restriction fragment length polymorphism method for Saccharomyces cerevisiae and Saccharomyces bayanus identification in enology

ELSEVIER

FEMS Microbiology Letters 138 (I 996) 239-244

Development of a polymerase chain reaction/restriction fragment length polymorphism method for Saccharomyces cerevisiae and Saccharomyces bayanus identification in enology Isabelle Masneuf a3*, Michel Aigle ‘, Denis Dubourdieu

a

d Fuculte’ d’CFnologie de Bordeaux. 351, Cours de la Libe’ration, 33400 Talence, France ’ IBGC, I, me Camille Saint Sai;ns. 33077 Bordeaux, France

Received 23 February 1996; revised I9 March 1996; accepted I9 March 1996

Abstract S. bayanus and S. paradoxus, first identified by Several yeast strains of the species Saccharomyces cerecisiae, hybridization experiments and measurements of DNA/DNA homology, were characterized using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the MET2 gene. There was no exception to the agreement between this method and classical genetic analyses for any of the strains examined, so PCR/RFLP of the MET2 gene is a reliable and fast technique for delimiting S. cerwisiae and S. bayanus. Enological strains classified as S. bayanus, S. checalieri, and S. capensis gave S. cerevisiae restriction patterns, whereas most S. uc~arum strains belong to S. hayanus. Enologists should no longer use the name of S. bayanus for S. cerer,isiae Gal strains, and should consider S. bayanus as a distinct species.

Keywords: Molecular taxonomy; Saccharom.vces cerel,isiae; Saccharomyces

bayanus; PCR/RFLP;

charomyces

1. Introduction

Electrophoretic

cerecisiae

strains, are at present used to between four species of the genus Saccharomyces sensu strict0 [6,7]: S. cereoisiae, S. bayanus, S. paradoxus and S. pastorianus. Until now, these four species have been differentiated by hybridization experiments and measurements of DNA/DNA homology [8-l I]. Wine yeasts belong to a few species and physiological races. Enologists usually designate them as varieties of S. cerecisiae, i.e. S. cerecisiae var. cerecisiae, var. bayanus, var. ucarum, var. chevalieri, var. cupensis, etc., differentiated by ferrnentation tests of various sugars, although Yarrow [12] considered all these yeasts to be synonyms of S. cerecisiae. However, this designation is inconsistent discriminate

For the last 10 years, genetic characterization of wine yeast strains has progressed by applying different molecular biology techniques: mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis [l,3], separation of yeast chromosomes by pulsed-field electrophoresis 13,181. DNA fingerprinting [2,5] and polymerase chain reaction @CR) [4,51. These methods, first applied to differentiate Sac-

* Corresponding 84 64 68.

author. Tel.: +33 56 84 64 90; Fax: +33 56

0378-1097/96/$12.00 0 1996 Federation PII SO378- IO97(96)001 17-6

of European

Microbiological

karyotype

Societies. All rights reserved

with a more recent classification [ 131. which distinguished 10 species in the genus S~~~c~hcrrom~c~rs, including S. cerel~isiur and S. ba~urtlrs. Thus. S. baymms is used by enologists to designate a physiological race of S. crrrl~isiue. whereas taxonomists now consider it a separate species. Hybridization experiments [l4] show that most wine yeast strains which ferment melibiose (Mel ’ ) belong to S. buyznus, although some Mel’ strains must be included in S. cerer,isiur. Moreover. according to Naumov and his colleagues. S. buyrllrs displayed a species-specific chromosomal pattern which could be easily distinguished from that of S. wrc~~isiae [ 141. In a recent study of the phylogenetic origin of S. carlsbergensis. the use of PCR/RFLP of the MET2 gene was proposed for differentiating S. crrer,isiar from S. buyanus [I s]. In the present study. we intended to verify the concordance between hybridization tests and PCR/RFLP analysis of MET2 gene for delimiting S. cem~isiue and S. bqanu.s species. in order to help characterize a large number of wine yeast strains.

