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Identification of Yeast Strains Using the Polymerase Chain Reaction
Journal of The Science of Food and Agriculture
Abstract
Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.
... Strains of Saccharomyces cerevisiae can be differentiated using PCR amplification with primers targeting the long terminal repeated (LTR) sequences found throughout the yeast genome in LTR-transposons such as Ty1 and Ty2. [12][13][14] The PCR primers bind to sites near the ends of the approximately 330 bp long LTR domain, also known as a delta element, and generate amplicons composed of sequences from adjacent LTR domains. [12,13] A highly reproducible approach is needed to consistently identify different strains based on properties of interdelta PCR amplicons. ...
... [12][13][14] The PCR primers bind to sites near the ends of the approximately 330 bp long LTR domain, also known as a delta element, and generate amplicons composed of sequences from adjacent LTR domains. [12,13] A highly reproducible approach is needed to consistently identify different strains based on properties of interdelta PCR amplicons. ...
... The match between the interdelta NGS fingerprint of the unknown Sample 12 to the reference fingerprint of Sample 8 supported the conclusion that Sample 12 represented a cross contamination by an in-house yeast, the Belle Saison strain. The lack of a match between the fingerprint of Sample 19 and any of the brewing yeast utilized in the brewery (i.e., Samples 1,8,13,14,15,16,and 17) supported the conclusion that Sample 19 represented contamination by a foreign wild yeast strain. ...
Identifying beer spoilage microbes is readily accomplished using PCR analyses targeting specific types of microbes, but the general classification of wild yeast versus brewing yeast using cultivation independent molecular methods has remained challenging. The approach presented in this study utilized genetic fingerprint matching to determine if an unknown yeast isolate matches the fingerprint of catalogued brewing yeast strains. Interdelta Next Generation Sequencing (NGS) fingerprints were produced using PCR amplification of delta elements, also known as long terminal repeat sequences of transposons of yeast. Fingerprints identifying different yeast strains were generated by processing reads produced by NGS of the interdelta PCR amplicons using open source software. The interdelta NGS fingerprint comprises DNA sequences that can be recorded, compared, and utilized as a fixed reference for yeast strain identification. Interdelta NGS fingerprints were shown to be reproducible and capable of distinguishing between strains of brewing yeast and wild yeast. Experimental yeast contamination demonstrated the utility of the approach for identifying in-house brewing yeast cross contamination versus foreign wild yeast contamination.
... Analysis by microsatellite loci is one of the most effi cient marker methods and therefore used in studying genetic diversity, genotyping, construction of genetic maps, population analysis and clarifying phylogenetic relationships at the species/subspecies/genus level of many organisms as humans, plants, animals and microorganisms (Weising et al., 1995;Webster and Reichart, 2005;Carneiro Vieira et al., 2016). Delta analysis is also a well-established method for the diff erentiation of yeasts (Lavallée et al., 1994;Legras and Karst, 2003) Th e used primers are homologous with the delta sequence of the Ty transposon (Ness et al., 1993). Th e delta elements are so-called LTRs (Long Terminal Repeats) which are present on retrotransposons as fl anking 'direct repeats', but are also distributed as individual LTRs in the genome (Lesage and Todeschini, 2005). ...
... Th e delta elements are so-called LTRs (Long Terminal Repeats) which are present on retrotransposons as fl anking 'direct repeats', but are also distributed as individual LTRs in the genome (Lesage and Todeschini, 2005). Th e number and position of the delta elements are diff erent for each yeast strain, allowing discrimination (Ness et al., 1993). PCR fi ngerprinting with primer M13 is a method that has already long been used for the diff erentiation of yeasts (Lieckfeldt et al., 1993;Andrighett o et al., 2000;Urso et al., 2008). ...
... PCR reactions were carried out in a 10 μl reaction volume consisting of 1,5 μl of template DNA, 2X GoTaq® Master Mix (Promega, Madison, USA) and 5 μM of forward and reverse primer, in the case of M13 10 μM primer. Th e used primers were δ1 (5'-CAA AAT TCA CCT ATA TCT CA -3'), δ2 (5'-GTG GAT TT T TAT TCC AAC A -3') described by Ness et al. (1993) and δ12 (5'-TCA ACG ATG GAA TCC CAA -3') designed by Legras and Karst (2003). PCR combinations δ1-2 and δ12-2 were described by Legras and Karst (2003). ...
In recent years an increasing trend towards terroir-emphasizing yeasts has emerged, and with that a need to distinguish local strains from commercial available wine yeasts. Therefore, in the present study, a database was set up with the genetic profi les of commercial wine yeasts used in Austria. This is an important tool for the selection and verifi cation of autochthonous wine yeasts from vineyards, wineries or must productions. Three diff erent molecular markers, namely microsatellites, interdelta analysis and M13 fingerprinting, were tested for their ability to differentiate 75 commercial wine yeast strains in a single approach and in combination. It turned out that at least two of the methods should be used for a meaningful comparison and distinct discrimination. Four autochthonous yeasts could be differentiated from their commercially propagated relatives. If a quick initial assessment has to be done, then microsatellite analysis is certainly the favored method.
Differenzierung von kommerziellen Weinhefestämmen mittels molekularer Marker.
In den letzten Jahren hat sich ein zunehmender Trend zu terroir-betonten Hefen herausgebildet, mit dem die Notwendigkeit verbunden ist, lokale Stämme von kommerziell erhältlichen Weinhefen zu unterscheiden. In der vorliegenden Studie wurde daher eine Datenbank mit den genetischen Profilen der in Österreich verwendeten kommerziellen Weinhefen erstellt. Dies ist ein wichtiges Instrument für die Auswahl und Überprüfung von autochthonen Weinhefen aus Weinbergen, Weingütern oder Mostproduktionen. Drei verschiedene molekulare Marker, nämlich Mikrosatelliten, Interdelta-Analyse und M13-Fingerprinting, wurden auf ihre Fähigkeit getestet, 75 kommerzielle Weinhefestämme in einem einzigen Ansatz und in Kombination zu unterscheiden. Es stellte sich heraus, dass mindestens zwei der Methoden für einen aussagekräft igen Vergleich und eine eindeutige Unterscheidung verwendet werden sollten. Vier autochthone Hefen konnten von ihren kommerziell vermehrten Verwandten unterschieden werden. Wenn eine schnelle Ersteinschätzung durchgeführt werden soll, ist die Mikrosatellitenanalyse mit Sicherheit die zu bevorzugende Methode.
... Идентификация штаммов дрожжей методом интердельта анализа, предложенным в 1993 году, основана на полиморфизме длин амплифицированных фрагментов, расположенных между дельта-элементами [6]. ...
... Первоначально для проведения интердельта анализа были разработаны праймеры δ1+δ2 для анализа внутривидовой изменчивости и для дифференциации штаммов S. cerevisiae [6,9]. Позже метод был дополнен новой парой праймеров δ12 +δ21 и показано, что использование сочетаний праймеров δ 12 +δ 21 и δ 12 +δ 2 позволяет установить больший полиморфизм у анализируемых штаммов. ...
... Individual yeast strains can be differentiated using genome sequencing or a variety of PCR-based methods. One method which is widely applied is through amplification of interdelta regions of the genome, giving rise to a unique genetic fingerprint by which yeast strains can be identified (Ness et al., 1993;Legras and Karst, 2003). This method is recommended for analysis of brewing 25 | P a g e strains by the American Society of Brewing Chemists ( Van Zandycke et al., 2008) and can also be used to evaluate the presence of mutations within a yeast strain genome, which can occur when yeast are exposed to environmental stresses and when a single yeast population is utilised for long periods of time (Powell and Diacetis, 2007) . ...
... Interdelta PCR is a routine method for identifying yeast strains, whereby multiple discriminatory transposon-flanking regions are amplified and revealed using gel electrophoresis (Ness et al., 1993;Legras and Karst, 2003). This method has been used for identification of a range of industrial strains (Lavalliée et al., 1994) and is the current recommended methods for yeast strain identification according to the ASBC methods of analysis (Zandycke et al., 2008). ...
As modern industrial efforts shift towards more economic and sustainable practices, the brewing industry is no different. Namely, the application of high gravity (HG) and very high gravity (VHG) brewing practices provide the means to increase brewery efficiency and reduce energy input. However, the conditions experienced throughout VHG fermentations exert an increased degree of stress on the fermenting yeast population, resulting in poor ethanol yields, impaired yeast quality and unbalanced beer flavour. The aim of this research was to investigate the influence of stress factors experienced by yeast during fermentation on yeast physiology and key quality indicators. The impact of osmotic stress, a key stress factor in relation to VHG practices, was highlighted to cause unwanted changes to yeast vacuolar and mitochondrial physiology, as well as plasma membrane damage and cell death, contributing to reduced fermentation performance. The alleviation of osmotic stress was found to be possible through the application of a sugar top-up regime to a VHG fermentation, preventing exposure of yeast to extreme osmolarity upon pitching. This optimisation procedure yielded positive results in terms of improving yeast quality and was confirmed to reduce the occurrence of characteristically stressed organelle morphologies and decrease stress exertion. This research not only provides a further understanding of the yeast physiological response to stress, but offers brewers a viable method to improve fermentation efficiency and harness the potential of VHG brewing.
