorphic DNA analysis (Molnár et al.,1995; Paffetti et al., 1995; Torriani et al., 1999), mitochondrial DNA restrictionanalysis (Querol et al., 1992; Guillamón 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 phenotypiclevel within the Saccharomyces sensu stricto complex (Vaughan-Martini andMartini, 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 otherrelated 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 indigenousstrains and to monitor the stability of used strains during fermentation process andafter storage. There is the need to control fermentation process and to ensure thatselected yeast strains conduct it to obtain a final product with specific characteri-stics

In this study total DNA and mtDNA restriction profile analysis, and mtDNASouthern hybridisation were applied in order to characterise and verify the taxo-nomic assignment of Italian wine yeast strains belonging to the Saccharomycessensu 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 wastested.

MATERIALS AND METHODS

Yeast strains. On the basis of optimum growth temperature, the strains studiedwere 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 temperatureof growth was <30 °C and as non-cryotolerant when its optimal temperature ofgrowth was > 30 °C. Strains investigated together with corresponding collectionnumbers, origin and optimal growth temperature are given in Table 1.

192

DNA extraction. 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 inexponential phase by centrifugation (5300 x g for 20 min at 4 °C) and washedtwice 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 about3 hours. To lyse spheroplasts SDS was added to a final concentration of 1% andthe mixture was incubated at 65 °C for 30 min. After the addition of 1/3 volumeof 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 tothe recovered supernatant to precipitate nucleic acids. RNAse treatment and DNArecovery were performed according to Giovannetti et al. (1990).

193

TABLE 1 - Strains used, their isolation source and optimal growth temperature

Ref.a Speciesb Species Strain Isolation source Topt.c

(original designationsepithet)

1 S. cerevisiae S. cerevisiae DBVPGd6173T Beer >30g

2 S. cerevisiae B7e Fermenting grape juice >30h

3 S. cerevisiae Cu11e Fermenting grape juice >30h

4 S. cerevisiae G1e Fermenting grape juice >30h

5 S. ellipsoideus PC80e Fermenting grape juice >30i

6 S. cerevisiae S. oviformis DBVPGd6254T Fermenting grape juice <30i

7 S. oviformis DBVPGd1589 Wine >30i

8 S. bayanus 1024f Fermenting grape juice >30j

9 S. bayanus 7541f Fermenting grape juice >30j

10 S. uvarum 11052f Fermenting grape juice >30k

11 S. bayanus S. bayanus DBVPGd6171T Beer <30g

12 S. bayanus 12130f Fermenting grape juice <30j

13 S. bayanus 12212f Fermenting grape juice <30j

14 S. bayanus S. uvarum DBVPGd6179T Currant juice <30i

15 S. uvarum 12138f Fermenting grape juice <30k

16 S. bayanus 12000f Fermenting grape juice <30k

aReference number in figures; bVaughan-Martini and Martini, 1998; coptimal growthtemperature; dCollection of Dipartimento di Biologia Vegetale, Università di Perugia,Italy; eCollection of Dipartimento di Biotecnologie Agrarie, Università di Firenze, Italy;fCollection of Ente per gli Studi e l’Assistenza Viticola ed Enologica dell’Emilia Roma-gna, Italy; gBerardi and Fatichenti, 1989; hDe Philippis et al., 1994; iour unpublishedresults; jCastellari (Ente per gli Studi e l’Assistenza Viticola ed Enologica dell’EmiliaRomagna, Italy) personal communication; k Castellari et al., 1994; Ttype strain.

