ORIGINAL ARTICLE. G.V. de Melo Pereira, C.L. Ramos, C. Galvão, E. Souza Dias and R.F. Schwan. Abstract

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Use of specific PCR primers to identify three important industrial species of Saccharomyces genus: Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianus G.V. de Melo Pereira, C.L. Ramos, C. Galvão, E. Souza Dias and R.F. Schwan Biology Department, Federal University of Lavras, Lavras, MG, Brazil Keywords HO genes, PCR method, Saccharomyces sensu stricto, specific primer. Correspondence Rosane Freitas Schwan, Departamento de Biologia, Universidade Federal de Lavras, 37Æ Lavras-MG, Brazil : received 27 February 2010, revised 7 April 2010 and accepted 26 April 2010 doi: /j x x Abstract Aim: To develop species-specific primers capable of distinguishing between three important yeast species in alcoholic fermentation: Saccharomyces bayanus, Saccharomyces cerevisiae and Saccharomyces pastorianus. Methods and Results: Two sets of primers with sequences complementary to the HO genes from Saccharomyces sensu stricto species were used. The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. The second pair of primers (LgHO) was also constructed, within the HO gene, composed of perfectly conserved sequences common for S. bayanus species, which generate amplicon with 700 bp. No amplification product was observed in the DNA samples from non-saccharomyces yeasts. Saccharomyces species have also been characterized via electrophoretic karyotyping using pulsed-field gel electrophoresis to demonstrate chromosomal polymorphisms and to determine the evolutionary distances between these species. Conclusions: We conclude that our novel species-specific primers could be used to rapidly and accurately identify of the Saccharomyces species most commonly involved in fermentation processes using a PCR-based assay. Significance and Impact of the Study: The method may be used for routine identification of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes in less than 3 h. Introduction Saccharomyces sensu stricto is a species complex that includes most strains commonly used in the food and beverage industries as well as species of scientific relevance (Rainieri et al. 2003). The Saccharomyces sensu stricto complex, currently includes the species: S. bayanus, S. cariocanus, S. cerevisiae, S. mikatae, S. paradoxus, S. pastorianus, S. kudriavzevii and the recently described S. arboricolus, isolated from oak trees in China (Naumov et al. 2000; Sampaio and Goncalves 2008; Wang and Bai 2008). Among the yeasts in the S. sensu stricto group, the species S. cerevisiae, S. bayanus and S. pastorianus are associated with anthropic environments because of their high fermenting capabilities (Naumov et al. 2000). The genomes of a few wine yeast strains appear to have arisen from a hybridization event between two species, S. cerevisiae and S. bayanus, similar to that of the lager yeast S. pastorianus (Dunn et al. 2005). These species include the most important strains in yeast-based industries, mainly in baking, brewing, winemaking, fruit wine production and fuel ethanol production applications. Traditionally, the characterization of yeast species has been based upon morphological, physiological and biochemical characteristics, in particular the fermentation and assimilation of different sugars. The methodology used to identify yeasts based on morphology, biochemical characteristics and sexual reproduction requires the evaluation of tests to obtain the correct species identification. This process is complex, laborious and time Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