... Die Anzahl und Lage dieser Delta-Sequenzen ist intraspezifisch variabel. Ness et al. (1993) de-signten die Primer δ 1 und δ 2 zur Unterscheidung von S. cerevisiae-Stämmen. Legras u. ...
... (Legras u. Karst, 2003;Ness et al., 1993;Schuller et al., 2004;OIV, 2011) Genotyping durch Mikrosatelliten Mikrosatelliten sind polymorph und erlauben eine Differenzierung auf Stammebene. Es handelt sich hierbei um kurze Nukleotidsequenzen aus Tandemwiederholungen von zwei bis zehn Nukleotiden Länge, welche im Genom oft wiederholt werden. ...
In der önologischen Praxis werden meist unpasteurisierte Moste verwendet. Dadurch kommt es zu einem vermehrten Eintrag von Nicht-Saccharomyceten, welche den Fermentationsprozess nachhaltig beeinflussen können. Störungen im Fermentationsprozess werden im Praxisbetrieb zumeist nur anhand von Auffälligkeiten ausgewählter Parameter wie Zuckergehalt oder Temperatur oder dem Auftreten von Fehltönen detektiert. Die fermentierende Hefepopulation kann zu diesem Zeitpunkt bereits so geschädigt sein, dass ein Eingreifen in die Fermentation das Auftreten von Fehltönen im Endprodukt oder eine unvollständige Fermentation nicht mehr verhindern kann. Mit Hilfe der Flusszytometrie wurde eine effiziente Methode entwickelt, um die gängigen Vertreter der Fermentationspopulation wie Saccharomyces cerevisiae und der Schadhefenpopulation wie Hanseniaspora uvarum, Dekkera bruxellensis und Pichia anomala im Fermentationsverlauf mittels FISH (Fluoreszenz in situ Hybridisierung) zu detektieren und quantitativ zu erfassen. Durch eine rasche Detektion können rechtzeitig Gegenmaßnahmen ergriffen werden, bevor eine Schadhefenpopulation zu großen Einfluss auf den Fermentationsverlauf und die gebildeten Stoffwechselmetabolite nehmen kann. Die Etablierung der Flow-FISH wurde in definiertem Medium (YPD) und pasteurisiertem weißen Traubenmost mit Reinkulturen durchgeführt. Die Probenentnahme und Fixierung erfolgt direkt aus Fermentationsansätzen. Zur Hybridisierung werden 18S- und 26S-rRNA-Sonden mit FITC-Markierung eingesetzt. Für die Auswertung der flusszytometrischen Daten wird in dieser Arbeit die Methode der Overton-Subtraktion angewendet. Diese ermöglicht eine genauere Beurteilung der hybridisierten Zellpopulation als das übliche Setzen eines Markers. Dazu wird eine effektive Negativkontrolle mit komplementärer Sequenz zu der universellen Eukaryontensonde (Euk516) eingeführt. Anschließend erfolgte die Optimierung der bereits aus der Literatur bekannten Methode hinsichtlich Hybridisierungsbedingungen und Zellfixierung und somit ihre Anpassung an die Anforderungen einer quantitativen flusszytometrischen Analyse. Mit der Fixierung in Formaldehyd oder in Ethanol wurden Fixiermethoden entwickelt, die die Ansprüche an sowohl eine schnelle und zuverlässige Fixierung im Labor, als auch an eine rasche Fixierung im Keller, erfüllen. Zur Erhöhung der Fluoreszenzintensität wurden Hilfssonden entworfen. Diese sind unmarkiert und binden in direkter Nachbarschaft der spezifischen Sonde. Bei allen untersuchten Hefespezies S. cerevisiae, H. uvarum, D. bruxellensis und P. anomala kann durch den Einsatz der Hilfssonden die Fluoreszenzintensität deutlich gesteigert werden. Bei D. bruxellensis und P. anomala ist erst durch den Einsatz der Hilfssonden ein Nachweis möglich. Durch die Hilfssonden kann der Flow-FISH-Assay in einem breiteren Wachstumsbereich der Hefekultur eingesetzt werden. Ohne Hilfssonden ist die quantitative Detektion auf die mittlere logarithmische Wachstumsphase beschränkt. Mit Hilfssonden können schon in der frühen logarithmischen Wachstumsphase bis in die stationäre Phase hinein hybridisierte Zellen zuverlässig detektiert werden. Damit wird die kritische Phase der fermentativen Aktivität abgedeckt, um so zunehmende Kontaminationen in der Fermentation erkennen zu können. Die Spezifizität der Sonden ist gegeben. Zum Teil kommt es zu leicht erhöhten Fluoreszenzintensitäten gegenüber der Negativkontrolle vor allem mit der D. bruxellensis-Sondenkombination und dafür unspezifischen Hefen, die wahrscheinlich auf vermehrte Bindungen auf Grund der Zusammenstellung dieser Sondenkombination zurückzuführen sind.Der Flow-FISH-Assay ist auch in Mischungen verschiedener Hefespezies und bis zu einer Zellzahl von 10³ Zellen/ ml in der Ausgangsfermentation zuverlässig. Dieses Detektionslimit wird auch von anderen molekularbiologischen Methoden zur Hefedetektion erreicht. Im Gegensatz zu den meisten dieser Methoden kann die Flow-FISH auch die Anzahl der vorhandenen Hefen quantifizieren. Der Einsatz des Flusszytometers ermöglicht zusätzlich eine einfache Variante die Gesamtzellzahl aller Hefen in der Fermentation zu ermitteln. Das Detektionslimit der Flow-FISH ermöglicht eine Detektion bevor die Schadschwellenwerte der untersuchten Hefen erreicht sind. Die in dieser Arbeit vorgestellte Flow-FISH-Methode ist auch bei anderen Hefestämmen, die teilweise auch aus Wildisolaten stammen, anwendbar. Eine Übertragbarkeit auf native Fermentationen aus der önologischen Praxis ist gegeben. Es gelang sowohl Spontanfermentationen als auch inokulierte Fermentationen in Praxisgärungen im Stahltank auf ihre Hefepopulationszusammensetzung hin zu untersuchen und ihre Entwicklung im Laufe der Fermentation zu verfolgen.Der optimierte Flow-FISH-Assay bietet durch den Einsatz der Flusszytometrie und der in dieser Arbeit verwendeten Hilfssonden und Negativkontrolle eine stabile Basis zur weiteren Entwicklung eines Testsystems für den Einsatz in der önologischen Praxis.
... Primer pairs 18S and 28S were purchased from Sigma and used to amplify ITS region (ITS1 and ITS2) and the 5·8S rRNA gene; this was performed in an internal transcribed spacer (ITS) PCR [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. Internal transcribed spacer PCR enables the modification according to conditions described by [29][30][31][32] as follows: initial denaturation at 95 • C for 5 min, followed by 40 cycles consisting of 30 s at 95 • C for denaturation, 1 min for primer annealing at 46 • C and 1 min for primer extension at 72 • C, followed by a final extension at 72 • C for 5 min. The restriction endonucleases HinfI and HaeIII were used to digest the amplified DNAs. ...
... Using Interdelta PCR (Thermo Fisher Scientific, Singapore), a TY1 retrotransposon region was amplified with δ12/δ21 primer pairs in order to identify 96 strains (taken from the first 4 grape varieties) within the same yeast species [28,32,34]. The amplification of the δ region was done by placing the PCR tubes (SSI Bio, California, CA, USA) in a SimpliAmp thermal cycler (Thermo Fisher Scientific, Singapore) for initial denaturation at 95 • C for 5 min. ...
... Primer pairs 18S and 28S were purchased from Sigma and used to amplify ITS region (ITS1 and ITS2) and the 5·8S rRNA gene; this was performed in an internal transcribed spacer (ITS) PCR [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. Internal transcribed spacer PCR enables the modification according to conditions described by [29][30][31][32] as follows: initial denaturation at 95 • C for 5 min, followed by 40 cycles consisting of 30 s at 95 • C for denaturation, 1 min for primer annealing at 46 • C and 1 min for primer extension at 72 • C, followed by a final extension at 72 • C for 5 min. The restriction endonucleases HinfI and HaeIII were used to digest the amplified DNAs. ...