2. Materials and methods 2. I. Yeust struins Yeast strains from different culture collections are listed in Table I (CBS: Centraalbureau voor Schimmelcultures, Delft, The Netherlands: VKM: National Collection of Microorganisms. Moscow. Russia: MUCL: Mycotheque de I’Universite de Louvain: DBVPG: culture collection at Dipartimento di Biologia Vegetale, Universit’a di Perugia, Italy). Yeast strains from CLIB collection (Collection de Levures d’IntCr&t Biotechnologique) VKM Y-502. VKM Y1146,581,SCU ll,SCU74.SCU 13.L 19.L99.L 490, DBVPG 1642. DBVPG 1643. DBVPG 1689. L 579. L 1425 are monosporic cultures of S. cvrec~isiue and S. buyanu.s [14]. Industrial or wild yeasts come from the collection of the Faculte d’(Enologie de Bordeaux. 2.2. Primers Synthetic oligonucleotide primers for MET2 am plification were defined by Hansen and Kielland-

Table

I

Reference htrains of Soc,c,h~i,v,,lf?t,~.\ apeciea (‘I.: type strain) Speck s.

c‘err!~i.Ykf,

Strain designation CBS 5287 = VKM MUCL

Y-502

2783 I

.s. ho~wrut

VKM

s. /‘c”‘/tlow.c

DBVPC

6466

DBVPG

6307

DBVPG

6389

Y-I 146

DBVPG

6490

DBVPG

649 I

DBVPG

6493

DBVPG

6494

s. //CdiC~U.S

CBS 459 r

s. drrlu/irri

CBS 500 T

s. uipw7.\ic

MUCL

27830

Brdndt [ 151. The sequences of primer I and 2 are T-CGAAAACGCTCCAAGAGCTGG and S-GACCACGATATGCACCAGGCAG respectively.

The amplification reaction was carried out on entire yeast cells, after cultivating them on solid YPG medium (20 g/l L-glucose, IO g/I bactopeptone. IO g/l yeast extract, 20 g/l agar) for 72 h until stationary phase [ 161. After two successive washes, cells were maintained at 95°C for IO min. Amplification reactions were performed with a Perkin Elmer DNA thermal Thermocycler 480, under the following conditions: a 100 ~1 reaction mixture was prepared with 2 units of APPLIGENE (Strasbourg, France) Taq DNA polymerase, IO ~1 APPLIGENE IO X C buffer (MgCI? I .S mM), 100 pmol of each primer. 200 PM of each dNTP and about 2-4 X lOh cells. The reaction was cycled 30 times using the following program: 95°C for 30 s to denature the DNA, 60°C for 30 s for the annealing cycles and 72°C for 2 min for the extension reaction. PCR products were precipitated and aliquot samples were restriction digested. Amplified fragments and restriction fragments were analyzed by electrophoresis on a I .8% agarose gel in 1 X TBE buffer (0.09 M Tris-borate, 0.002 M EDTA), visualized after ethidium bromide staining by short UV (524 nm) and photographed with Polaroid 665 film. Boehringer Mannheim DNA molecular mass marker VIII was used.

I. Masneufer al./ FEMS Microbiology Letters 138 (1996) 239-244

2.4.

CHEF

(contour

clamped

homogeneous

electric

Fig. PstI VKM (lane

lb respectively corresponding to EcoRI and cleavage. The reference strain S. cereuisiae Y-502 (lane 8) and S. bayanus VKM Y-l 146 9) and seven wine Mel+ yeasts named S. cerecisiae var. uuarum (lanes l-7) were analyzed. Among these strains, five were previously identified as S. bayanus (lanes l-5) and two as S. cerevisiae (lanes 6 and 7) by hybridization tests [14]. For all four restriction enzymes used, the restriction profiles of lanes l-5 were identical to that of lane 9 (reference strain S. bayanus), and those of lanes 6 and 7 to that of lane 8 (reference strain S. cerecisiae). Additionally, we verified that EcoRI cleaves the S. cerevisiae MET2 fragment (two bands of 219 bp and 369 bp) and does not cleave the S. bayanus MET2 fragment. The behavior of PstI is strictly contrary, since PstI cleaves the S. bayanus MET2 fragment but not that of S. cereuisiae. A Mae111 restriction pattern of two bands (246 bp and 334 bp) is obtained for S. cerevisiae strains identical to that of reference strain S. cerevisiae and a Mae111 cleavage pattern of three bands (390 bp, 130 bp and 60 bp) for S. bayanus strains identical to that of reference strain S. bayanus (data not shown). Finally, NcoI cleaves the MET2 fragment of S. cerevisiae and reference strain (two bands 466 bp and 114 bp) but does not cleave the MET2 fragment of S. buyanus and the reference strain (data not shown). Complete comparison between the results obtained by hybridization experi-

field) gel electrophoresis

Chromosomal DNA was prepared in agarose plugs [ 171 and separated on a 0.8% agarose gel in 1 X TBE, at 165 V, temperature lo”C, using the following program [ 181: Switch Switch

12.5 h 16.5 h

40 to 90 s 80to 120s

The gel was stained with ethidium photographed with Polaroid 665 film.

