Genotypic Identification of industrial Saccharomyces cerevisiae Strains using Random Amplified Polymorphic DNA analysis

Orsolya Molnár1), Robert Messner1), Hansjörg Prillinger1), Ulf Stahl2), Hans Silberhumer3), Wilhelm Wunderer4)

1) Institut für Angewandte Mikrobiologie
Universität für Bodenkultur,Nußdorfer Lände 11 A- 1190 Wien

2) T.U. Berlin, Mikrobiologie und Genetik; D-13355 Berlin 65

3) Österreichisches Getränkeinstitut; A-1180 Wien, Michaelerstr. 25

4) HBLA u. BA Klosterneuburg; A-3400 Klosterneuburg

Introduction

Based on hybridization studies, serological observations, the base composition of nuclear DNA, and some traditionally used phenotypic characteristics like ascospore morphology and fermentative and assimilative activities seven Saccharomyces species (S. castellii, S. cerevisiae, S. dairensis, S. exiguus, S. kluyveri, S. servazzii, S. unisporus) are commonly accepted in modern text books of yeast taxonomy (Yarrow, 1984; Barnett et al., 1990). Seventeen further species (like S. bayanus, S. pastorianus (carlsbergensis), S. lindneri, S. mangini, and S. uvarum) originally described by phenotypic criteria were included within the species S. cerevisiae.

Studying nuclear (n)DNA/nDNA homologies within Saccharomyces cerevisiae sensu stricto, Vaughan-Martini and Martini (1987), Vaughan-Martini (1989) reestablished three further species, S. bayanus, S. pastorianus, and S. paradoxus. This concept has been corroborated further with mating experiments (Naumov, 1987) and different molecular approaches like electrophoretic karyotyping (Naumov et al., 1993; Vaughan-Martini et al., 1993), restriction polymorphisms in the internal transcribed spacers (Molina et al., 1992), PCR-fingerprinting with non-random oligonucleotides (Lieckfeldt et al., 1993), sequencing of ribosomal RNA (Peterson and Kurtzman, 1991) and Random Amplified Polymorphic DNA - Polymerase Chain Reaction (RAPD-PCR) analysis (Molnár et al., 1995) recently.

In the present study we used RAPD-PCR analysis and type strains of S. cerevisiae, S. bayanus, S. pastorianus, and S. paradoxus to identify Saccharomyces strains obtained from: RBF Tulln, BLVA Klosterneuburg, Österreichisches Getränkeinstitut, Institut für Gärungsgewerbe Berlin.

Materials and Methods

All strains investigated are listed in Table 1. They are maintained in the culture collection of the Institute of Applied Microbiology (IAM) in Vienna.

DNA-extraction:
Strains were grown overnight at 30°C on GYP agar (2% glucose, 0.5% yeast extract, 1% peptone, 2% agar). Extraction and purification of nuclear DNA was achieved according to Messner et al. (1994) by a modified CTAB method.

RAPD-PCR analysis:
The RAPD-PCR analysis was performed as described in Messner et al. (1994). 2 µl of the diluted DNA preparation corresponding to 10 to 20 ng was subjected to RAPD-amplification in a final volume of 25 µl containing 10 mM KCl, 10 mM (NH4)2SO4, 20mM Tris pH 8.8, 4.5 mM MgSO4, 0.1 % [w/v] Triton X-100, 0.2 mM of each nucleotidetriphosphate, 15 ng primer, 0.2 µg/ml bovine serum albumin fraction V and 0.4 units Taq-DNA polymerase. The mixture was overlayed with 50 µl light mineral oil and processed in a thermocycler. Three primers were used: GAGGGTGGCGGTTCT named M13, ACGGTCTTGG named decamer 1, and TGCCGAGCTG named decamer 2. The programs used were: 98°C/15 sec, 50°C/60 sec, 72°C/100 sec; 40 cycles for M13, 98°C/15 sec, 32°C/90 sec, 72°C/100 sec; 35 cycles for both decamers. The reactions were stopped by adjusting an EDTA-concentration of 10 mM and 20 µl of the PCR products were analysed in an 1.3 % [w/v] agarose gel in 0.5 x TBE buffer with 0.5 µg/ml ethidium bromide at 4°C and 5 V/cm. Lambda DNA digested with Pst I or 28S (a sequenced gen digested with Hinf I) was used as length standard. DNA-fragments were visualised by transillumination and photographed by a polaroid system.

Table 1. Strains investigated

Fig. 1. RAPD patterns of strains belonging to Saccharomyces sensu stricto primed by decamer 2 (TGCCGAGCTG). Lanes 1 and 2, S. cerevisiae, HA 258T and HA 290; lane 3, S. steineri HA 284T; lane 4, S. oleaginosus HA 313T; lane 5, S. bayanus HA 266T; lane 6, S. uvarum HA 326T; lane 7, S. globosus HA 229T; lane 8, lambda DNA digested with Pst I; lane 9, S. pastorianus HA 452T; lane 10, S. carlsbergensis HA 305T; lane 11, S. monacensis HA 271T; lanes 12 and 13, S. paradoxus, HA 451T and HA 220. Note the similarity in the patterns of the strains redesignated as S. cerevisiae (lanes 1 through 4), S. bayanus (lanes 5 through 7), S. pastorianus (lanes 9 through 11) and S. paradoxus (lanes 12 and 13), respectively. The strain HA 229T (lane 7) has the lowest similarity value (83%) with the S. bayanus type strain.