... Using Interdelta PCR (Thermo Fisher Scientific, Singapore), a TY1 retrotransposon region was amplified with δ12/δ21 primer pairs in order to identify 96 strains (taken from the first 4 grape varieties) within the same yeast species [28,32,34]. The amplification of the δ region was done by placing the PCR tubes (SSI Bio, California, CA, USA) in a SimpliAmp thermal cycler (Thermo Fisher Scientific, Singapore) for initial denaturation at 95 • C for 5 min. ...
Wine production depends on the fermentation process performed by yeasts, especially (but not solely) strains of the species Saccharomyces cerevisiae, which is a technique that has been practiced from the Middle Ages till modern days. Selecting indigenous starters offers a beneficial technique to manage alcoholic grape juice fermentation, conserving the particular sensory qualities of wine produced from specific regions. This paper investigated yeast biodiversity of four grape varieties (Carignan, Syrah, Grenache, and Aswad Karesh) grown in the pedoclimatic western semi-desert Bekaa Valley. Further research identified, characterized, and selected strains with the most industrial wine interest and economic value to Lebanon. By using molecular methods and by the ITS PCR analysis, the isolates belonging to the Saccharomyces and non-Saccharomyces genus were identified. These isolates taken from four varieties were further characterized by amplification with Interdelta and δ12/δ21 primer pairs, permitting the identification of 96 S. cerevisiae strains. Forty-five genomically homogenous groups were classified through the comparison between their mtDNA RFLP patterns. Based on physiological characterization analysis (H2S and SO2 production, killer phenotype, sugar consumption, malic and acetic acid, etc.), three strains (NL28629, NL28649, and NL28652) showed interesting features, where they were also vigorously fermented in a synthetic medium. These strains can be used as a convenient starter for typical wine production. In particular, Carignan and Syrah had the highest percentage of strains with the most desirable physiological parameters.
... The number and chromosomal position is strain-dependent 57 . Primers δ12 and δ21 were used since larger polymorphic differences can be identified, compared to the original δ1 and δ2 primers designed by Ness and co-workers 58 . www.nature.com/scientificreports/ ...
... Genetic characterization of Saccharomyces cerevisiae strains using delta PCR analysis. The genetic diversity within 111 strains isolated from spontaneously fermented Negro Saurí juice and one commercial wine Saccharomyces cerevisiae (Cross Evolution, Lallemand) was evaluated by PCR amplification of inter-delta (δ) regions, which flank Ty elements in the yeast genome 58 . DNA isolation was performed according to Liu et al. 103 . ...
‘Microbial terroir’ relates to the influence of autochthonous yeasts associated with a grape cultivar on the resultant wine. Geographic region, vineyard site and topography, climate and vintage influence the biodiversity of these microbial communities. Current research focus attempts to correlate their ‘microbial fingerprint’ to the sensorial and chemical characteristics of varietal wines from distinct geographical wine regions. This study focuses on the minor red grape variety, Negro Saurí, which has seen a resurgence in the León Appellation of Origin in Spain as a varietal wine. An experimental vineyard at Melgarajo S.A. (42° 15′ 48.68_N 5° 9′ 56.66_W) was sampled over four consecutive vintages, with autochthonous yeasts being isolated from grapes, must and pilot-scale un-inoculated fermentations, and identified by ITS sequencing. Forty-nine isolates belonging to Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora uvarum and Torulaspora delbrueckii were isolated from grapes and must, and early stages of fermentation dependent on seasonal variation. Saccharomyces cerevisiae predominated throughout fermentation, as a heterogeneous and dynamic population, with seven major biotypes identified amongst 110 isolates across four consecutive vintages. Twenty-four S. cerevisiae isolates representing five strains dominated in two or more vintages. Their persistence through fermentation warrants further validation of their oenological properties as starter cultures.
... Oui Oui Méthode moléculaire Fellgett, 1951Holland et al ., 1996 Non Oui IR-TF MADI-TOF-MS mt-RFLP Non Liu et al ., 1997 T-RFLP Caryotypage RISA DGGE CE-SSCP Lieckfeldt et al ., 1993 MSP-PCR ISS-PCR ERIC-PCR Sharples et Lloyd, 1990de Barros Lopes et al ., 1996 Microsatellites Ness et al ., 1993-Legras et Karst, 2003Field et Wills, 1998 RAPD Williams et al ., 1990 PCR-Interdelta Dubourdieu et al ., 1987Carle et Olson, 1985Stern et al ., 1984Vos et al ., 1995Guillamón et al ., 1998Fisher et Triplett, 1999Fischer et Lerman, 1983 Niveau d'identification déductif après analyse (Naumann et al., 1991). L'absorption des différents composants cellulaires (acides gras, protéines, peptides, glucides et composés phosphatés) génère un spectre unique qui ensuite sera comparé avec une base de données. ...
... En effet, au sein du génome de l'espèce S. cerevisiae, un grand nombre (Kim et al., 1998). Cette méthode a premièrement été décrite par Ness et al., (1993) (Xufre et al., 2011 ;Šuranská et al., 2016 ;Tra Bi et al., 2016 ;David-Vaizant et Alexandre, 2018). En comparaison avec les autres méthodes moléculaires, la PCR-Interdelta est la méthode la plus utilisée grâce à sa rapidité, sa reproductibilité et sa haute sensibilité (Schuller et al., 2004). ...
La biodiversité fongique interspécifique (Illumina Mi-Seq) et la dynamique des espèces Saccharomyces cerevisiae et Brettanomyces bruxellensis ont été étudiées au sein d’une nouvelle cuverie et/ou dans 3 caves d’élevage, plus particulièrement sur le sol, les murs, le matériel vinaire et l’extérieur des fûts. Dans la nouvelle cuverie, un consortium fongique (levures et moisissures) de départ est déjà présent sur tous les environnments étudiés avant l’arrivée de la première vendange. Ce consortium est constitué de genres tels que Aureobasidium, Alternaria, Didymella et Filobasidium. Ces genres qui persistent pendant deux millésimes, ne sont pas spécifiques de l’environnement de la cuverie et semblent être adaptés à tous les environnements naturels ou anthropiques au regard de leur caractère ubiquiste. Le consortium de départ est enrichi par des genres œnologiques (exemple : Hanseniaspora, Saccharomyces) qui sont introduits dans la cuverie soit par les vendanges, soit par des transferts potentiels entre les différents environnements de la cuverie. Cependant, ces genres ne semblent pas persister ou s’implanter probablement dû à leur faible adaptation aux conditions stressantes de l’environnement de la cuverie. La dynamique de la flore indigène S. cerevisiae dans la nouvelle cuverie a été également étudiée. Aucun isolat appartenant à cette espèce n’a été retrouvé avant l'arrivée de la première vendange confirmant que cette espèce n’est pas spécifique de l'environnement de la cuverie et que sa présence est en lien avec l'activité des fermentations alcooliques. Cependant, les résultats obtenus suggèrent une colonisation potentielle de l’environnement de la nouvelle cuverie par certaines souches de S. cerevisiae. Ces souches dites « colonisatrices » ont présenté une capacité plus élevée à former des biofilms comparée à celle de souches non implantées. Cette étude met en évidence l’importance de l’environnement de la cuverie qui constitue une véritable niche écologique pour les populations fongiques capables de s’implanter au cours du processus de vinification. Dans l’environnement des 3 caves d’élevage, le matériel vinaire et l’extérieur des fûts (en contact direct avec le vin) sont les environnements qui semblent favorables au développement et à l’installation des populations microbiennes cultivables (levures totales et bactéries lactiques) et des microorganismes d’altération (bactéries acétiques et B. bruxellensis), contrairement au sol et aux murs où des populations faibles ont été trouvées. Des souches récurrentes de B. bruxellensis ont été retrouvées sur le matériel et sur l’extérieur des fûts et pourraient être à l’origine de la contamination de vins au cours de l’élevage. Ces souches récurrentes présentent des capacités de formation de biofilms et de résistance plus importantes qui pourraient expliquer la persistance de B. bruxellensis dans des caves d’élevage. Ces résultats soulignent l’importance du nettoyage du matériel vinaire et du suivi microbien régulier des vins au cours de l’élevage afin de limiter les contaminations.
... In order to discriminate the yeast isolates at strain level, a preliminary identification of the best performing molecular technique was carried out on five isolates, using the following fingerprinting techniques: Restriction Fragment Length Polymorphism analysis of mitochondrial DNA (RFLP mtDNA) (Agnolucci et al., 2007(Agnolucci et al., , 2009), mitochondrial COX1 gene introns amplification (Lopez et al., 2003), repetitive sequence Polymerase Chain Reaction (Lattanzi et al., 2013), Random Amplified Polymorphic DNA (RAPD) (Succi et al., 2003) and inter-delta regions amplification (Ness et al., 1993). ...
