Abstract

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5′-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3′-SSU end) and the 3′-specific primers were located in the LSU gene (positions 560–590 from the 5′-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.

Introduction

The taxonomy of the genus Saccharomyces has undergone significant changes in the last two decades. However, recent progress in molecular biology has led to the development of new techniques which have made significant advances in the taxonomy of this group. DNA relatedness and fertility experiments have established that Saccharomyces sensu stricto complex [1] comprises four sibling species: Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus and Saccharomyces paradoxus [2,3]. These species include the most important strains in yeast-based industries, such as brewing and winemaking. Traditionally, their identification and characterisation have been based on morphological traits and, especially, on physiological properties [4,5]. However, these characteristics tend to be influenced by cultural conditions and can give ambiguous results [6]. By contrast, molecular biology techniques are an alternative to the traditional methods of identification and characterisation of yeasts and an important tool in solving industrial problems. These techniques include: DNA–DNA reassociation [2,3], chromosomal DNA profiles [6,7] mitochondrial DNA RFLPs [8], rDNA restriction analysis of the internal transcribed spacers and 5.8S rDNA [9], rDNA sequencing [10]. However, some of these methods are sophisticated and they cannot be easily employed in industrial laboratories.

For this reason, we have developed a rapid and reliable method to differentiate species within the Saccharomyces sensu stricto group. This method is based on the design of species-specific primers to distinguish S. cerevisiae from the S. bayanus and S. pastorianus species.

Materials and methods

Yeasts strains

In this study we used collection strains of S. cerevisiae, S. bayanus and S. pastorianus and 15 wild strains isolated from different wine fermentations, previously identified as S. cerevisiae [11] (Table 1).

1

Collection strains of Saccharomyces used in the study

Species Origin Other designations 
S. cerevisiae CECT 1942T ATCC 18824, CBS 1171 
S. bayanus CECT 1941T CBS 380 
S. bayanus CECT 1969 ATCC 76514, CBS 395 
S. bayanus CECT 1991 DSMZ 70411 
S. pastorianus CECT 1940NT ATCC 12752, CBS 1538 
S. pastorianus CECT 1970 CBS 1503 
S. pastorianus CECT 11037 ATCC 76529, CBS 1513 
S. cerevisiae FMR Ia  
S. cerevisiae FMR IIa  
S. cerevisiae FMR IIIa  
S. cerevisiae FMR IVa  
S. cerevisiae FMR Va  
S. cerevisiae FMR VIa  
S. cerevisiae FMR VIIa  
S. cerevisiae FMR VIIIa  
S. cerevisiae FMR IXa  
S. cerevisiae FMR Xa  
S. cerevisiae FMR XIa  
S. cerevisiae FMR XIIa  
S. cerevisiae FMR XIIIa  
S. cerevisiae FMR XIVa  
S. cerevisiae FMR XVa  
ATCC, American Type Culture Collection, Rockville, MD, USA. CBS, Centraalbureau voor Schimmelcultures, Delft, The Netherlands. CECT, Spanish Type Culture Collection, University of Valencia, Valencia, Spain. DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. FMR, Faculty of Medicine Reus, Tarragona, Spain. T: Type strain. NT: Neotype strain. 
Species Origin Other designations 
S. cerevisiae CECT 1942T ATCC 18824, CBS 1171 
S. bayanus CECT 1941T CBS 380 
S. bayanus CECT 1969 ATCC 76514, CBS 395 
S. bayanus CECT 1991 DSMZ 70411 
S. pastorianus CECT 1940NT ATCC 12752, CBS 1538 
S. pastorianus CECT 1970 CBS 1503 
S. pastorianus CECT 11037 ATCC 76529, CBS 1513 
S. cerevisiae FMR Ia  
S. cerevisiae FMR IIa  
S. cerevisiae FMR IIIa  
S. cerevisiae FMR IVa  
S. cerevisiae FMR Va  
S. cerevisiae FMR VIa  
S. cerevisiae FMR VIIa  
S. cerevisiae FMR VIIIa  
S. cerevisiae FMR IXa  
S. cerevisiae FMR Xa  
S. cerevisiae FMR XIa  
S. cerevisiae FMR XIIa  
S. cerevisiae FMR XIIIa  
S. cerevisiae FMR XIVa  
S. cerevisiae FMR XVa  
ATCC, American Type Culture Collection, Rockville, MD, USA. CBS, Centraalbureau voor Schimmelcultures, Delft, The Netherlands. CECT, Spanish Type Culture Collection, University of Valencia, Valencia, Spain. DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. FMR, Faculty of Medicine Reus, Tarragona, Spain. T: Type strain. NT: Neotype strain. 

