Financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004, POCTI/AGR/56771/2004), and AGRO (ENOSAFE, Nº 762)

Casal, Margarida

Dellinger, B.

Graça, Magda Maria Couto da Silva

Schuller, Dorit Elisabeth

Universidade do Minho: RepositoriUM

References[1] López V., Querol A., Ramón D., and Fernández- Espinar M.T. 2001. Int J Food Microbiol 68:75-81[2] P. Chatonnet, D. Dubourdieu, J. Boidron, M. Pons. 1992. J Science Food Agriculture 60: 165-178[3] L. Dias, S. Dias, T. Sancho, H. Stender, A. Querol, M. Malfeito-Ferreira, V. Loureiro. 2003. Int J Food Microbiol 20: 567-574[4] P. Martorell, A. Querol, M. T. Fernández-Espinar. 2005. Appl Environ Microbiol 71: 6823-6830[5] B. Esteve-Zardoso, T. Zorman, C. Belloch, A. Querol. 2003. Systematic and Applied Microbiology 26: 404 - 411.[6] C. C. Tebbe, A. Schmalenberger, S. Peters, F. Schweiger. 2001. Environmental Molecular Microbiology, UK, 161-175B. Dellinger, M. Silva Graça, M. Casal and D. SchullerDepartamento / Centro de Biologia, Universidade do Minho, Braga, Portugal ConclusionsWe identified a 164 bp fragment inside the D1/D2 domain of the 26S rDNA, that shows sufficient nucleotide divergence amongspecies and can be used for the distinction of wine related yeast species based on their CE-SSCP mobility values.The range of mobility values (156 – 187 for primer SSCPF and 158 - 202 for primer SSCPR ) creates a sufficiently high number of combinations for the unequivocal distinction of 16 from 22 wine related yeast species. However, three pairs of species (Dekkeraanomala and Candida vini; Sacchromyces cerevisiae and Saccharomyces paradoxus; Zygosaccharomyces bailii and Zygosaccharomyces bisporus) were not distinguishable. Intraspecific standard deviations increased with the capillary electrophoresis temperature. At 25ºC, standard deviations associatedto run-to-run variations, evaluated for 8 strains, were less than 0.7 mobility values, showing that CE-SSCP is a  reproducible and portable method for wine yeast identification.Materials and MethodsCE-SSCP data acquisitionFor CE-SSCP data acquisition PCR products were diluted 1:40 in ultrapuresterile water. 1 µl diluted PCR product was combined with 11,25 µldeionized formamide, 0,25 µl internal DNA molecular weight standard Genescan-500 TAMRA (PE, Applied Biosystems) and 0.5 µl 0.3 N NaOH. Samples were mixed thoroughly and incubated at 95 ºC for 5 min, followed by incubation on ice for denaturation and subsequent formation of single stranded conformations.CE-SSCP analysis was performed using an ABI Prism 310 genetic analyzer (PE Applied Biosystems) with a 47 cm length, 50 µm inner diameter capillary. The non denaturing polymer used consisted of 3 % (w/w) Gene Scan Polymer (PE Applied Biosystems) and 10 % (w/w) glycerol. As electrophoresis buffer 1x TBE containing 10 % glycerol was used. The injection time and voltage were set to 5 sec and 15 kV respectively, while electrophoresis voltage was 12 kV. The syringe pump time was 300 sec, and data collection was performed during 25 min. As the electrophoresis temperature has a great influence on the mobility values, analysis was carried out at three different constant temperatures (35, 30 and 25 ºC), in order to compare the results. A matrix file was created following the manufacturer’s instructions to account for spectral overlap of the various fluorescent molecules. To obtain comparable results, Gene Scan Analysis Software 3.5 (PE Applied Biosystems) was used.Identification of Wine Related Yeast Species by Capillary Electrophoresis Single Strand Conformation Polymorphism Analysis (CE-SSCP) of the 26S rRNA GeneYeast strainsGenomic DNA of 22 wine related yeast species was used. The strains were obtained from the Instituto Superior de Agronomia, Portugal (ISA), the Portuguese Yeast Culture Collection, Portugal (PYCC), the American Type Culture Collection, USA (ATCC), the Centraalbureau voor Schimmelcultures, The Netherlands (CBS) and the Colección Española de Cultivos Tipo, Spain (CECT).IntroductionIn wine industry yeast spoilage presents a severe problem related to great economic loss. Dekkerabruxellensis is described as the most serious spoilage yeast, due to its ability to produce high amounts of volatile phenols which cause off-flavours [2]. Pichia guilliermondii has the ability to produce the same phenols with efficiencies as high as those found in D. bruxellensis [3]. Besides its beneficial effect in wine fermentation Saccharomyces cerevisiae is also able to cause spoilage after fermentation as it resists high ethanol concentrations [4]. Yeasts of the genus Zygosaccharomyces also cause spoilage in wines and among them Zygosaccharomyces bailii is considered one of the most dangerous and frequently found yeasts in spoiled food and beverages [].It is important to detect spoilage yeasts quickly to allow wineries to intervene rapidly and effectively. In recent years there has been a great effort to develop rapid identification techniques. In comparison to traditional culture dependent methods, these new PCR-based methods allow faster detection and identification. CE-SSCP presents a powerful analysis technique that separates DNA fragments of the same length according to their sequence [6]. It is based on the heat denaturation of PCR amplified DNA where single stranded fragments are formed. These fragments are subjected to capillary electrophoresis under non denaturing conditions, where they form folded conformations due to their sequence. In this study, a CE-SSCP assay was developed based on a 164 bp fragment of the D1/D2 domain of the 26S rRNA gene in order to distinguish between various wine yeasts.RESULTSIntraspecific variation and influenceof electrophoresis temperature on the mobility valuesCE-SSCP data analysisMobility values of the internal standard Genescan – 500 TAMRA (red dye) at 25ºC, were assigned to peaks 1-9 of the internal standard, corresponding to 50, 100, 150, 194, 251, 266, 300, 340 and 350.The software then aligned the sample peaks in reference to the internal standard peaks (red peaks resulting from TAMRA labeled standard) using the Global Southern Alignment method.The four species analyzed in this preliminary study can be clearly distinguished by their peak patterns.Primers and PCR amplificationBased on sequence polymorphisms of the D1/D2 domain of the 26 S rDNA primers were designed for the amplification of a 164 bpfragment (SSCPF and SSCPR). The 5’ ends of the forward and reverse primer were labelled with the fluorescent dyes HEX and 6-FAM, respectively.The PCR reactions contained 20 ng template DNA, 0,4 µM of each primer, 2 mM MgCl2, 0,2 mM of each deoxynucleoside triphospha-te, 1x Taq polymerase buffer (MBI Fermentas) and 0,5 U Taqpolymerase (MBI Fermentas), all adjusted to a final volume of 6 µlwith MIliQ water. PCR was carried out in a icycler thermal cycler(Biorad). An initial denaturation step of 94 ºC for 4 min was followed by 36 cycles of 30 sec at 94 ºC, 30 sec at 50 ºC and 30 sec at 72 ºC, with a final extension at 72ºC for 10 min. Correct amplification was checked by agarose gel electrophoresis. Samples were stored at -20 ºC until CE-SSCP analysis.A single DNA extraction and quantification was performed for twostrains of each of the four species mentioned in the Table. Duplicate PCR amplifications per strain were carried out and fromeach PCR product two CE–SSCP samples were prepared and analyzed in three CE-SSCP runs at 25 ºC. Average and standard deviation were calculated between the results of the