Saccharomyces uvarum, a proper species within Saccharomyces sensu stricto

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1 FEMS Microbiology Letters 192 (2000) 191^196 Saccharomyces uvarum, a proper species within Saccharomyces sensu stricto e Abstract Andrea Pulvirenti a; *, Huu-Vang Nguyen b, Cinzia Caggia c, Paolo Giudici a, Sandra Rainieri d, Carlo Zambonelli e a Facolta di Agraria, Universita di Modena e Reggio Emilia, Viale Kennedy 17, Reggio Emilia 42100, Italy b Collection de Levures d'intëreªt Biotechnologique, Laboratoire de Microbiologie et Gënëtique Molëculaire INA-PG INRA, BP01, Thiverval-Grignon, France c Istituto di Industrie Agrarie, Universitä di Catania, Via Santa So a, 98, Catania 95123, Italy d Institute for Wine Biotechnology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita di Bologna, Villa Levi, via F.lli Rosselli 107, Reggio Emilia 42100, Italy Received 21 July 2000; received in revised form 7 September 2000; accepted 11 September 2000 Saccharomyces uvarum is proposed as a proper species within the complex Saccharomyces sensu stricto. Molecular characteristics including the similarity of the restriction profile of the non-transcribed spacer 2 (NTS2) and of the D1/D2 sequences of the rdna, as well as other genotypic and phenotypic characteristics confirm that this group of strains is highly homogeneous and distinguishable from other species of the Saccharomyces sensu stricto group. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Molecular typing; Ribosomal DNA; Taxonomy; Saccharomyces sensu stricto; Saccharomyces uvarum 1. Introduction The Saccharomyces sensu stricto group is composed of four species: Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus and Saccharomyces pastorianus [1]. This grouping, mainly based on DNA reassociation analysis and on some physiological characteristics does not seem to be natural for the species S. bayanus. This species, in fact, contains strains that possess heterogeneous phenotypic and genotypic characteristics [2]. The S. bayanus type strain has been con rmed to be a natural inter-speci c hybrid between Saccharomyces uvarum and S. cerevisiae [3]. It possesses characteristics that are not encountered in any other natural isolate and thus it is not representative of the species [2,3]. Several studies have recently demonstrated the existence of a homogeneous group of strains within the species * Corresponding author: Tel.: +39 (522) ; Fax: +39 (522) ; andrea.pulvirenti@tiscalinet.it S. bayanus [2^4]. These strains were referred to as S. uvarum because they possess the phenotypic characteristics of strains originally described for the species S. uvarum by van der Walt in 1970 [5]. They are widely distributed in nature and are commonly isolated from fermented beverages, especially if maintained at low temperature (0^2³C) [6^8]. The present study was carried out to give further evidence of the existence of a separate natural group, within the Saccharomyces sensu stricto, that can be considered a proper species; S. uvarum. To this end we have investigated the ribosomal DNA (rdna) of strains that were grouped under S. uvarum according to some common characteristics described in a previous study [2,9,10]. In particular these strains exhibit (i) the typical sugar fermentation pattern of the former S. cerevisiae physiological race uvarum [11]; (ii) an electrophoretic karyotype characterised by the presence of two bands in the area between 365 and 225 kb; (iii) a de ned fermentation pro le in grape juice that di ers from that of S. cerevisiae and S. paradoxus strains in the amount of glycerol, succinic acid, and acetic acid produced, as well as whether they synthesise or degrade malic acid; and (iv) a high sporulation / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (00)