Fig. 2. RAPD patterns of strains primed by M13 (GAGGGTGGCGGTTCT). Lanes 1 through 5, S. cerevisiae, HA 247, HA 248, HA 249, HA 250, HA 290; lane 6, S. carlsbergensis HA 305T; lane 7, S. bayanus HA 266T; lane 8, S. uvarum HA 326T; lanes 9 through 12, S. cerevisiae, HA 251, HA 252, HA 253, HA 254; lane 13, 28S (a sequenced gen digested with Hinf I). Note the similarity in the patterns of the strains redesignated as S. cerevisiae (lanes 1 through 5 and lanes 9 through 12).

Fig. 3. RAPD patterns of strains primed by decamer 2 (TGCCGAGCTG). Lanes 1 through 10, S. cerevisiae, HA 285, HA 286, HA 287, HA 288, HA 297, HA 289, HA 290, HA 293, HA 294, HA 295; lane 11, S. pastorianus HA 452T; lane 12, S. uvarum HA 326T; lane 13, lambda DNA digested with Pst I. Note the similarity in the patterns of the strains redesignated as S. cerevisiae (lanes 1 through 10).

Fig. 4. RAPD patterns of strains primed by M13 (GAGGGTGGCGGTTCT). Lanes 1 through 4, S. cerevisiae, HA 293, HA 294, HA 295, HA 290; lane 5, S. carlsbergensis HA 216; lane 6, S. uvarum HA 304; lane 7, S. pastorianus HA 452T; lanes 8 and 9, S. uvarum, HA 327 and HA 326T; lane 10, S. bayanus HA 266T; lane 11, 28S (a sequenced gen digested with Hinf I). Note the similarity in the patterns of the strains redesignated as S. cerevisiae (lanes 1 through 4 and lane 6) and S. pastorianus (lanes 5 and 8), respectively.

Results and Discussion

The investigated and redesignated Saccharomyces strains are shown in Table 1. RAPD-PCR patterns of the four investigated Saccharomyces species (S. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus) using the decamer 2 are shown in Fig. 1. All the four species are represented with two to four strains - former type strains and others. These groups are representative samples for the genetic variability within each species (S. cerevisiae: 0-12 %; S. bayanus: 0-17 %; S. pastorianus: 0-13 %; S. paradoxus: 0-14 %; Molnár et al., 1995). All species are separated down to the level of the background noise. A higher percentage of DNA similarity was found between the strains of S. bayanus and S. pastorianus only (Molnár et al., 1995). With an estimate of DNA similarity of 45 %, however, both species are genetically separable and remain distinct with the RAPD-PCR method too.

It was commonly supposed that S. paradoxus is found only in nature and S. cerevisiae, S. bayanus, and S. pastorianus have been isolated mostly from artificial, man-created fermentation environments (Vaughan-Martini et al., 1993). Not surprisingly we found only these last three species among the industrial strains investigated. Our RAPD analysis showed that it is impossible to separate the beer yeast from yeast fermenting grape and apple juices as originally suggested by Meyen (1838). Although all strains isolated from wine (Table 1.; HA 247- 254 Fig. 2: lanes 1 through 4 and 9 through 12; HA 295 Fig. 3: lane 10 and Fig. 4: lane 3; HA 284T, original epithet S. steineri, Fig. 1: lane 3; other data not shown) and one from grape must (HA 280, original epithet S. carlsbergensis, data not shown) belonged to S. cerevisiae, two strains fermenting fruit juices (HA 229, original epithet S. globosus, Fig. 1: lane 7; HA 270, original epithet S. uvarum, data not shown) proved to be S. bayanus. The yeast strains isolated from beer occurred within each of the three species. But it is impossible to assign top fermenting yeasts exclusively to S. cerevisiae and bottom fermenting yeasts to S. pastorianus. We could not find any top fermenting yeasts within the yeast strains redesignated as S. pastorianus. But the species S. cerevisiae included both top (HA 217, data not shown; HA 218, data not shown; HA 258T Fig. 1: lane 1; HA 287 Fig. 3: lane 3; HA 288 Fig. 3: lane 4; HA 290 Fig. 1: lane 2, Fig. 2: lane 5, Fig. 3: lane 7, Fig. 4: lane 4; HA 297 Fig. 3: lane 5) and bottom fermenting yeast strains (HA 285 Fig. 3: lane 1; HA 286 Fig. 3: lane 2; HA 304, original epithet S. uvarum, Fig. 4: lane 6) using RAPD analysis.

In conclusion, we tried to show that the isolation sources of the yeasts and phenotypic characteristics like fermenting and assimilation patterns are not sufficient for an unequivocal identification of yeasts. The RAPD analysis makes comparable the whole chromosomal DNA exhibiting bands of genotypically specific value. This method turned out to be a fast, reliable, highly sensitive, and convenient method. It may replace time-consuming nDNA/nDNA hybridization experiments for species identification, characterization, and delimitation, especially if type strains are included.

Abbreviations:

HA = Hefe Ascomycet.
IFG = Institut für Gärungsgewerbe, Berlin, Germany.
CCY = Institute of Chemistry of the Slovak Academy of Sciences, Bratislava, Slovakia.
RBF = Raiffeisen Bioforschung, Tulln, Austria.
W = Österreichisches Getränkeinstitut (Collection Weihenstephan).
IMB M = Institut für Milchwirtschaft und Bakteriologie, BOKU Wien
K = HBLA u. BA Klosterneuburg
lyo = with lyophilisation conserved culture
T indicates type strain of the epithet.

Acknowledgements

For sending strains we are indepted to Dr. E. Slavikova, K. Scheide, and Dr. H. Rohm.

References

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