resulting 12 runs for each strain.Standard deviations were less than 1 mobility value.DNA isolationTotal yeast genomic DNA was isolated from cultures grown in 1 ml YPD medium for 48 hours at 30 ºC. Cells were harvested by centrifugation and DNA isolation was performed as previously described Yeast cells were cultivated in 5 ml of YPD medium (24 h, 28ºC, 160 rpm) and DNA isolation was performed using a previously described method [1]. DNA was quantified, diluted to 20 ng/µl using 10 mM TrisHCl 1 mM EDTA pH 8,0 and stored at -80ºC until use.6 –FAMHEX5’ - fluorochormeTACCACCCACTTAGAGCTCGAGTTGTAATTTGGAGASequence (5’ – 3’)225 – 24278 - 95Position in D1/D2 domain (S. cerevisiae) [bp]SSCPRSSCPFPrimerDekkera bruxellensisHEX 6-FAMPichia guilliermondiiSaccharomyces cerevisiaeZygosaccharomyces bailii1 3 4 5 67 8 92X-axis is peak mobility, Y-axis is relative fluorescence intensity. Peaks resulting from PCR amplification with the labeled primers SSCPF and SSCPR, showed as filled peaks, were assigned mobility values relative to the internal standard (not visible at this scale).The influence of electrophoresis temperature on the mobility values CE-SSCP analysis was evaluated at (35, 30 and 25 ºC). A single DNA isolation, PCR amplification and CE-SSCP analysis was performed for each strain of the 4 species. The standard deviation decreased with the temperature and showed values of 0.05 – 0.30 at 25 ºC, compared to 0.15 – 1.14 at 30ºC. The intraspecific variation showed standard deviations of less than 1 mobility value between strains belonging to the same species. An exception is Z. bailii, that presented higher variation in the 6-FAM labeled strand at 35 and 30 ºC. This was due to strain ISA1214, that differed in mobility values from the other strains. Sequencing of the D1/D2 domain of these strains revealed that strain ISA1214 has one base pair difference in comparison to the other strains.Subsequent analysis were always performed at 25 ºC.Reproducibility - Determination of run to run variationAnalysis of other wine related yeast speciesDistinct strains of 22 wine related yeast species were analyzed. Due to the high reproducibility of the method a single DNA isolation, PCR amplification and CE-SSCP run were performed.It was possible to separate yeast species based on their mobility values of both strands. However, some species showed very similar mobility values: Dekkera anomala and Candida vini; Sacchromyces cerevisiae and Saccharomyces paradoxus; Zygosaccharomyces bailiiand Zygosaccharomyces bisporus. Within the Saccharomyces sensu stricto complex it was possible to distinguish S. bayanus from S. cerevisiae and S. paradoxus, showing a difference of 3 mobility values in the HEX labeled strain.Candida famata, Candida stellata, Issatchenkia orientalis and Schizosaccharomyces pombe strains showed a more complex peak patternconsisting of several peaks. This might be due to several stable single strand conformations for both strands. Metschnikowia pulcherrima was the only species where the HEX labeled strand showed higher mobility values than the 6-FAM labeled strand.Dorit SchullerCentro de Biologia, Campus de GualtarUniversidade do Minho4710-057 Braga, Portugal  Tel: 253 - 60 40 10/17Fax: 253 – 67 89 80dschuller@bio.uminho.ptThis poster is available athttp://repositorium.sdum.uminho.ptAcknowledgementsThis study was financially supported by the programs POCI 2010 (FEDER/FCT, POCI/AGR/56102/2004, POCI/AGR/56771/2004), and AGRO (ENOSAFE, Nº 762). X-axis is peak mobility, Y-axis is relative fluorescence intensity.188,70161,48189,79158,60190,60155,44ISA 1600Dekkera bruxellensis0,700,301,250,261,140,58SD (+/-)183,12162,12186,55159,06189,58160,89averageIntraspecific variation182,80162,33186,15159,25189,06160,87PYCC 