bromide

241

and

3. Results Hansen and Kielland-Brandt [ 153 have studied the following strains by PCR/RFLP: S. cerevisiae S288C, S. bayanus CBS 380 and S. uuarum CBS 395. We have extended the PCR/RFLP analysis of the MET2 gene to 12 Mel+ strains (ex. ucarum) which had already been identified as S. cerecisiae and S. bayanus by hybridization experiments by Naumov et al. [14]. The enzyme PstI was associated with EcoRI, NcoI and MaeIII, because while there is a PstI site in the MET2 sequence of S. bayanus, there is none in the MET2 sequence of S. cereuisiae. Some results obtained are shown in Fig. la and

tG92pb

+ t

404 pb 242 pb

Fig. 1. MET.2 DNA from Mel+ Saccharomyces wine yeast strains, identified by Naumov et al. [14]. Digestion by EcoRl (a) and PsrI (b). Lane I: S. bayanus SCU 11; lane 2: S. bayanus SCU 13; lane 3: S. bayanus SCU 73; lane 4: S. bayanus L 19; lane 5: S. ba.yanus L490; lane 6: S. cerecisiae L 579; lane 7: S. cerevisiae L 1425; lane 8: S. ceredsiae VKM Y-502; lane 9: S. bayanus VKM Y -1146; M: molecular mass marker.

ments [14] and PCR/RFLP of the MET2 gene for the strains analyzed are listed in Table 2. The concordance between these two methods for S. cerec,isiae and S. bayanu.s species delimitation do not have any exceptions for the 14 strains analyzed. PCR/RFLP of the MET2 gene has been applied to strains identified as S. paradoxus by DNA/DNA hybridization experiments [ 191 and electrophoretic karyotyping [20]. In the absence of data on the S. paradoxus MET2 sequence, the same restriction enzymes were tested for these analyses. Results obtained for four S. paradoxus strains are shown in Fig. 2a and Fig. 2b, corresponding to the EcoRI and Mae111 cleavage, respectively. For each of these figures, the restriction patterns of the reference strains S. bayanus (lane 5) and S. cerecisiae (lane 6) are shown. Although of the same size as those of S. cerecisiae and S. bayanus. the S. paradoxus amplified MET2 fragment differs in that it is not cleaved by EcoRI or PstI (data not shown). The restriction by Mae111 gives a two band pattern (334 bp and about 220 bp) (Fig. 2b). Lastly, the amplified MET2 gene is not cleaved by NcnI (results not shown). The same results were obtained for all the S. paradoxus strains studied (Table I ). By the same methods, we analyzed 17 industrial dry yeast strains, designated as S. cerelisiae, four fermenting galactose (Gal+) (VLI; VL3c; 7 I B; WET 136) and I3 not fermenting galactose (Gall ) (VL3a; FIO: KD; B0213; CH 158; N 96; Vin 13: QA23; IOC; SBI; 016: CIVC 8130: DVIO). The usual enological designations for those two cate1

2

3

4

5

6

Table 2 Specica delimitation of S. c~rrr~~i.sitrr/S. hqurw by hybridizing and PCR/RFLP of the MET2 gene tests (R: reference strain) Strain

Hybridization

CLIB 2 19: VKM Y-502 R GLIB 2 18: VKM Y- 1 146 R CLIBIOI: SCU I I CLIB 103: SCU 73 GLIB 102: SCU I? CLIB 108: L I9 CLIB I IO: L 490 GLIB 94: L 579 CLIB 95: L 1425

S. S. s. s. s. s. s. S. S.

cvrwisiar ha~unus htr~trnu.\ htr~trr1u.s hqww.s htr!unu.s hqcmus

test

PCR-RFLP S. S. S. s. s. s. s.

test

crrerisiar hrryatw

tqwul.\ hawlilu.~ hu,wrnu.s htr~clnu.s htrwnus

crrec~isitre

S. crrwisirw

crrrf,isiae

S. cem~isitrr

The smx results were obtained for the S. hcrwnus strains 5x1, CLIB 109: L 99. CLIB 113: DBVP 1642, CLIB 114: DBVPG 1633 and CLIB I 15: DBVPG 1689.

gories are S. cerelisiae var. cerecisiae (Gal’) and S. cerecisiae var. bayanus (Gal-). Results for five strains are given in Fig. 3a,b. All the strains, Gal+ or Gal-, have an EcoRI and PstI restriction pattern of the amplified MET2 gene typical of S. cerecisiae. We also analyzed many wild wine yeast strains. isolated from spontaneous fermentations: 8 Gal ’ strains, 45 Galstrains, 2.5 Mel’ strains, 2 S. cerecisiae var. capensis, Gall and Mall strains (which do not ferment maltose), 3 S. cerecisiae var. cheralieri Gal + and Mal strains and one S. italicus strain (Table 1). Restriction digests of the MET2 PCR/amplified DNA produced by EcoRI, Mae111 and PstI are typical of S. cererisiae. On the other hand. most Mel+ strains. named S. cerecisiae var. w’arum (22 strains/25), gave a restriction digest