... The intraspecific diversity of the 78 yeast isolates, along with the collection strains and two commercial baker's yeasts (Table 1), was carried out by inter-delta regions analysis, as it resulted the best performing technique. Amplification reaction was performed in a final volume of 50 μL, containing 5 μL of 10× DyNAzyme Buffer Mg 2+ -free (Finnzymes, Thermofisher, Milan, Italy), 4 mM MgCl 2 , 0.2 mM of each dNTP (EuroClone), 5 μM of δ 1 (5′-CAAAATTCACCTATA/TTCTCA-3′) and δ 2 (5′-GTGGATTTTTATTCCAACA-3′) (Ness et al., 1993) primer (Eurofins Genomics, Ebersberg Germany), 1.25 U of Taq DyNAzyme II DNA polymerase (Finnzymes) and 160 ng of DNA. PCR amplification was carried out with an iCycler-iQ Multicolor Real-Time PCR Detection System (Bio-Rad, Milan, Italy), using the following conditions: 97°C initial denaturation for 4 min; 30 amplification cycles of 30 s at 94°C, 1 min at 45°C, 2 min at 72°C; final extension at 72°C for 10 min. ...
The increasing demand for healthy baked goods boosted studies on sourdough microbiota with beneficial metabolic traits, to be used as potential functional starters. Here, yeast populations of traditional sourdoughs collected from four Tuscan bakeries were investigated. Among 200 isolated strains, 78 were randomly selected and molecularly characterized. Saccharomyces cerevisiae was dominant, representing the only species detected in three out of the four sourdoughs. The fourth one harbored also Kazachstania humilis. Inter-delta regions analysis revealed a high intraspecific polymorphism discriminating 16 biotypes of S. cerevisiae isolates, which clustered based on their origin. Representative isolates from each biotype group were individually used to ferment soft and durum wheat flour, aiming at evaluating their pro-technological, nutritional and functional features. During fermentation under standardized conditions, all strains were able to grow of ca. 2 log cycles, but only S. cerevisiae L10Y, D18Y and D20Y had a significantly shorter latency phase in both flours. Overall, the highest volumes were reached after 16 h of fermentation in both soft and durum fermented dough. S. cerevisiae D2Y produced the highest dough volume increase. K. humilis G23Y was the only strain able to increase the total free amino acids concentration of the doughs. Overall, values of phytase activity were significantly higher in durum compared to the corresponding soft fermented dough. K. humilis G23Y and S. cerevisiae D20Y, D24Y showed a threefold higher phytase activity than spontaneously fermented control, and the highest concentration of total phenols. Almost all the strains led to increases of antioxidant activity, without significant differences among them. Investigations on the resistance of the strains to simulated gastric and intestinal conditions, that is considered a pre-requisite for the selection of probiotics, revealed the ability to survive in vitro by many of the strains considered. This study proposed the best performing yeast strains selected among autochthonous sourdough yeasts based on their pro-technological, nutritional and functional traits to be used as starters for making sourdough baked goods or functional cereal-based beverages. Although some yeast strains combined several technological and nutritional traits, the association of more selected strains seemed to be a requisite to get optimal sourdough characteristics.
... Around 100 δ copies are present in the yeast genome of S. cerevisiae as part of retrotransposons Ty1 or as isolated elements. The number and localization of these elements demonstrate certain intraspecific variability, which Ness et al. (1993) took advantage of to develop specific primers (δ 1 and δ 2 ) that are useful to differentiate strains of S. cerevisiae. Later Legras and Karst (2003) optimized the technique by designing two new primers (δ 12 and δ 21 ) located very near δ 1 and δ 2 . ...
... Consequently, new primers were able to differentiate more strains, and 53 commercial strains were unequivocally differentiated (Legras and Karst, 2003). Schuller et al. (2004) confirmed this later by showing that the δ 2 and δ 12 combination could identify twice as many strains as the set of primers designed by Ness et al. (1993). ...
The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the availability of rapid and simple molecular techniques that show genetic polymorphisms at species and strain levels have enabled the study of yeast diversity during wine fermentation. This review will summarize the mechanisms involved in generating genetic polymorphisms in yeasts, the molecular methods used to unveil genetic variation, and the utility of these polymorphisms to differentiate strains, populations, and species in order to infer the evolutionary history and the adaptive evolution of wine yeasts, and to identify their influence on their biotechnological and sensorial properties.
... Ce nombre très élevé de copies couplé au changement de position de ces éléments delta en fonction des souches de levures en font de très bons marqueurs du polymorphisme. La méthode a été proposée pour la première fois par Ness et al. (1993). Legras et al. (2003) Le caryotypage permet par l'utilisation de techniques d'électrophorèse en champs pulsé, une séparation de l'ADN chromosomique non digéré en fonction de sa taille . ...
... La discrimination des souches de levure appartenant à l'espèce Saccharomyces cerevisiae a été réalisée par PCR-delta. Cette méthode utilise des amorces ciblant les éléments delta présents en plusieurs copies dans le génome de la levure Saccharomyces cerevisiae et dont la position change en fonction des souches (Ness et al., 1993). L'amplification et la séparation des fragments amplifiés sur gel d'agarose permettent ainsi l'obtention de profils spécifiques en ...
The effects of different anthropogenic activities (vineyard, winery) on fungal populations from grape to wine were studied. To characterize these effects, it was necessary to access to the overall diversity of populations (pyrosequencing and spectroscopy FT-IR) but also to intra-specific diversity (FT-IR). Spectroscopy FT-IR has been validated for their ability to characterize the global population and to discriminate the strains for three species of non-Saccharomyces yeasts (NS). For the first time, it is shown that the grape berry is a limited source for NS yeasts while the winery seems to be a significant source; the air is an important vector for dissemination of these yeasts. In addition, persistence of NS yeast strains from year to year in the winery has been demonstrated. The studied anthropogenic activities modify the fungal diversity. Thus, lower biodiversity of grapes from organic modality was measured for the three vintages considered. The pressing / clarification step revises strongly fungal populations and the influence of the winery flora is confirmed. The addition of SO2 changes the population dynamics and favors the dominance of the species S. cerevisiae. The non-targeted chemical analysis shows, for the first time, that these wines can be distinguished at the end of the alcoholic fermentation (with or without SO2) depending on plant protection. Thus, the existence in wines of chemical and microbiological signatures associated with vineyard protection mode is highlighted.
... The latter solo elements are numerous in some genomes, and this feature has been used to differentiate isolates within a single species by amplifying the regions between LTRs, using primers in the conserved regions of LTRs (inter-LTR PCR). This approach has been applied to various species, including S. cerevisiae (Legras and Karst, 2003;Ness et al., 1993), Debaryomyces hansenii and Kluyveromyces marxianus (Sohier et al., 2009). ...
... We were able to improve strain differentiation by developing inter-LTR PCR for G. candidum, which successfully differentiated all but nine of the strains that were not separated by MLST. Inter-LTR PCR is simple, reliable and fast, as demonstrated previously for other species (Ness et al., 1993;Legras and Karst, 2003;Sohier et al., 2009) and may therefore be useful for strain differentiation, for example, in such applications as analysis of yeast population dynamics during cheese ripening. ...
Geotrichum candidum is an ubiquitous yeast, and an essential component in the production of many soft cheeses. We developed a Multi-Locus Sequence Typing (MLST) scheme with five retained loci (NUP116, URA1, URA3, SAPT4, PLB3) which were sufficiently divergent to distinguish 40 Sequence Types (STs) among the 67 G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a PCR inter-LTR scheme, a fast and reproducible RAPD-like method for G. candidum, to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analyzed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species.
... Genomic DNA extraction of yeast strains, both parental strains as well as putative hybrids, was performed using a fast extraction protocol proposed by [7]. Genomic DNA was used for PCR amplification with the inter-delta transposon primer set MLD1 5 -CAAAATTCACCTAAATTCTCA-3 and MLD2 5 -GTGGATTTTTATTCCAACA-3 [23]. PCR products were loaded on a 1.5% (w/v) agarose gel in TBE buffer 1X (40 mM Trisacetate, 1 mM EDTA) and visualized by means of a UV transilluminator after staining with ethidium bromide 5 µg/mL. ...