2 Specific PCR primers to identify Saccharomyces genus G.V. de Melo Pereira et al. consuming (Deak 1995). Moreover, members of S. sensu strict species are very close phylogenetically to S. cerevisiae and cannot be differentiated on the basis of conventional tests. This group of yeast consists of very closely related species that, in some cases, never seem to show a clearcut separation (Rainieri et al. 2003). Molecular techniques have been developed as an alternative to traditional techniques for the identification and characterization of yeasts (Bernardi et al. 2008). Different approaches, such as genetic analysis, DNA DNA reassociation, electrophoretic karyotyping, rrna sequencing, rdna restriction analysis and PCR-DGGE, have proven useful for distinguishing between strains of the S. sensu stricto group (Lopes et al. 1998; Torriani et al. 1999; Fernández-Espinar et al. 2000; Naumova et al. 2003; Manzano et al. 2004; Bernardi et al. 2008; Duarte et al. 2009; Campos et al. 2010). However, some of these methods are complicated and cannot be easily used in industrial laboratories (Huang et al. 2008). PCR amplification of specific sequences for the identification of organisms has become common because of the relative ease of manipulation and the increasing availability of PCR thermal cyclers. Some authors have suggested that specific primers can be used to identify yeasts belonging to the Saccharomyces genus (Josepa et al. 2000; Huang et al. 2008). The selection of sequences of DNA that is intended to amplify by PCR is a critical point of this technique. The specificity of PCR derives from the precision, with which the initiators perform this task, i.e., hybridize with the target DNA. For detection of yeast Saccharomyces sensu stricto group, the region chosen to be common to most strains encodes for proteins which are important for this group and has no homology with other micro-organisms. In Saccharomyces sensu stricto, HO genes have been cloned and sequenced (Kodama et al. 2003). The action of HO leads to high-efficiency switching between a and a cell types, which occurs by transposition of a block of information from a genomic storage position to the mating type locus, where this information is expressed and determines cell type (Russell et al. 1986). In this study, we have developed two new species-specific PCR primer pairs homologous to the HO genes from S. bayanus, S. cerevisiae and S. pastorianus species. Using these primers, a method is proposed that could quickly and unambiguously identify these species by a simple and rapid PCR amplification. Materials and methods Yeast strains The 27 S. sensu stricto group reference strains used in this study belong to the Microbial Physiology Lab collection (DBI UFLA, Lavras, Brazil) and are listed in Table 1. Strains of non-saccharomyces yeast were obtained from sugar cane and coffee fermentation and from tropical fruits (Schwan et al. 2001; Silva et al. 2005, 2008; Duarte et al. 2009) and were identified via conventional biochemical tests according to the methods of Kurtzman and Fell (1998) and Barnett et al. (2000), and (Table 1). Electrophoretic karyotyping analysis Analysis of yeast chromosome polymorphisms was performed generally as described by Bernardi et al. (2008). Yeast cells were pregrown in YPG (1% yeast extract (Merck), 2% peptone (Himedia), 2% glucose (Merck)), ph 5Æ6 at28 C for 48 h. PFGE was performed on a PFGE (pulsed-field gel electrophoresis) CHEF-DRIII unit (Bio- Rad, CA, USA). After electrophoresis, gels were stained with 1% (v v) ethidium bromide for 1 h and rinsed twice with MilliQ water (Millipore, JaVrey, USA) for 15 min. The gels were visualized via UV transillumination, and images were captured using a Polaroid camera (Concord, USA). DNA extraction and PCR conditions Yeast cells were pregrown in YPG, ph 5Æ6 at 28 C for 48 h. The yeast DNA was extracted from the pure cultures according to the method described by Duarte et al. (2009). The 25 ll PCR mixture contained 12Æ5 ll de Mix GoTaq Ò Green Master 2X (Promega), 2 ll of DNA diluted to 10 ng ll )1 and 0Æ5 lmol l )1 of each primer. The sequences of the primers utilized in this study and the amplification conditions are shown in Table 2. Amplification products were separated by electrophoresis on a 0Æ8% (w v) agarose gel and stained with Syber Green (Invitrogen, USA). DNA fragments were visualized by UV transillumination, and images were captured using a Polaroid camera. A ladder marker (GeneRuler 100 bp DNA Ladder Plus) was used as a size reference. Primer selection To select the primers used, sequences of the HO gene regions were downloaded from the NCBI nucleotide database and aligned using the multiple sequence alignment program clustal_x (1Æ8) (Thompson et al. 1997). Based on these alignments, the S. cerevisiae S. pastorianus (ScHO-F and ScHO-R)- and S. bayanus S. pastorianus (LgHO-F LgHO-R)-specific primers were designed (Table 2). The specificity of these primers was verified by searching for homologous nucleotide sequences in the GenBank sequence database using the blast search program ( 132 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