2 192 A. Pulvirenti et al. / FEMS Microbiology Letters 192 (2000) 191^196 ability. We have analysed the RFLP patterns carried out on PCR fragments obtained using primers that could amplify speci cally the non-transcribed spacer sequence (NTS2 or IGS2, Inter Genic Spacer), that was demonstrated to be suitable for distinguishing sibling yeast species. We have also analysed the nucleotide sequence from the 5P end of the 26S rdna gene (D1/D2 region) that has been demonstrated to be instrumental in the identi cation and the segregation of ascomycetous yeast species. 2. Materials and methods 2.1. Yeast strains The yeast strains that were used in the present study are listed in Table 1. S. uvarum strains belonging to the Dipartimento di Protezione e Valorizzazione Agroalimentare (DIPROVAL) culture collection (Universitä di Bologna, Reggio Emilia, Italy) were isolated from cold-stored grape juice obtained from wineries of di erent areas of Italy. Strains CLIB 103, CLIB 115 and SRc 55 were isolated from French wineries. The strains were described in previous studies [2,4,10,12]. The natural isolates were compared with the type strains of S. cerevisiae, S. pastorianus, S. paradoxus, and the type strains of the ancient species S. uvarum [5] and Saccharomyces abuliensis [13] Isolation of genomic DNA Yeast was grown overnight in yeast peptone dextrose broth (YPD: yeast extract 2%, w/v; peptone 2%, w/v; dextrose 4%, w/v) with shaking at 28³C. rdna was extracted from 3-ml cultures using the method described by Ho man and Winston [14] PCR/RFLP condition of the NTS2 region The ampli cation procedure of the rdna region NTS2 was carried out using the method described by Nguyen and Gaillardin, [4]. Enzymatic digestion was carried out on 6 Wl of ampli ed DNA to a nal volume of 20 Wl with AluI, (Gibco BRL), BanI (Biolabs), SmaI (Biolabs) and TaqI (Biolabs). Restriction fragments were separated for 2 h in 2% agarose (NuSieve 3:1) in 0.5UTBE at 130 V. Gels were stained with ethidium bromide, destained in sterile water and photographs were taken with a Bio-print 5.08 camera system (Vilber Lourmat) Partial sequencing of the 26S of rdna and sequences analysis DNA for sequencing was ampli ed as described by Kurtzman and Robnett [15], with the forward and reverse primers (NL1-NL4) two times each. The ampli ed D1/D2 DNA fragments were puri ed with a QIAquick PCR puri cation kit (Qiagen). Sequences obtained were analysed and compared using a Staden package (Dear and Staden), a GCG Wisconsin package (Genetics Computer Group, Madison, WI, USA), and to sequences in GenBank. 3. Results 3.1. RFLP of the NTS2 region of rdna By comparing the nucleotidic sequences of the area NTS2 of the Saccharomyces sensu stricto type strains, of the ancient type strain of S. uvarum (CBS 395) as well as strain CLIB 218 a monosporic culture obtained from S. abuliensis CBS 7001, it was possible to establish a map for AluI, BanI, TaqI and SmaI restriction sites (results not shown). This map con rmed the divergence of the NTS2 region for all the strains tested and showed their charac- Table 1 List of Saccharomyces strains used in the present study Species CBS number a Other culture collection numbers b S. uvarum DIPROVAL S32 S. uvarum DIPROVAL C11 S. uvarum DIPROVAL C12 S. uvarum DIPROVAL M31 S. uvarum DIPROVAL S31 S. uvarum DIPROVAL S12 S. uvarum DIPROVAL M32 S. uvarum DIPROVAL M12 S. uvarum DIPROVAL C32 S. uvarum DIPROVAL S22 S. uvarum DIPROVAL M11 S. uvarum DIPROVAL C31 S. uvarum DIPROVAL S. uvarum DIPROVAL S. uvarum SRc 55 S. uvarum CLIB 115 S. uvarum CLIB 103 S. uvarum c CBS 395 CLIB 251 S. bayanus d CBS 380 CLIB 181 S. abuliensis (derivative CBS 7001) CLIB 218 S. bayanus CBS 1505 S. bayanus CBS 1604 CLIB 253 S. cerevisiae CBS 5635 S. cerevisiae CBS 4054 S. cerevisiae d CBS 1171 CLIB 227 S. cerevisiae DIPROVAL 7070 S. paradoxus CBS 5829 S. paradoxus UCD 51^186 S. paradoxus d CBS 432 CLIB 228 S. pastorianus CBS 1513 S. pastorianus d CBS 1538 CLIB 218 UCD: Department of Viticulture and Enology University of California, Davis, CA, USA. a CBS: Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. b CLIB: Collection de Levures d'intëreªt Biotechnologique, INRA, Thiverval-Grignon, France. c Former type strain from the time when S. uvarum was recognised as a species [5]. d Type strain [1].