There is growing interest in yeast selection for industrial fermentation applications since it is a factor that protects a wine’s identity. Although it is strenuous evaluating the oenological characteristics of yeasts in selection processes, in many cases the most riveting yeasts produce some undesirable organoleptic characteristics in wine. The aim of the present work is to improve an industrial yeast strain by reducing its hydrogen sulfide (H2S) production. To accomplish this, two different improvement approaches were used on said yeast: hybridization by mass mating and adaptive laboratory evolution, both performed through spore generation and conjugation, thus increasing genetic variability. Three evolved variants with lower H2S production were obtained and used as starters to carry out fermentation at an industrial level. Wine quality was analyzed by its principal oenological parameters and volatile aroma compounds, which were both corroborated by sensory evaluations. Significant differences between the produced wines have been obtained and a substantial improvement in aromatic quality has been achieved. Both hybrids were the most different to the control due to terpenes and esters production, while the evolved strain was very similar to the parental strain. Not only have organoleptic defects been reduced at an industrial level, more floral and fruitier wines have been produced.
... The reaction mix was incubated for 2 h and analyzed on 2% agarose gel. Customized oligonucleotides were used to amplify some regions of the yeast genome between the elements that provide an amplified sequence polymorphism, useful in differentiating S. cerevisiae at the strain level (Legras and Karst 2003;Ness et al. 1993). Amplification reactions were performed on a GeneAMP PCR System 2004 thermal cycler (Applied Biosystems, Foster City, California), using primers d12 and d21 and using the same experimental conditions set by Legras and Karst (2003). ...
The aim of this research was to acquire new strains of Saccharomyces cerevisiae exhibiting opposite characteristics of cell wall adsorption: very high adsorption activity toward the ochratoxin A, very low adsorption activity toward the pigmented phenolic compounds contained in musts from black grapes. For this purpose, starting from 313 strains of Saccharomyces cerevisiae, 12 strains were pre-selected and used to obtain 27 intraspecific hybrids. Eleven crosses out of 27 were validated as hybrids; the best five hybrids were used in guided winemaking at four Calabrian wineries. The employed experimental protocol has allowed to select yeast strains for their different adsorption activity, improving the strains by spore clone selection and construction of intraspecific hybrids. These results suggest an efficacious way to improve the characteristics of interest in wine yeasts.
... We also verified that our CRISPR--Cas9/sgRNA system did not result in non--specific genome targeting by sequencing 9 of the targeted strains (Supplementary file 1B). We also tested efficacy of our approach in the polyploid industrial Saccharomyces cerevisiae strain ATCC4124 that has superior tolerance and productivity phenotypes (Ness et al. 1993). The non--tRNA promoter previously used in haploid yeast , PSNR52, functioned in diploid S288C yeast but failed to result in targeting in the polyploid industrial yeast ATCC4124; in contrast, the tRNA Pro promoter enabled highly-efficient barcode insertion into all copies of the URA3 locus in ATCC4124, which we term cis--multiplexing, i.e. introducing a double--stranded DNA break at a single genomic locus across all chromosomes ( Figure 3C and Supplementary file 1A). ...
The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.
... The separation by size of these bands can be used to differentiate S. cerevisiae strains. This method was developed for Ness et al. (1993) and Masneuf and Dubourdieu (1994) to genotype strains of S. cerevisiae. The facility to perform the PCR analysis without extraction of the DNA (using directly the colony) has made this technique the most widely used to differentiate S. cerevisiae strains. ...
... The intraspecific diversity of such isolates, along with the two commercial baker's yeasts, was carried out by inter-delta region analysis. Amplification reaction was performed using δ 1 (5′-CAA AAT TCA CCT ATA/TTC TCA -3′) and δ 2 (5′-GTG GAT TTT TAT TCC AAC A-3′) 61 primers (Eurofins Genomics, Ebersberg Germany) and 160 ng of DNA, as reported in Palla et al. 19 . All gels were visualized and captured as previously described. ...
The increasing demand for healthy baked goods boosted studies on sourdough microbiota with beneficial metabolic traits, to be used as potential functional starters. Here, 139 yeasts isolated from cereal-based fermented foods were in vitro characterized for their phytase and antioxidant activities. The molecular characterization at strain level of the best 39 performing isolates showed that they did not derive from cross contamination by baker’s yeast. Afterwards, the 39 isolates were in vivo analyzed for their leavening ability, phytase activity and polyphenols content using five different wholegrain flours, obtained from conventional and pigmented common wheat, emmer and hull-less barley. Combining these findings, through multivariate permutation analysis, we identified the 2 best performing strains, which resulted diverse for each flour. Doughs singly inoculated with the selected strains were further analyzed for their antioxidant capacity, phenolic acids, xanthophylls and anthocyanins content. All the selected yeasts significantly increased the total antioxidant activity, the soluble, free and conjugated, forms of phenolic acids and anthocyanins of fermented doughs. This study revealed the importance of a specific selection of yeast strains for wholegrain flours obtained from different cereals or cultivars, in order to enhance the pro-technological, nutritional and nutraceutical traits of fermented doughs.
... La composition de la solution de bleu de méthylène utilisée est la suivante : 2 g de citrate de sodium (C 6 H 5 Na 3 O 7 , 2H 2 0) ; 10 mL de bleu de méthylène à 1 g/L et de l'eau (qsp 100mL). La migration sur gel après amplification des régions interδ donne un profil différent selon les souches de S. cerevisiae (Ness et al., 1993). ...
La caractérisation phénotypique de l’espèce Torulaspora delbrueckii en conditions œnologiques, à partir de l’étude d’un grand nombre de souches, a permis de mettre en évidence une grande variabilité au sein de cette espèce. En effet, les souches de T.delbrueckii présentent des différences au niveau des durées de phase de latence et de fermentation, des capacités biotiques mais aussi des productions d’éthanol (maximum 12% vol.). Cette variabilité se retrouve également pour la production d’acidité volatile, de glycérol et de certains arômes. Ce travail confirme les faibles productions d’acidité volatile et de glycérol de cette espèce et met en évidence une réponse au stress osmotique différente de celle de l’espèce Saccharomyces cerevisiae. Au final, l’espèce T. delbrueckii présente une grande « pureté » de fermentation et produit peu de composés indésirables comme le sulfure d’hydrogène, les phénols volatils, l’acetoïne, l’acétaldéhyde et le diacétyle. La réalisation de co-inoculations T. delbrueckii / S. cerevisiae sur moûts liquoreux, permet une réduction systématique de l’acidité volatile des vins, en comparaison à une fermentation pure de l’espèce S. cerevisiae, quelque soit la souche de T. delbrueckii utilisée. De plus, la souche T. delbrueckii OXT1 1//2 a permis de complexifier la composition aromatique d’un moût sec issu du cépage Sauvignon blanc (esters fermentaires +25%, phényl-2-éthanol +51% et thiols volatils +31%). Enfin, la mise au point d’un fermenteur à double compartiment, avec une séparation physique des levures tout en conservant l’homogénéité du milieu de culture, a permis d’aborder l’étude des interactions entre ces 2 espèces. Des inhibitions de type « cell-cell contact » ont ainsi été mises en évidence.
... The genetic diversity of S. cerevisiae strains has been analyzed by several methods such as karyotyping by pulse field gel electrophoresis [27], mitochondrial DNA restriction analysis (mtDNA RFLP) [28][29][30][31], fingerprinting based on repetitive delta sequences [32,33] and microsatellite genotyping [34][35][36]. Schuller et al. [37] have recently shown that microsatellite typing, using 6 different loci [36], an optimized interdelta sequence analysis [33] and RFLP of mitochondrial DNA generated by the enzyme HinfI had the same discriminatory power. ...
One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.
... Plates with glycerol as the carbohydrate source and 14% v/w ethanol, (inhibitory to the S. paradoxus strain), allowed selection of hybrid mitochondrial sufficient and ethanol tolerant colonies. Confirmation of the hybrid nuclear genome of each strain was by multiplex PCR using both Transposon primers 10 and Intron primers 11 (Figure 1). Lanes 2 (AWRI 838) and 3 (52-153) display parental, strain-specific, fingerprints. ...
When we think of Saccharomyces cerevisiae, fermentation immediately comes to mind, but this is not the only trait that makes this yeast the organism of choice for bread, beer and wine production. The winemaking industry, for example, requires robust strains, capable of converting sugar to ethanol in challenging conditions; high osmotic stress and low pH in the initial grape must, followed by high ethanol concentration at the later stages of fermentation. Winemakers also look for ways of using fermentation to introduce aroma and flavour diversity to their wines as a means of improving style and for product differentiation. Choice of wine yeast from the plethora of strains available to winemakers is one way of achieving this, particularly with the new breed of interspecific hybrid yeast strains currently being generated.
... The discovery of DNA amplification by polymerase chain reaction (Saiki et al. 1988) has allowed the development of a second generation of methods targeting polymorphic regions. The polymorphism of the distances between insertion sites of delta element dispersed in the genome of strains has been used as the ground for a fingerprint technique (Ness et al. 1993;, that has become very popular too. Despite the similar resolution obtained by mtDNA RFLP, PFGE, and interdelta typing (Schuller et al. 2004), these three methods present different weaknesses: low resolution of PFGE, the exclusive focus on mitochondrial DNA for mtDNA RFLP, and the small number of bands and possible homoplasy for interdelta typing event for the improved version proposed by . ...