5167T182,81162,19185,74159,14188,73161,48ISA 1265184,16161,68188,41158,67191,26160,11ISA 1214182,69162,28185,88159,18189,26161,10ISA 1149Zygosaccharomyces bailii0,080,110,220,130,310,81SD (+/-)194,67168,66193,90174,85193,06176,71averageIntraspecific variation194,73168,72194,06174,85193,00176,87PYCC 4455T194,60168,49193,60174,66192,66177,78L 196194,60168,68194,06174,96193,22175,98L170194,74168,74193,88174,92193,36176,20L169Saccharomyces cerevisiae0,050,170,280,200,190,33SD (+/-)197,30174,87197,80180,89197,42186,79averageIntraspecific variation197,27174,98197,57180,72197,30186,36ISA 2145197,28174,62198,20181,19197,42186,69ISA 2131197,38174,89197,72180,83197,69187,04ISA 2126197,28174,99197,70180,83197,26187,07ISA 2105Pichia guilliermondii0,170,090,080,080,150,18SD (+/-)188,75161,46189,84158,62190,79155,21averageIntraspecific variation188,58161,58189,81158,54190,74155,05ISA 2104188,72161,43189,95158,61190,91155,10ISA 1791188,98161,36189,80158,74190,91155,25ISA 16496-FAMHEX6-FAMHEX6-FAMHEXStrainSpecies123425ºC30ºC35ºCCandida vini6-FAMHEXStrainSpecies190,64156,04IGC 4380190,49155,91IGC 3294190,51156,02IGC 2495190,69156,05IGC 4121 Type/CBS 5759Pichia anomala157,57160,88IGC 4384158,72160,95PYCC 5625Metschnikowia pulcherrima202,09185,26ISA 1308202,23186,37ISA 1421Lodderomyces elongisporus186,53169,35IGC 2902186,65169,41IGC 3286186,63169,47ATCC 10022/CBS 6432186,37169,40IGC 3886 Type/CBS 712Kluyveromyces marxianus193,32184,40MT1/BG/10193,32184,40ISA 1189/CBS 276Hanseniaspora uvarum187,71162,09ISA 2117187,70162,08ISA 1703187,82162,13ISA 1700188,70161,48ISA 1600Dekkera bruxellensis193,59161,22IGC 5133193,86161,16ISA 1652 TypeDekkera anomala192,61161,56PYCC 2597192,61161,42ISA 1007189,15160,81PYCC 3664Candida veronae193,72161,39PYCC 3671Candida vanderwaltii197,55177,52192,63161,45CBS 157Candida stellata170,59189,34161,00PYCC 3056Candida famata190,06166,21PYCC 3073Candida cantarelliSaccharomyces bayanus197,09174,63ISA 2105Pichia guilliermondii6-FAMHEXStrainSpecies189,17163,39IGC 3694/CBS 5717189,09163,32IGC 3693/CBS 5714189,15163,28PYCC 5276 TypeZygosaccharomyces rouxii182,78162,37IGC 5381Zygosaccharomyces bisporus 182,78162,21IGC 5337182,79162,38IGC 5336182,75162,26IGC 5335 Type182,32162,79CBS 2856182,90162,35IGC 4806182,19162,66ISA 1022/IGC 4267182,71162,16IGC 5167 Type/ISA 1149 TZygosaccharomyces bailii190,44155,84ISA 1083Saccharomycodes ludwigii194,89166,63IGC 4656194,88166,59IGC 4578/CBS 5829195,04166,54IGC 4576/CBS 406194,88166,63IGC 4570 TypeSaccharomyces paradoxus193,18184,10ISA 1088193,05183,92ISA 1089Saccharomyces ludwigii194,00168,09IGC 3931194,00167,95IGC 3983193,86167,80IGC 2608/CBS 1782194,29168,07IGC 4455 Type/CBS 1171Saccharomyces cerevisiae195,94163,55IGC 4568/CBS 424195,48163,37IGC 4569/CBS 425195,63163,46IGC 4565/CBS 378195,48163,26IGC 4456 Type/CBS 380193,48163,46IGC 5166Rhodotorula mucilaginosa197,09174,65ISA 2286197,07174,64ISA 2145197,08174,64ISA 2126187.96 (0.32)162,00 (0.32)ISA 1649196.73 (0.27)174.55 (0.67)ISA 2105P. guilliermondii196.89 (0.15)174.54 (0.43)ISA 2126 194.39 (0.14)167.86 (0.49)L 169S. cerevisiae194.28 (0.12)168.07 (0.46)L 170 6-FAMHEXPYCC 4531ISA 1265ISA 1600Strain182.48 (0.30)162.36 (0.29)182.39 (0.41)162.38 (0.21)Z. bailii187.60 (0.42)162.08 (0.23)D. bruxellensisAverage (SD)Species

Identification of wine related yeast species by capillary electrophoresis single strand conformation polymorphism analysis (CE-SSCP) of the 26S rRNA Gene

http://repositorium.sdum.uminho.pt/bitstream/1822/5972/1/2006_Bioquimica_SSCP_Poster.pdf

Identification of wine related yeast species by capillary electrophoresis single strand conformation polymorphism analysis (CE-SSCP) of the 26S rRNA Gene

Abstract

Similar works

Full text

Available Versions

Universidade do Minho: RepositoriUM