3 A. Pulvirenti et al. / FEMS Microbiology Letters 192 (2000) 191^ teristic restriction patterns. The combined use of these restriction enzymes allowed the separation of strains belonging to the Saccharomyces sensu stricto species (Fig. 1). In particular; AluI could separate S. cerevisiae type strain and S. paradoxus type strain from S. pastorianus and S. uvarum strains, moreover it could separate S. pastorianus type strain from S. uvarum type strain. BanI could separate S. cerevisiae type strain from the other strains used in this experiment. TaqI could separate S. cerevisiae type strain and S. paradoxus type strain from S. pastorianus and S. uvarum type strains as well as separating S. pastorianus type strain from S. uvarum type strain. SmaI could separate S. paradoxus type strain from the other strains tested. The RFLP of the NTS2 region was carried out on S. uvarum strains isolated from cold-stored grape juice and was then compared to that of Saccharomyces strains analysed in previous studies. The S. uvarum strains showed a very homogeneous RFLP pattern corresponding to that showed by the ancient type strain CBS 395 (Fig. 2, lane 15) and of strains CBS 1604 (Fig. 2 lane 17) and CBS 7001 (Fig. 2 lane 23). Fig. 1. Restriction sites in the NTS2 area of strains Sequencing of the D1/D2 region of the rdna We ampli ed and sequenced the domain D1/D2 large sub-unit (26S) rdna from seven S. uvarum strains isolated from cold-stored grape juice, strains CBS 1505 and CBS 1604 as well as the two ancient type strains, S. uvarum CBS 395 and S. abuliensis CLIB 218 derivative of CBS 7001 (Table 2). When the sequences were compared, a 100% homology was found among all these strains except for the ancient S. uvarum type strain CBS 395 which shared only 98% of homology with respect to this majority group. Compared to D1/D2 sequences from the currently recognised Saccharomyces sensu stricto in GenBank, the S. uvarum group showed an homology degree higher than 99% with S. bayanus and S. pastorianus type strains. As expected this group showed a much lower degree of homology with S. cerevisiae and S. paradoxus type strains, 97 and 98% respectively. The D1/D2 sequence comparison showed that S. abuliensis is in fact a strain of S. uvarum as already suggested [2,3], while the the D1/D2 region of the S. uvarum type strain CBS 395 diverged from other S. uvarum strains.

4 194 A. Pulvirenti et al. / FEMS Microbiology Letters 192 (2000) 191^196 Fig. 2. A, B, C and D: Restriction by AluI; BanI; TaqI; SmaI enzymes: from left to right: X Marker V pbr 322 with the ampli ed fragment; 1 = S32; 2 = C11; 3 = C12; 4 = M31; 5 = S31; 6 = S12; 7 = M32; 8 = M12; 9 = C32; 10 = S22; 11 = M11; 12 = C31; 13 = DIPROVAL 11204; 14 = DIPROVAL 12233; 15 = CBS 395; 16 = CBS 1505; 17 = CBS 1604; 18 = CLIB 251; 19 = DIPROVAL 7070; 20 = CBS 5635; 21 = CBS 4054; 22 = CBS 1171; 23 = CBS 7001; 24 = UCD 51^186; 25 = CBS 1513.