DNA digestion. Each µg of total DNA was digested with 3 units of restrictionendonuclease. 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 toselect enzymes giving electrophoretic patterns with several well separated lowmolecular weight bands suitable for computer analysis. SacI and StuI were selec-ted to perform SDS-PAGE of DNA of all yeast strains studied. Ten µg of totalDNA of each sample were digested with the chosen endonucleases. SDS-PAGEof total DNA digests was performed as described by Giovannetti and Ventura(1995) and Ventura et al. (1993). Band patterns were recorded using a LKBUltroscan laser densitometer, using the procedure described by Giovannetti andVentura (1995) and Ventura et al. (1993). The similarity between all pairs of pat-terns was determined using the Dice similarity coefficient (SD) (Sneat and Sokal,1973). Strains were clustered by analysing the matrix of SD 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 analysisperformed 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 DNA restriction profile remained stableover time, DNA of the strains G1 and 12138, obtained from two cultures, weredigested with StuI and submitted to electrophoretic run. DNA samples from asame strain gave identical electrophoretic profiles (data not shown).

mtDNA restriction profile and mtDNA Southern hybridisation. Restrictionendonucleases such as AluI, DdeI, HaeIII, HinfI, MaeI, MaeII, Sau3A1, RsaIrecognise a high number of sites in yeast nDNA, but few sites in mtDNA. As aresult, restriction fragments of high molecular weight can be obtained frommtDNA sequences that are easily distinguishable by gel electrophoresis fromfragments of low molecular weight obtained from nDNA (Querol et al., 1992).This makes it possible to obtain mtDNA restriction profiles without previouslyisolating mitochondria and/or purifying mtDNA.

Five µg of total DNA were digested with AluI or RsaI following the manu-facturer’s instruction (Roche Diagnostics, Switzerland). DNA fragments wereseparated by 0.8% (wt/vol) agarose gel electrophoresis in Tris-borate buffer (90mM 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 30min) and neutralisation (0.5 M Tris-HCl, 1.5 M NaCl, pH 7.5 for 40 min), DNAfragments were transferred under low vacuum (5 inches of Hg) for 90 min in 10x SSC (1.5 m NaCl, 0.15 M Na3citrate) 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 DNALabelling and Detection Kit (Roche Diagnostics, Switzerland).

Hybridisation and the immunoenzymatic detection of hybridised fragments

194

were performed, in accord to the Nonradiactive DIG DNA Labelling and Detec-tion Kit handbook (Roche Diagnostics Switzerland), inside the glass tube of ahybridisation oven. Hybridisation was performed at 42 °C for 16 h.

RESULTS

Total DNA restriction profile analysis The restriction endonucleases StuI or SacI gave electrophoretic profiles with wellseparated bands in the range between 298 and 1230 bp and between 394 and1033 bp, respectively. As an example the profiles obtained with StuI are reportedin 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 bandsfrom all of the others. After the determination of the SD coefficient between eachpair of profiles, the genomic relationship among strains was evaluated with

195

strains

bp

FIG. 1 – Total DNA restriction profiles of Saccharomyces strains performed by SDS-PAGE and silver staining. DNA was digested with StuI. Reference numberslisted in Table 1 designate strains. Lanes M contain DNA molecular weightmarker VI (Roche Diagnostics, Switzerland).

UPGMA. The dendrograms obtained from UPGMA analysis indicated a strikingcorrespondence 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 strainsincluding the type strain of S. cerevisiae (DBVPG 6173) and the cryotolerant

196

FIG. 2 – Dendrogram based on UPGMA clustering of total DNA restriction profile dataobtained with StuI. Reference numbers listed in Table 1 designate strains.

FIG. 3 – Dendrogram based on UPGMA clustering of total DNA restriction profile dataobtained with SacI. Reference numbers listed in Table 1 designate strains.

strain DBVPG 6254; the other (B) comprised cryotolerant strains including thetype strain of S. bayanus (DBVPG 6171). The two clusters joined, with bothenzymes, at SD value around 0.60. Inside each group the strains showed SD levelshigher than 0.70. Differences in the degree of genomic relationship among strainsin the two dendrograms were modest.