3 G.V. de Melo Pereira et al. Specific PCR primers to identify Saccharomyces genus Table 1 Collection of species used in this study and their identification using each primers pairs +, PCR-amplified product with each primer pair detected; ), PCR-amplified product with each primer pair not detected. Numbers between brackets correspond to size in bp of amplified products of PCR Species Designation Primers pairs and amplified product (bp) ScHO-F ScHO-R Saccharomyces cerevisiae *CBS (400) S. cerevisiae *WT (WILD) + (400) S. cerevisiae *UFLA CA116 + (400) S. cerevisiae *SA-1 + (400) S. cerevisiae *CAT-1 + (400) S. cerevisiae *PE-2 + (400) S. cerevisiae *BG-1 + (400) S. cerevisiae *VR-1 + (400) S. cerevisiae *UFLA CA16 + (400) S. cerevisiae *CCT (400) S. bayanus *CBS 1604 ) + (700) S. bayanus *CCT789 ) + (700) S. bayanus UFLA 08 ) + (700) S. bayanus UFLA 09 ) + (700) S. pastorianus *CBS (300) ) S. pastorianus *ATCC (300) ) S. pastorianus UFLA 19 + (300) ) S. pastorianus UFLA 20 + (300) ) S. paradoxus *CBS 432 ) ) S. paradoxus UFLA 05 ) ) S. paradoxus UFLA 06 ) ) S. paradoxus UFLA 07 ) ) S. mikatae *CBS 1816 ) ) S. mikatae *SS1 ) ) S. cariocanus *CBS ) ) S. cariocanus UFLA 453 ) ) S. kudriavzevii *CBS 1802 ) ) Dekkera bruxellensis UFLA CA 26 ) ) D. bruxelensis UFLA CAF 5 ) ) S. kudriavzevii *SS5 ) ) Debaryomyces hansenii *FAB 5 ) ) Candida sake *FAB 1 ) ) C. bombi *CCT 1689 ) ) C. rugopelliculosa UFLA CAF115 ) ) C. rugopelliculosa *CCT 1702 ) ) Pichia guilliermondii UFLA IC-38 ) ) Zygoascus hellenicus UFLA 185 ) ) Stephanoascus smithiae *SCF 631 ) ) Torulaspora delbrueckii UFLA CAC 5 ) ) T. pretoriensis *CCT 1688 ) ) Arxula adeninivorans UFLA AA 23 ) ) Citeromyces matritensis *CCT 2374 ) ) Kluyveromyces marxianus *CCT 3172 ) ) Lodderomyces elongisporus *CCT1690 ) ) LgHO-F LgHO-R *Type strain. Results Characterization of the Saccharomyces sensu stricto group by PFGE Characterization using the PFGE technique involved comparing the karyotypes of species from the Saccharomyces sensu stricto group (Fig. 1). The species S. cerevisiae and S. pastorianus showed clear differences in their banding patterns; however, certain similarities were also observed, which made the proximity between the two species evident, even when considering different strains of each species. In spite of the differences, there were no difficulties in grouping all of the strains into their respective Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

4 Specific PCR primers to identify Saccharomyces genus G.V. de Melo Pereira et al. Table 2 List of PCR primer pairs designed and used for this study Primers Gene region chromosome GenBank accession number Sequence Amplification conditions ScHO LgHO HO gene chromosome IV AM Lg-HO and HO genes chromosome IV AB and AB GTTAGATCCCAGGCGTAGAACAG-3 5 -GCGAGTACTGGACCAAATCTTATG-3 5 -TGGAAAGTCTACGAGAACAAGCC-3 5 -CCTCTATGTGAAGTCCGTATACTG-3 35 cycles 94 C 30s 61 C 30s 72 C 2 min 35 cycles 94 C 30s 55 C 30s 72 C 2 min Figure 1 Yeast strain electrophoretic karyotype analysis. Line 1, Saccharomyces bayanus UFLA 08; lines 2, 3, 6 and 8, Saccharomyces pastorianus ATCC 2366, S. pastorianus UFLA 19, S. pastorianus UFLA 20, S. pastorianus CBS 1538; line 4, Saccharomyces cariocanus UFLA 453; lines 5, 7 and 9 Saccharomyces cerevisiae CA116, S. cerevisiae UFLA 16, S. cerevisiae WT; line 10, S. cariocanus CBS ; line 11, Saccharomyces paradoxus UFLA 05; line 12, Saccharomyces mikatae SS1. S. bayanus UFLA 08 S. mikatae SS1 S. paradoxus UFLA 05 S. cariocanus CBS S. cariocanus SS3 S. pastorianus ATCC 2366 S. pastorianus UFLA 19 