5 A. Pulvirenti et al. / FEMS Microbiology Letters 192 (2000) 191^ Table 2 Homology percent of the nucleotides of the D1/D2 region of the rdna Strain 4. Discussion S. abuliensis a CBS 7001 S. bayanus b CBS 380 S. uvarum strains are currently classi ed as S. bayanus [1]. In this study we demonstrate using molecular methods that S. uvarum is a homogeneous taxonomic group, distinct from S. bayanus. A separation of S. uvarum from S. bayanus strains had already been pointed out by Gouliamova and Hennabert, by analysis and comparison of the sequence of the ITS region [16]. Nevertheless, their study was limited to the type strain of S. bayanus (CBS 380, see also Fig. 3) and the ancient type strain of S. uvarum (CBS 395) and therefore could not provide full evidence of the existence of a separate species for S. uvarum strains. S. bayanus type strain CBS 380, in contrast with S. uvarum strains does not ferment melibiose; presents a fermentation pro le in grape juice and an electrophoretic karyotype typical for hybrid cultures [17,18] and does not sporulate. By combining the physiological characteristics of S. uvarum strains with the sequencing data reported in the present study, we are now able to state that S. uvarum S. cerevisiae b CBS 1171 can be considered a proper species. The name S. uvarum was chosen because this group of strains possess the same physiological characteristics typical of the S. uvarum species described by van der Walt in 1970 [5]; namely the ability to ferment melibiose and the inability to grow at temperatures above 37³C. However, this species is not fully typi ed by the ancient S. uvarum type strain CBS 395. This strain, in fact, possesses the same electrophoretic karyotype and the same NTS2/RFLP banding patterns as the other S. uvarum strains but it does not share the fermentation pro le in grape juice and shows a relatively low homology in the nucleotide sequence of the region D1/D2 of the rdna. As already observed, the most representative strain of the group is the ancient S. abuliensis type strain CBS 7001 [2]. S. uvarum is a species made up of strains widely distributed in nature that possesses homogeneous and de ned genetic as well as physiological characteristics. On the contrary, S. bayanus type strain is an old isolate whose characteristics are not present in yeast strains currently isolated. For these reasons we believe that it is appropriate to re-establish the species S. uvarum and maintain it separate from S. bayanus. References S. pastorianus b CBS 1538 S. paradoxus b CBS 432 DIPROVAL S DIPROVAL C DIPROVAL S DIPROVAL S DIPROVAL M DIPROVAL DIPROVAL CBS CBS a Ancient type strain. b Type strain. Fig. 3. A: Restriction by AluI enzyme: from left to right 1 = CBS 1538 (S. pastorianus); 2 = CBS 380 (S. bayanus); X = 100 bp marker. B: Restriction by BanI enzyme: from left to right X = 100 bp marker; 1 = CBS 1538 (S. pastorianus); 2 = CBS 380 (S. bayanus) S. uvarum a CBS 395 [1] Vaughan-Martini, A. and Martini, A. (1998) Saccharomyces Meyen ex Reess. In: The Yeasts, A Taxonomic Study (Kurtzman, C.P. and Fell, J.W., Eds.), 4th edn., pp. 358^371. Elsevier, Amsterdam. [2] Rainieri, S., Zambonelli, C., Hallsworth, J.E., Pulvirenti, A. and Giudici, P. (1999) Saccharomyces uvarum, a distinct group within Saccharomyces sensu stricto. FEMS Microbiol. Lett. 177, 177^185. [3] Nguyen, H.-V., Lëpingle, A. and Gaillardin, C. (2000) Molecular typing demonstrates homogeneity of Saccharomyces uvarum strains and reveals the existence of hybrids between S. uvarum and S. cerevisiae, including the S. bayanus type strain CBS 380. Syst. Appl. Microbiol. 23, 71^85. [4] Nguyen, H.-V. and Gaillardin, C. (1997) Two subgroup within the Saccharomyces bayanus species evidenced by PCR ampli cation and restriction polymorphism of the non-trascribed spacer 2 in the ribosomal DNA unit. Syst. Appl. Microbiol. 20, 268^294. [5] van der Walt, J.P. (1970) Genus Saccharomyces Meyen emend. Reess.

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