mtDNA restriction profile and mtDNA Southern hybridisationThe mtDNA restriction profiles, obtained with enzymes RsaI or AluI, are reportedin figures 4 and 5. Inside the group of cryotolerant strains, except for the strainDBVPG 6254, a high level of similarity of mtDNA profiles was evident. Thestrains DBVPG 6171 (type strains of S. bayanus), DBVPG 6179 and 12212, andthe strains 12138 and 12000 (Fig. 4a, 5a) showed identical profiles. On the otherhand the non-cryotolerant strains showed more mtDNA divergence. Most ofstrains exhibited a specific profile with only the pair DBVPG 6173 (type strain ofS. cerevisiae) and DBVPG 1589 showing the same profile. The mtDNA profilesof non-cryotolerant strains, independently of the enzyme used, were characterisedby the presence of some larger bands than those found in non-cryotolerant strains.To further test the effectiveness of mtDNA profiles to distinguish cryotolerantfrom non-cryotolerant strains, mtDNA profiles obtained with RsaI or AluI, werehybridised with purified mtDNA of S. cerevisiae. While, independently from theenzyme used, the polymorphism of non-cryotolerant strains was confirmed, allcryotolerant strains, except DBVPG 6254, did not hybridise with the S. cerevisiaemtDNA probe (Fig. 4b, 5b).

DISCUSSION

The application of total DNA restriction profile analysis, independently from therestriction endonuclease used, permitted a clear typing of strains tested andshowed 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 1designate strains. Lanes M contain digoxigenin-labelled DNA molecular weightmarker II (Roche Diagnostics, Switzerland).

strains

bpbp

ded the type strain of S. cerevisiae, all the other non-cryotolerant strains plus thecryotolerant strain DBVPG 6254. The other taxon comprised the type strain of S.bayanus and all remaining cryotolerant strains. The degree of genotypic diversityfound inside each cluster and between the two clusters (Fig. 2, 3) was much lowerthan that found by Barberio et al., (1994) who applied the same molecularapproach to strains identified, by classical methods, as S. cerevisiae or S. baya-nus. These authors reported that strains attributed to S. cerevisiae or S. bayanusshowed a value SD of 0 (no DNA restriction fragment in common). This is verypuzzling since it would be expected that strains belonging to the same or highlycorrelated species should have at least some DNA bands in common. It is likelythat the apparent absence of similarity found by these authors was due to the useof restriction endonucleases that yield a very small number of fragments (notmore than sixteen with HpaI and eight with KspI). On the contrary the advantageof total DNA restriction profile obtained using polyacrylamide gel and stainedwith silver nitrate is that of giving finely resolved restriction profiles with a largenumber of well separated low molecular weight bands (Giovannetti and Ventura,1995). This is advantageous because the analysis of a large number of restrictionsites allows the genome structure to be randomly assayed at many loci.

The analysis of mtDNA restriction profiles and mtDNA Southern hybridisa-tions provided further evidence that the strains studied constitute two distinctgenomic groups, one including all cryotolerant strains (except DBVPG 6254),and the other with non-cryotolerant strains. Restriction fragments of mtDNAhigher than 6557 bp were only found in non-cryotolerant strains and DBVPG6254. These bands could be typical of the species S. cerevisiae (Guillamón et al.,1994; Guillamón et al., 1996). Moreover no hybridisation signal was obtained incryotolerant strains when the mtDNA of a S. cerevisiae strain was used as aprobe. 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 DNArestriction 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 1designate strains. Lanes M contain digoxigenin-labelled DNA molecular weightmarker II (Roche Diagnostics, Switzerland).

strains

bpbp

199

TAB

LE

2 -

Tax

onom

ic p

ositi

on o

f st

rain

s st

udie

d

Stra

inO

rigi

nal e

pith

etSp

ecie

saT

optb

Taxo

nom

ic p

ositi

on b

ased

on:

Iden

tific

atio

nde

sign

atio

nsba

sed

onth

is s

tudy

Tota

l DN

Am

tDN

Am

tDN

ASo

uthe

rn

rest

rict

ion

prof

ilere

stri

ctio

nhy

brid

isat

ion

DB

VPG

617

3TS.