S. pastorianus UFLA 20 S. pastorianus CBS 1538 S. cerevisiae WT S. cerevisiae CA116 S. cerevisiae UFLA Distances Figure 2 Dendogram of Saccharomyces sensu stricto group strains based on electrophoretic karyotypes (profiles). species (Fig. 2). Similar results have been observed between the species S. mikatae and S. paradoxus, which have clearly shown band sharing despite having a certain degree of polymorphisms. Because these two species seem to share most of the bands found in S. bayanus, as seen in Fig. 2, these three species can be grouped together. These results show that once cultures are obtained to generate the electrophoretic profiles for each species, it is possible to use PFGE as a complementary technique for species classification. Therefore, the PFGE technique may be of great use in study of species within the Saccharomyces genus; yet, this must be carried out carefully, as the occurrence of polymorphisms should not always be interpreted as evidence of a distinct species. Even greater care must be taken with S. cerevisiae, in which it is quite common to observe a certain degree of chromosomal polymorphism among different strains. On the other hand, when the chromosomal profiles are identical, the identification of the species can be confirmed with a great degree of confidence. Identification via PCR The specificity of the primers (listed in Table 2) was validated via PCR using genomic DNA from 44 yeast strains (Table 1).The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. On the other hand, PCR with the LgHO primers produced a single amplificon of c. 700 bp only in the type strain of S. bayanus (Fig. 3). No PCR product was detected from other related species or groups with these primers. To simplify the amplification reaction and shorten the time required for identification, PCR was performed directly from colonies of yeast instead of using purified genomic DNA, which made the technique rapid and lowcost (Bernardi et al. 2008). No false-positive results were seen. The DNA from the species S. cerevisiae and S. pastorianus were amplified with an annealing temperature of 55 and 61 C, respectively, indicating a high specificity of hybridization between the target region of these species and the tested ScHO primer. When amplifying S. bayanus DNA with the LgHO primer, only a temperature of 55 C could be used for the annealing step. Discussion The dynamics of micro-organisms in industrial fermentation affects the sensory quality and chemical composition 134 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

5 G.V. de Melo Pereira et al. Specific PCR primers to identify Saccharomyces genus 400 bp 300 bp (a) bp Figure 3 Agarose gel 0,8% of the products amplified by PCR. Specific primers of the genes OH of Saccharomyces cerevisiae S. pastorianus and S. bayanus were used. (a) ScHO-F ScHO-R amplification: 1 Ladder marker (GeneRuler 100 bp DNA Ladder Plus), 2 Saccharomyces cerevisiae CBS 1171, 3 Saccharomyces pastorianus CBS. (b) LgHO- F LgHO-R amplification: 4 Ladder marker (GeneRuler 100 bp DNA Ladder Plus), 5 Saccharomyces bayanus CBS of the produced wine. Controlling bacterial contamination during fermentation with selected yeast is relatively easy by using antibiotics or reducing the ph value. Such control becomes difficult when contaminant microorganisms are phylogenetically similar to the industrial ones. When discussing the sensu stricto species, it must be kept in mind that they may represent a special case in yeast speciation, because these fermentation yeasts have had the opportunity to replicate far more often than groups not utilized in industrial fermentation (Vaughan- Martini and Martini 1993). This is a group characterized by a high fermentative ability and other physiological and molecular similarities that suggest a close phylogenetic relationship. Bioethanol and distilled beverage industries need rapid, reliable and simple methods for yeast identification. This work proposes the use of primers that allow the clear and direct identification of three of the most important yeast species in alcoholic fermentation. When using the ScHO