Multiple studies have revealed the richness of the fungal microbial community of grape must and wine which are under the influence of environmental and anthropogenic factors. However, until recently, we had no evidence of how historical processes may have impacted the diversity of the yeasts found on grapes and developing during wine alcoholic fermentation. The different generations of molecular methods have provided progressively more and more information for Saccharomyces cerevisiae, and recently for other yeast species of the grape must microbial communities, gradually revealing how the development of wine technology has shaped the diversities of wine yeasts. This chapter presents the recent advances made on the knowledge of the diversities of yeast species of the grape must and wine microbial community. For the model species S. cerevisiae, the successive generations of genotyping methods and population genomics have offered the most comprehensive view of its evolution including population structure, genomic specificities, and demographic history. The development of molecular methods revealed as well the importance of other Saccharomyces species and their related interspecies hybrids with S. cerevisiae. More recently, the exploration of the diversities of non-Saccharomyces yeast species members of the grape must and wine community such as Hanseniaspora uvarum, Torulaspora delbrueckii, Brettanomyces bruxellensis, Lachancea thermotolerans and Starmerella bacillaris revealed specific population structures.
... These last decades, several studies have been focused on genetic diversity and biogeographical distribution of diverse S. cerevisiae strains. Molecular techniques used for this purpose among many others are pulsed field gel electrophoresis (PFGE) (Vezinhet et al., 1990), restriction analysis of the mitochondrial DNA (mtDNA-RFLP) (Aigle et al., 1984;Querol et al., 1992), amplified fragment length polymorphism (AFLP) (Azumi and Goto-Yamamoto, 2001), amplification of interdelta regions by PCR (Ness et al., 1993;Legras and Karst, 2003) and microsatellite markers (Techera et al., 2001;Legras et al., 2005). Microsatellite analyses have offered significant advances in understanding distribution and dynamics of S. cerevisiae population from different terroirs. ...
... Yeast strains were identified by interdelta PCR fingerprinting using interdelta primers δ2 (5 ′ -GTGGATTTTTATTCCAACA-3 ′ ), δ12 (5 ′ -TCAACAATGGAATCCCAAC-3 ′ ), and δ21 (5 ′ -CAT CTTAACACCGTATATGA-3 ′ ) (Ness et al., 1993;Legras and Karst, 2003). Primer pairs selected for further amplification and analysis were δ2 + δ12 and δ12 + δ21, which both yielded the greatest range of well-resolved bands. ...
The widespread production of fermented food and beverages has resulted in the domestication of Saccharomyces cerevisiae yeasts specifically adapted to beer production. While there is evidence beer yeast domestication was accelerated by industrialization of beer, there also exists a farmhouse brewing culture in western Norway which has passed down yeasts referred to as kveik for generations. This practice has resulted in ale yeasts which are typically highly flocculant, phenolic off flavor negative (POF-), and exhibit a high rate of fermentation, similar to previously characterized lineages of domesticated yeast. Additionally, kveik yeasts are reportedly high-temperature tolerant, likely due to the traditional practice of pitching yeast into warm (>28°C) wort. Here, we characterize kveik yeasts from 9 different Norwegian sources via PCR fingerprinting, whole genome sequencing of selected strains, phenotypic screens, and lab-scale fermentations. Phylogenetic analysis suggests that kveik yeasts form a distinct group among beer yeasts. Additionally, we identify a novel POF- loss-of-function mutation, as well as SNPs and CNVs potentially relevant to the thermotolerance, high ethanol tolerance, and high fermentation rate phenotypes of kveik strains. We also identify domestication markers related to flocculation in kveik. Taken together, the results suggest that Norwegian kveik yeasts are a genetically distinct group of domesticated beer yeasts with properties highly relevant to the brewing sector.
... In order to check dominance of the inoculated strains, 20 different clones of each sample were characterized by two methods: the pro®le of Ty insertions obtained by PCR, and karyotype obtained by PFGE. PCR was carried out according to the method described by Ness et al. (1992). Ampli®cation was performed using a Gene Cycler TM (Bio-Rad) thermal cycler using the following primers: delta 1 (5 H CAAATTCACCTATA/TTCTCA3 H ) and delta 2 (5 H GTGGATTTTTATTCCAACA3 H ). PCR conditions were: (i) 95 C for 10 min; (ii) four cycles at 95 C for 30 s, 45 C for 30 s and 72 C for 2 min; (iii) 30 cycles at 95 C for 30 s, 42 C for 30 s and 72 C for 2 min. ...
... The SC preparation was a lyophilized flocculent strain of Saccharomyces cerevisiae plus growth me- dium. This specific strain of S. cerevisiae has been genetically defined by using PCR test associated with the δ1 (5′CAAATTCACCTATATCTCA3′) and δ2 (5′GTGGATTTTTATTCCAACA3′) primers (Ness et al., 1993). It has been deposited and registered in an official culture collection under the following reference: CBS 493.94. ...
Eight horses were allotted into pairs consisting of one cecum- and right ventral colon-fistulated animal and one cecum-fistulated animal. They were fed daily at the same level of intake either a high-fiber (HF) or a high-starch (HS) diet without or with 10 g of a Saccharomyces cerevisiae preparation, in a 4 × 4 Latin square design. The HS diet provided a starch overload (i.e., 3.4 g starch•kg⁻¹ BW•meal⁻¹) while maintaining a high amount of fiber intake (i.e., dietary NDF/starch ratio was 1.0). A 21-d period of adaptation to the treatments occurred before cecal and colonic contents were withdrawn 4 h after the morning meal to count total anaerobic, cellulolytic, and lactic acid-utilizing bacteria, lactobacilli, and streptococci. Lactic acid, volatile fatty acids, ammonia concentrations, and pH were measured on cecal and colonic fluid samples collected hourly during the first 12-h postfeeding. When the HS diet was fed, the concentration of total anaerobic and lactic acid-utilizing bacteria increased (P < 0.001), whereas that of cellulolytic bacteria decreased (P < 0.05) in the cecum. The concentration of lactobacilli and streptococci increased (P < 0.001) in the cecal and colonic contents. These alterations of the microbial profiles were associated with decreases (P < 0.001) of pH, (acetate + butyrate)/propionate ratio and with an increase (P < 0.001) of lactic acid concentration. Supplementing the S. cerevisiae preparation increased (P < 0.01) the concentration of viable yeast cells, averaging 4.3 × 10⁶ and 4.5 × 10⁴ cfu/mL in the cecal and colonic contents, respectively. Yeast supplementation had almost no effect on microbial counts in the cecum and colon. The supplementation of S. cerevisiae appeared to modify (P < 0.05) pH, concentrations of lactic acid and ammonia, molar percentages of acetate and butyrate with the HS diet and [(acetate + butyrate)/propionate] ratio when the HF diet was fed. The effects of the S. cerevisiae preparation were greater in the cecum than in the colon, which coincided with the abundance of yeast cells. When the digestion of starch in the small intestine was saturated, the effect of the addition of a S. cerevisiae preparation appeared to limit the extent of undesirable changes in the intestinal ecosystem of the horse.
... region (see above). S. cerevisiae isolates were characterized by means of delta element amplification as previously described 45 . The primers d1 (5′-CAAAATTCACCTATWTCTCA-3′) and d2 (5′-GTGGATTTTTATTCCAACA-3′) were used. ...
Saccharomyces cerevisiae is a common yeast with several applications, among which the most ancient is winemaking. Because individuals belonging to this species show a wide genetic and phenotypic variability, the possibility to identify the strains driving fermentation is pivotal when aiming at stable and palatable products. Metagenomic sequencing is increasingly used to decipher the fungal populations present in complex samples such as musts. However, it does not provide information at the strain level. Microsatellites are commonly used to describe the genotype of single strains. Here we developed a population-level microsatellite profiling approach, SID (Saccharomyces cerevisiae IDentifier), to identify the strains present in complex environmental samples. We optimized and assessed the performances of the analytical procedure on patterns generated in silico by computationally pooling Saccharomyces cerevisiae microsatellite profiles, and on samples obtained by pooling DNA of different strains, proving
... With the initial aim of checking a significant number of S. cerevisiae isolates with oenological interest, 880 S. cerevisiae isolates were checked for inter-delta polymorphisms by targeting the δ elements which are repeat sequences that flank the TY1 retrotransposon (Ness et al., 1993). In addition, 11 strains that were isolated from lysine agar plates (D.O. ...