M

-692 pb 4-404 pb 242 pb

Fig. 2. MET2 DNA restriction analysis of S. pwtiduxm strains with the endonucleases Ec‘coRI(a) and Mae111 (b). Lanes l-4: 6466, 6489, 6490, 6493; lane 5: S. huwnus VKM Y-l 146. lane 6: S. crrwisiue VKM Y-502; M: molecular mass marker,

S. pcwudo.rus

I. Masneuj’et al. / FEMS Microbiology

Letters 138 (1996) 239-244

243

(b)

(4

Fig. 3. MER DNA restriction analysis of industrial wine yeast strains with restriction endonucleases EcoRI (a) and PHI (b). Lane 1: FlO; lane 2: KD: lane 3: B0213; lane 4: DVIO; lane 5: QA23; lane 6: S. buyanus VKM Y-l 146; lane 7: S. cerevisiae VKM Y-502; M: molecular mass marker.

typical of S. buyanus species; three strains, however, must be included in the species S. cerecisiae (data not shown). In previous works, many authors have shown that electrophoretic karyotyping analyses can be used to differentiate species belonging to the genus Succharomyces sensu strict0 [20], especially S. cerecisiae and S. bayanus species [ 14,21,22]. Karyotype analyses of wine yeast strains studied in the present work corroborate results obtained by PCR/RFLP of the MET2 gene. Chromosomal DNA patterns of S. buyanus strains previously identified by PCR/RFLP of the MET2 gene displayed band patterns similar to 1

2

3

4

5

6

7

8

9

10

Fig. 4. Karyotypes of S. cerevisiae and S. buyanus wild wine yeast strains. Lanes 1, 3, 4, 5, 7, 8, 9: S. cereL,isae; lanes 2, 6, IO: S. bapznus.

one another cereuisiue

but different from band patterns strains (Fig. 4).

of S.

4. Discussion and conclusion Hybridization experiments and DNA/DNA hybridization, which are difficult to implement and time consuming, are hardly relevant to fast identification of wine yeast species. On the other hand, PCR/RFLP of the MET2 gene technique is useful and fast. Direct amplification on entire cells and the use of PstI enzyme have enabled us to improve this technique for delimiting S. cerevisiue and S. buyunus. The findings obtained are perfectly consistent with the results of hybridization experiments. They are also in concordance with differentiation based on karyotype analysis, but this method takes much longer and its results are tedious to interpret because of the intraspecific polymorphism of wine yeast karyotypes [3,18]. The first results show that S. paradoxus species can be identified by PCR/RFLP of the MET2 gene. Nevertheless, the MET2 gene sequence of S. purudoxus species has to be established in order to improve the restriction enzyme choice for the RFLP analyses. From a taxonomic point of view, this method gives some new information for identifying enological yeast species. After analyzing a large number of industrial and wild strains, we show that enological strains classified as S. cereuisiue var. buyunus, S. cereuisiae var. cupensis, S. cerevisiue var. chevulieri are in fact S. cerevisiue that have probably lost

I. M~istwrfrt

244


certain characteristics: the ability to ferment galactose in the case of S. cerecisiue var. buyunus and S. cerersisiae var. cqwnsis, maltose in the case of S. var. cerei,isiue var. cupensis and S. cerecisiae cher’alieri. However, the majority of wine Mel’ yeast strains (ex. S. cerevisiae var. ucarum) are S. ba.wznu.s, in accordance with Naumov’s and Kielland-Brandt’s findings and the Mel ’ phenotype can no longer be used to differentiate S. cereuisiae from S. bayanus. To avoid confusion in enological terminology, the name S. bqunus should no longer be used to refer to S. cerecisiae Galstrains, but should be applied only to strains belonging to the distinct species S. bagnus.

Letter.\ 138 (IYY6I

239-244

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[I

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( 1984)

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Acknowledgements

[I31 Barnett,

J.A..

Payne,

R.W.

Yeasts: Characteristics

We wish to thank R.K. Mortimer (Berkeley), LALLEMAND Inc. (Canada), H.V. Nguyen (GLIB, Paris) and G.I. Naumov (Moscow) for strains and J.C. Gandar (IBGC, CNRS, Bordeaux) for the synthesis of oligonucleotide primers. This research was in part supported by SARCO (Bordeaux, France).

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(1990)

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[t61

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