primer, it is possible to distinguish S. cerevisiae from S. pastorianus by the size of the bands. Moreover, a new primer was also presented (LgHO) for the identification of S. bayanus, with an amplification fragment of about 700 bp. Both primers used in this study have been tested with several other yeast species commonly found in bioethanol and cachaca industries, and amplification was not observed for any of them (Table 1). (b) Saccharomyces cerevisiae is found almost exclusively in man-made fermentation environments (Vaughan-Martini and Martini 1987; Redzepovic et al. 2002), and because it is universally preferred for initiating wine fermentation, it became known as the wine yeast (Pretorius 2000). However, some wine yeast starter culture strains also belong to the S. bayanus species. In contrast to the strong link between winemaking and the latter two members of the Saccharomyces sensu stricto group, strains of S. pastorianus are usually associated with the production of lager beer (Vaughan-Martini and Martini 1987; Turakainen et al. 1993; Rodrigues de Sousa et al. 1995). There are contradictory observations regarding the genetic classification of these three species. The species S. cerevisiae and S. pastorianus showed similar phenotypes by the molecular techniques used in this work, and clearly shared several bands by the PFGE technique and hybridization with primer ScHO, which only differed in the length of the amplified region. Yet, the species S. bayanus showed the most distinct chromosomal profile and was the only species whose DNA was amplified by the LgHO primer. Such discrepancies can be justified by the peculiar physiological characteristics of S. bayanus, such as the ability to grow well at low temperatures (below 30 C). Saccharomyces bayanus grows at around 1 2 C and does not grow above 35 C, while S. cerevisiae shows a different behaviour, as it does not grow at low temperatures such as 1 2 C, but grows at 35 C and often at C. When compared to S. cerevisiae, S. bayanus has a higher fermentative rate at low temperatures (7 C) and shows a reduction in the ethanol yield at intermediate temperatures (22 30 C) (Kishimoto and Goto 1995). Another characteristic of this species is its ability to ferment melibiose. Of the 21 species of the sensu stricto group isolated in France and Italy, only two were genetically identified as S. cerevisiae; the others were designated S. bayanus (Naumov et al. 1993). The use of yeasts in biotechnology is of key importance to many industrial sectors. In these circumstances, the maintenance and confirmation of the strain used in the various processes is fundamental. The currently available steps to identify members of the sensu stricto group are slow compared to the production flow in industries manufacturing bread, beer, cachaça, wine and other alcoholic beverages, as well as in the production of biofuels. As a result of this work, the developed PCR technique seems to be very useful in accurately identifying important species of the S. sensu stricto group in less than 3 h. Acknowledgements The Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico do Brasil (CNPQ), Fun- Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010)

6 Specific PCR primers to identify Saccharomyces genus G.V. de Melo Pereira et al. dação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) are acknowledged for financial support and scholarships for GVMP and CLR. References Barnett, J.A., Payne, R.W. and Yarrow, D. (2000) Yeasts: Characteristics and Identification, 4th edn. Cambridge, UK: Cambridge University Press. Bernardi, T.L., Pereira, G.V.M., Cardoso, P.G., Dias, E.S. and Schwan, R.F. (2008) Saccharomyces cerevisiae strains associated with the production of cachaca: identification and characterization by traditional and molecular methods (PCR, PFGE and mtdna-rflp). World J Microbiol Biotechnol 43, Campos, C.R., Silva, C.F., Dias, D.R., Basso, L.C., Amorim, H. and Schwan, R.F. (2010) Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage, cachaça in Brazil. J App Microbiol 108, Deak, T. 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