aIsolated from spontaneous fermentation in Porrera (Spain) in 1995.

Design of primers

The different S. cerevisiae, S. bayanus and S. pastorianus rDNA sequences used were obtained from the EMBL (Table 2) and aligned with the Clustal W, version 1.5 of multiple sequence alignment computer program [12]. From these alignments, the S. cerevisiae (SC1 and SC2)- and S. bayanus/S. pastorianus (SB1 and SB2)-specific primers were designed (Fig. 1). The respective sequence of these primers are:

2

Sequence data of the strains used in the primers design

Saccharomyces species Abbreviation in Fig. 1 Isolate number DNA fragment Size (bp) GenBank accession number 
S. cerevisiae S-cere-1 IFO10217 18Sp*, ITS-1, 5.8S, ITS-2, 28Sp 840 D89886 
S. cerevisiae S-cere-2 CBS 1171 28Sp 319 L20827 
S. cerevisiae S-cere-3 clone py1ra3 18Sp, ITS-1, 5.8S, ITS-2, 28S 3911 J01355 
S. pastorianus S-past-1 IFO1167 18Sp, ITS-1, 5.8S, ITS-2, 28Sp 827 D89889 
S. bayanus S-baya-1 IFO1127 18Sp, ITS-1, 5.8S, ITS-2, 28Sp 791 D89887 
S. bayanus S-baya-2 IFO1127 28Sp 132 D83573 
p*: Partial sequence. 
Saccharomyces species Abbreviation in Fig. 1 Isolate number DNA fragment Size (bp) GenBank accession number 
S. cerevisiae S-cere-1 IFO10217 18Sp*, ITS-1, 5.8S, ITS-2, 28Sp 840 D89886 
S. cerevisiae S-cere-2 CBS 1171 28Sp 319 L20827 
S. cerevisiae S-cere-3 clone py1ra3 18Sp, ITS-1, 5.8S, ITS-2, 28S 3911 J01355 
S. pastorianus S-past-1 IFO1167 18Sp, ITS-1, 5.8S, ITS-2, 28Sp 827 D89889 
S. bayanus S-baya-1 IFO1127 18Sp, ITS-1, 5.8S, ITS-2, 28Sp 791 D89887 
S. bayanus S-baya-2 IFO1127 28Sp 132 D83573 
p*: Partial sequence. 
1

Diagrammatic representation of position of the rDNA primers used for PCR amplification.

1

Diagrammatic representation of position of the rDNA primers used for PCR amplification.

  • SC1: 5′-AACGGTGAGAGATTTCTGTGC-3′

  • SC2: 5′-AGCTGGCAGTATTCCCACAG-3′

  • SB1: 5′-GCTATTCCAAACAGTGAGACT-3′

  • SB2: 5′-CAGTTGGCAGTATTCCCACTA-3′

The expected PCR results and the thermal conditions were checked with the amplify version 1.2 program.

PCR conditions

Each strain was analysed with both pairs of primers (SB1/SB2 and SC1/SC2). The thermal cycling parameters were an initial denaturation at 94°C for 5 min and 30 s for subsequent cycles, primer annealing at 50°C for 1 min and primer extension at 72°C for 1 min. There were 30 cycles followed by a final extension at 72°C for 7 min. The amplified products were electrophoresed on 1.4% Multipurpose agarose gel (Boehringer) and stained with ethidium bromide. A 100-bp DNA ladder marker (Gibco BRL) was used as the size standard.