cer

evis

iae

S. c

erev

isia

e>

30D

BV

PG62

54T

S. o

vifo

rmis

S. c

erev

isia

e<

30S.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

e

B7

S. c

erev

isia

e>

30S.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

e

Cu1

1S.

cer

evis

iae

>30

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

G1

S. c

erev

isia

e>

30S.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

e

PC80

S. e

llip

soid

eus

>30

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

DB

VPG

158

9S.

ovi

form

is>

30S.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

e

1024

S. b

ayan

us>

30S.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

e

7541

S. b

ayan

us>

30S.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

e

1105

2S.

uva

rum

>30

S. c

erev

isia

eS.

cer

evis

iae

S. c

erev

isia

eS.

cer

evis

iae

DB

VPG

617

1TS.

bay

anus

S. b

ayan

us<

30

DB

VPG

617

9T

S. u

varu

mS.

bay

anus

<30

S. b

ayan

usS.

bay

anus

Non

-S. c

erev

isia

eS.

bay

anus

1213

0S.

bay

anus

<30

S. b

ayan

usS.

bay

anus

Non

-S. c

erev

isia

eS.

bay

anus

1221

2S.

bay

anus

<30

S. b

ayan

usS.

bay

anus

Non

-S. c

erev

isia

eS.

bay

anus

1213

8S.

uva

rum

<30

S. b

ayan

usS.

bay

anus

Non

-S. c

erev

isia

eS.

bay

anus

1200

0S.

bay

anus

<30

S. b

ayan

usS.

bay

anus

Non

-S. c

erev

isia

eS.

bay

anus

a Vau

ghan

-Mar

tini a

nd M

artin

i, 19

98; b o

ptim

al g

row

th te

mpe

ratu

re; T

type

str

ain.

The high genetic similarity found, with molecular approaches applied in thisstudy, between the type strain of S. cerevisiae (DBVPG 6173) and the non-cryo-tolerant strains originally identified as S. uvarum (11052) and S. bayanus (1024and 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 thesestrains, have been demonstrated to be inadequate to clearly distinguish strainsbelonging to S. cerevisiae from those belonging to S. bayanus. In regard to theother 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-cryotolerantgenomic group (S. cerevisiae cluster) is consistent with its attribution to the spe-cies S. cerevisiae on the basis of nDNA/nDNA relatedness (Vaughan-Martini andMartini, 1987). The behaviour of this strain in respect to growth temperature isatypical 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 thatDBVPG 6254 could be a thermosensitive mutant, since Madeira-Lopes and VanUden (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 DNA restric-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 areanalysed. However, recently the use of AFLP (Amplified Fragment Length Poly-morphism), a molecular method comparable for time requirement, specialisedequipment and software to total DNA restriction profile performed on polyacry-lamide gel and stained with silver nitrate, has been proposed for the taxonomyand the genome fingerprinting of bacteria and yeasts (Janssen et al., 1996; deBarros 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 oftenrequires the use of several primers to generate profiles which discriminate yeaststrains at the interspecific level (Fernández-Espinar et al., 1998; Xufre et al.,1998). Moreover, it has been shown that co-migration of RAPD-PCR bands ofalmost identical size but different sequences occurs (Oakey et al., 1998). Thissuggests that caution must be used when very similar RAPD profiles areemployed for strain typing. Therefore, total DNA restriction profile, for its highreproducibility, resolution and ability to point out subtle variations in genomestructure, can be useful for typing yeast strains which appear indistinguishable byother molecular methods.

AcknowledgementsThe authors tank A. Vaughan-Martini for a critical review of the manuscript. Weare also grateful to E. Casalone (Dipartimento di Biologia Animale e Genetica,Firenze, Italy) for providing the purified mtDNA of S. cerevisiae.

200

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