Saccharomyces cerevisiae is the most important yeast species for the production of wine and other beverages. In addition, nowadays, researchers and winemakers are aware of the influence of non-Saccharomyces in wine aroma complexity. Due to the high microbial diversity associated to several agro-food processes, such as winemaking, developing fast and accurate methods for microbial identification is demanded. In this context, MALDI-TOF MS mass fingerprint provides reliable tool for fast biotyping and classification of microorganisms. However, there is no versatile and standardized method for fungi currently available. In this study, an optimized sample preparation protocol was devised for the biotyping of yeasts of oenological origin.
... In order to check dominance of the inoculated strains, 20 different clones of each sample were characterized by two methods: the pro®le of Ty insertions obtained by PCR, and karyotype obtained by PFGE. PCR was carried out according to the method described by Ness et al. (1992). Ampli®cation was performed using a Gene Cycler TM (Bio-Rad) thermal cycler using the following primers: delta 1 (5 H CAAATTCACCTATA/TTCTCA3 H ) and delta 2 (5 H GTGGATTTTTATTCCAACA3 H ). PCR conditions were: (i) 95 C for 10 min; (ii) four cycles at 95 C for 30 s, 45 C for 30 s and 72 C for 2 min; (iii) 30 cycles at 95 C for 30 s, 42 C for 30 s and 72 C for 2 min. ...
Dada la importancia de la composición nitrogenada del mosto, por sus consecuencias en el aroma del vino, es imprescindible conocer el efecto que las diferentes prácticas agronómicas tienen en estas sustancias, tanto en el aspecto cualitativo como en el cuantitativo. En la campaña 2012 se estudiaron, en vendimia, los efectos de diferentes tratamientos de riego (dosis y momento de aplicación) en la composición nitrogenada de cuatro variedades blancas de vid: Airén (Ai), en Albacete; Cigüente (Ci) en Badajoz, Moscatel de Alejandría (Mo) en Valencia y Verdejo (Ve) en Valladolid. En todos los casos, son viñedos conducidos en espaldera, mediante posicionamiento vertical de la vegetación, podados en cordón Royat bilateral y sometidos a similares prácticas de cultivo. En cada localización, la fertilización nitrogenada fue idéntica para todos los tratamientos. Independientemente del régimen hídrico impuesto, Arginina y Prolina fueron los aminoácidos mayoritarios en todos los casos. Los mayores valores de TAC (Contenido Amínico Total) y NFA (Nitrógeno Fácilmente Asimilable) se hallaron en la variedad Mo, seguida de Ci, Ai y, finalmente Ve. Las cantidades de Prolina (aminoácido no asimilable por las levaduras) siguieron la misma tendencia. Las variedades Ci y Mo presentaron respectivamente los mayores y los menores valores de Asparagina, Treonina y Fenilalanina, aminoácidos importantes como precursores de aromas fermentativos. En general, los diferentes regímenes hídricos apenas incidieron en los perfiles amínicos de Ai y Mo, pero sí en Ci y Ve, si bien la tendencia observada fue distinta para cada variedad.
... La distribuzione statistica di questi elementi è di circa uno ogni 150 kb ed essi sono spesso concentrati nelle regioni genomiche adiacenti ai geni del tRNA (Eigel e Feldmann, 1982). Di conseguenza, gli elementi δ, ripetuti direttamente o inversamente, sono separati da segmenti genomici amplificabili: poiché la posizione e/o il numero degli elementi delta sono differenti nei vari ceppi, i prodotti generati dalla amplificazione saranno differenti a seconda dei ceppi (Ness et al., 1993). 78 Figura 84: La struttura di un elemento trasponibile del lievito. ...
Il laboratorio di microbiologia
LE ATTREZZATURE DEL LABORATORIO . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 1
I microscopi ottici . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 1
Le tecniche di analisi microscopica . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 2
Gli strumenti di incubazione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 3
Le bilance tecniche ed analitiche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 4
Gli agitatori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 4
Microcentrifuga . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 5
pH-metri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 5
Anse e pipette sterili monouso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 5
Pipette automatiche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 6
Vetreria specifica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 6
Puntali sterili . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 6
Capsule Petri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 7
LA CREAZIONE DI UN AMBIENTE STERILE . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 8
L’uso del Bunsen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 8
Le cabine a flusso laminare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 8
La sterilizzazione di materiali e substrati . . . . . . . . . . . . . . . . . . . . . . . . .pag. 9
LE ANALISI DEL DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 10
L’elettroforesi su gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 10
Le apparecchiature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 13
Il termociclatore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 16
LA CLASSIFICAZIONE DEI SUBSTRATI NUTRITIVI . . . . . . . . . . . . . . . . . . . .pag. 16
Gli ingredienti dei substrati nutritivi . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 18
Esame, ricerca e conta dei microrganismi del vino
L’ESAME MICROSCOPICO DI UNA COLTURA DI LIEVITO . . . . . . . . . . . . . . .pag. 23
Scheda 1: I preparati microbiologici a goccia schiacciata . . . . . . . . . . . . .pag. 23
LE COLORAZIONI PER MICROSCOPIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 24
Scheda 2: La colorazione di Schaeffer e Fulton . . . . . . . . . . . . . . . . . . . .pag. 24
Scheda 3: La colorazione di Gram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 25
Scheda 4: Determinazione della vitalità cellulare del lievito
mediante colorazione con blu di metilene . . . . . . . . . . . . . . . . . . . . . . . .pag. 26
L’ESAME MACROSCOPICO DI UNA COLTURA DI LIEVITO . . . . . . . . . . . . . . .pag. 27
I METODI DI CONTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 28
Scheda 5: Conta diretta delle cellule di lievito
con vetrino contaglobuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 28
Scheda 6 : I metodi di conta diretti:
descrizione delle camere di conta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 30
I metodi di conta indiretti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 31
Scheda 7: Il conteggio dei lieviti totali . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 32
Scheda 8: Determinazione del contenuto di lieviti contaminanti
durante la fermentazione mediante coltura su agar-lisina . . . . . . . . . . . . .pag. 33
Scheda 9: Determinazione del contenuto di lieviti
e batteri acetici nei mosti e nei vini . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 35
Scheda 10: Determinazione del contenuto di batteri lattici di mosti e vini .pag. 37
Scheda 11: Ricerca dei lieviti del genere Dekkera/Brettanomyces
e di specie cicloeximide-resistenti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 38
Scheda 12: Filtrazione su membrana: controllo di sterilità . . . . . . . . . . . .pag. 39
I METODI DI CONTA DIRETTI ALTERNATIVI . . . . . . . . . . . . . . . . . . . . . . . . .pag. 40
Le gallerie API . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 40
Il riconoscimento dei diversi lieviti di interesse enologico
STRISCI A SINGOLA COLONIA DI LIEVITI DI COLLEZIONE . . . . . . . . . . . . . .pag. 43
Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 44
Metschnikowia pulcherrima . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 45
Hanseniaspora uvarum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 46
Hanseniaspora guilliermondii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 47
Saccharomycodes ludwigii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 48
Issatchenkia terricola . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 49
Pichia membranaefaciens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 50
Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 51
Saccharomyces bayanus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 52
Torulaspora delbrueckii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 53
Zygosaccharomyces rouxii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 54
Zygosaccharomyces bailii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 55
Candida stellata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 56
Kluyveromyces thermotolerans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 57
Zygoascus hellenicus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 58
Brettanomyces spp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 59
Il sughero ed il suo controllo
LA QUALITÀ MICROBIOLOGICA DEL SUGHERO ED IL SUO CONTROLLO . . .pag. 63
Scheda 13: Controllo microbiologico dei tappi . . . . . . . . . . . . . . . . . . . .pag. 65
Le analisi molecolari
LE ANALISI MOLECOLARI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 69
La determinazione delle diverse specie di lievito tramite PCR-RFLP
delle regioni ITS degli rDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 73
Scheda 14: analisi tramite PCR-RFLP della regione 5.8S-ITS rDNA . . . . . .pag. 75
Scheda 15: analisi tramite PCR-RFLP della regione 26S rDNA . . . . . . . . .pag. 77
Distinzione dei diversi ceppi di S. cerevisiae tramite amplificazione
delle sequenze inter-δ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 78
Scheda 16: analisi tramite amplificazione delle sequenze inter-δ . . . . . . . .pag. 79
Scheda 17: analisi del polimorfismo della lunghezza dei frammenti
di restrizione del DNA mitocondriale (mt-DNA) . . . . . . . . . . . . . . . . . . . .pag. 80
Bibliografia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 83
Note Biografiche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pag. 87
... Another commonly used molecular approach relies on sequencing the interdelta element, whose amplification by PCR allows differentiating at the strain level S. cerevisiae strains [33,48]. Other powerful molecular tools for S. cerevisiae strain differentiation are the amplification of polymorphic microsatellite loci, also called simple sequence repeats (SSRs) [40,42], the multilocus sequence typing (MLST) and yeast killer virus (virus dsRNA) [43,44,49,50]. ...