Results and discussion

The small (18S) and large (25S) rRNA sequences from the species of the Saccharomyces sensu stricto display high identity. Kurtzman and Robnett [10] reported that only 12 nucleotide sites were found to be variable when 900 nucleotide sequences from these species were compared. Furthermore, the 900-base sequences from S. pastorianus and S. bayanus were identical. The alignment of the rRNA sequences of S. cerevisiae, S. bayanus and S. pastorianus has us allowed to design species-specific primers for S. cerevisiae and S. bayanus/S. pastorianus. We were not able to find enough nucleotide variation to design specific primers for these latter species. Despite the postulated hybrid origin of S. pastorianus from the cross between S. cerevisiae and S. bayanus, the difficulty in differentiating this species from S. bayanus on the basis of other molecular markers [8] and phenotypic characters [13] has been widely reported.

ITS regions are characterised by a lesser degree of evolutionary conservation than 18S and 25S rRNA genes and they exhibit far greater interspecific differences [14]. As expected, the multiple sequence alignments showed a higher level of nucleotide variation in this region than 18S gene (SSU) and the 5′-primers were designed based on the sequences ranging from 152 to 172 (SB1) and 161 to 181 (SC1) of the ITS-1 spacer. However, ITS-2 alignment did not reveal enough differences to design the 3′-primers based on these sequences. These primers were selected from positions 562–582 (SC2) and 564–585 (SB2) of the 25S gene (LSU).

The use of SB1/SB2 primers produced an amplification product of approximately 1170 bp in the S. bayanus and S. pastorianus reference strains but the S. cerevisiae type strain (CECT 1942) was never amplified (Fig. 2). On the other hand, the PCR process with the SC1/SC2 primers yielded an amplification product of the same size in the type strain of S. cerevisiae only. Non-specific amplification occasionally appears in S. bayanus or S. pastorianus strains with the S. cerevisiae-specific primers. This non-specific amplification, when it occurred, showed a constant pattern of five bands ranging from 360 to 1290 bp, and would not be confused with the specific product obtained for S. cerevisiae. This unexpected amplification disappeared when the DNA concentration was adjusted to 1–10 ng in the PCR mixture.

2

PCR amplification of the studied strains. A: SC1/SC2 amplifications. B: SB1/SB2 amplifications. The selected molecular-size marker (100-bp DNA ladder) is indicated on the left and right.

2

PCR amplification of the studied strains. A: SC1/SC2 amplifications. B: SB1/SB2 amplifications. The selected molecular-size marker (100-bp DNA ladder) is indicated on the left and right.

Recently, Sabaté et al. [11] studied the dynamics of the natural Saccharomyces strains present in wine fermentation. All the strains isolated in that work were identified as S. cerevisiae on the basis of the mtDNA restriction patterns [8]. In order to check the efficiency of the species-specific primers, 15 of these strains were tested. All of the isolates yielded an amplification product of 1170 bp with SC1/SC2 primers and no amplicon was detected with SB1/SB2. The utility of this technique for the identification of natural Saccharomyces isolates, was therefore demonstrated.

The availability of specific primers is a useful tool for rapid identification of species of the genus Saccharomyces that could not be differentiated on the basis of phenotypic characteristics [15]. Moreover, our method allowed processing of the amplification reaction without DNA extraction. An aliquot of a growth plate colony was directly added to the PCR mixture and the same results were obtained in 3 h. However, it should be taken into account, as mentioned above, that the lack of DNA quantification sometimes yielded unspecific amplifications. Therefore the cellular concentration used for the process should be tested.

Unambiguous identification of natural and commercial yeast has always been a concern for the industry. We should keep in mind that, generally, industry cannot arrange sophisticated techniques for routine work. We have provided an easy and quick technique to employ in these laboratories.

Acknowledgements

This work was supported by Grant PM95-0160 from CICYT (Ministerio de Educación y Ciencia) and the Fundació Ciència i Salut, Reus, Spain.

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