The sparkling wine market has expanded in recent years, boosted by the increasing demand of the global market. As for other fermented beverages, technological yeasts and bacteria selected to design commercial starter cultures represent key levers to maximize product quality and safety. The increasing economic interest in the sector of sparkling wine has also implied a renewed interest in microbial resource management. In this review, after a brief introduction, we report an overview of the main characterization criteria in order to select Saccharomyces cerevisiae strains suitable for use as starter cultures for the production of base wines and to drive re-fermentation of base wines to obtain sparkling wines. Particular attention has been reserved to the technological characterization aspects of re-fermenting phenotypes. We also analysed the possible uses of selected non-Saccharomyces and malolactic strains in order to differentiate specific productions. Finally, we highlighted the main safety aspects related to microbes of enological interest and underlined some microbial-based biotechnological applications helpful to pursue product and process innovations. Overall, the sparkling wine industry may find a relevant benefit from the exploitation of the wide resources associated with vineyard/wine microbial diversity.
... Se ha reportado que los elementos delta están concentrados en regiones genómicas contiguas a los genes ARNt (Eigel & Feldmann, 1982). La variabilidad intraespecífica que otorga el número y la localización de estos elementos fue aprovechada por Ness et al., (1993) para diseñar iniciadores específicos (δ1 y δ2) para la amplificación por PCR de las regiones delta, útiles para diferenciar cepas de S. cerevisiae. Estos autores muestran que la estabilidad de los elementos δ es suficiente para aplicar esta técnica como método de identificación de cepas de S. cerevisiae a nivel industrial, afirmaciones que fueron confirmadas posteriormente por numerosos autores. ...
Las levaduras, además de ser un modelo de la investigación biomédica, tienen diversas aplicaciones en la industria alimentaria, en agricultura y la producción de etanol combustible. Dado que la calidad y la cantidad del producto dependen de la dinámica y la frecuencia de los microorganismos presentes en la fermentación, el uso de herramientas de caracterización molecular se ha incrementado y popularizado en las industrias que emplean levaduras. Estas técnicas se basan en la amplificación o análisis por enzimas de restricción de una porción del ADN genómico de levadura y se clasifican de acuerdo a su capacidad de resolución taxonómica para discriminar a nivel inter o intra-específica. La primera parte de la revisión incluye pruebas interespecíficas tales como, análisis de restricción o RFLP para las regiones ITS2, ITS1-5.8, D1 / D2 de los genes 26S ribosomal DNA. La segunda parte incluye, pruebas de uso común para caracterización nivel de cepa, tales como: la amplificación aleatoria del ADN polimórfico (RAPD), análisis cromosómico por electroforesis en gel de campo pulsado (PFGE), análisis de restricción del ADN mitocondrial (ADNmt- RFLP) análisis por mini / micro satélites y la huella genética de ADN por amplificación de regiones interdelta de los transposones Ty. Esta revisión describe y discute los detalles técnicos de los métodos más utilizados para la caracterización molecular de las levaduras y algunos ejemplos de sus aplicaciones en el contexto industrial.
Palabras clave: Levaduras, caracterización molecular, identificación intraespecífica especies, Saccharomyces cerevisiae.
... In case of food spoilage outbreaks, discrimination of the strain responsible for the spoilage may also be important in order to Face back possible sources and routes of cont¿mination in the food indusny. Nucleic acid-based techniques are sensitive and have the advantage of not being influenced by ttre environmental conditions of the cells because DNA is kept constant during growth (146). These techniques are DNA fingerprinting (60,63,82,109,154,155,207), elecEophoretic karyotyping (35,51,97,98,156,191,(200)(201)(202) and mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis (2,63,77,201). ...
... Esteve-Zarzoso et al. 1999). Strain typing of S. cerevisiae isolates were performed by amplification of inter-region, using the primers pair d2 (5 0 -GTGGATTTTTATTCCAAC-3 0 ) (Ness et al. 1993) and d12 (5 0 -TCAACAATGGAATCCCAAC-3 0 ) (Legras and Karst 2003). DNA extraction from yeast isolates was carried out using the synthetic resin Instagene Matrix (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instruction. ...
In an effort to implement principles of traditional concentrated grape must fermentation to the production of new generation balsamic vinegars (BVs), the specific goals of the study were the isolation and molecular identification of the predominant yeasts in concentrated grape must (cv. Xinomavro), their technological characterization and the evaluation of the fermentative aptitude of the selected strains. Tolerance against 5-hydroxymethyl-furfural (HMF) and furfural, acetic acid and glucose concentration was examined by appropriate methods and tests. The enological characteristics studied were acetic acid and H2S production, foaming and flocculation ability and key enzymatic activity. PCR–RFLP analysis revealed only the presence of Saccharomyces cerevisiae and Hanseniaspora uvarum among the 14 predominant osmophilic yeast isolates. Tolerance to both HMF and furfural was found strain- and dose-dependent and was suggested as a critical factor in the pre-selection of yeast starters. The most tolerant yeasts to these stress factors, a S. cerevisiae and a non-Saccharomyces strains, showed satisfactory growth in the presence of high glucose and acetic acid content (up to 600 g/L and 2 % w/w, respectively) and desirable enological characteristics. Results from the comparative evaluation of the fermentative aptitude of these strains with a commercial wine strain highlighted that the isolates had glucophilic behaviour and ability to produce desirable amounts of ethanol (100–120 g/kg) in short time (~20 d). The key volatiles useful for varietal discrimination and differentiation between the BVs and the traditional ones were also evaluated.
... Numerous molecular methods have been developed to study the ecology and population dynamics of S. cerevisiae strains (reviewed in Ramon 1996, Fernández Espinar et al. 2006). Interdelta sequencing typing uses the variation of the number and position of the delta element, a repeated sequence that flanks the Ty1/Ty2 retrotransposon (Ness et al. 1993), and allows for interpreting strain similarities and evolutionary or adaptive distance Karst 2003, Liu et al. 2014). ...
Interdelta sequence typing was used to investigate the genetic diversity of 54 Chinese indigenous wine strains of Saccharomyces cerevisiae selected on the basis of preliminary sequence analysis from 349 strains that were isolated previously from 15 spontaneous fermentations in Shanshan, Xinjiang, and Qing Tongxia, Ningxia, China. Of the 54 strains tested, 78% (42/54) were confirmed as genetically distinct. Dendrograms based on strain similarity revealed differences in the genetic relationships of Xinjiang yeast populations between table and wine grape varieties, in addition to differences between red and white grape varieties in Ningxia (Dice coefficients of 0.448 and 0.674, respectively). When data from Saccharomyces strains collected from California, France, Italy, northern Europe, and Spain were included in the analysis, the dendrogram revealed five groups containing 51, 4, 48, 3, and 1 strain, respectively. Ningxia and Xinjiang provinces displayed local specific S. cerevisiae biotas that show a clear separation from other strains. Cluster XJ19 isolated from Xinjiang displayed a high level of similarity with UCD587, UCD2515, and UCD2516 from California. Clusters XJ2, XJ7, XJ20, and XJ3, also isolated from Xinjiang, had a lower degree of similarity with other Chinese indigenous genotypes and strains from other regions. This study compares, for the first time, the genetic diversity and relationships between indigenous S. cerevisiae wine strains collected from Xinjiang and Ningxia provinces in China with wine strains from different geographic regions.
... The amplification PCR reactions were performed on a Thermal Cycler (Bio-Rad, Hercules, CA, USA), with the PCR mixture added directly to these unpurified DNA suspensions. Interdelta sequences were amplified according to the procedure described by Gobbi et al. (2013) using δprimers proposed by Ness et al., (1993) and optimized by Legras and Karst, (2003). The amplification products were separated by electrophoresis on 1.5% (w/v) agarose gels at 75 V for 1 h in 0.5× TBE buffer. ...
Over the last few years the use of multi-starter inocula has become an attractive biotechnological practice in the search for wine with high flavour complexity or distinctive characters. This has been possible through exploiting the particular oenological features of some non-Saccharomyces yeast strains, and the effects that derive from their specific interactions with Saccharomyces. In the present study, we evaluated the selected strain Zygotorulaspora florentina (formerly Zygosaccharomyces florentinus) in mixed culture fermentations with Saccharomyces cerevisiae, from the laboratory scale to the winery scale. The scale-up fermentation and substrate composition (i.e., white or red musts) influenced the analytical composition of the mixed fermentation.
Most alcoholic fermentations in winemaking are nowadays performed by commercially produced yeast strains that are inoculated into the fermentation vessel. Although many yeast strains are available on the global market, the generation of new strains that display different and/or new traits continues to attract significant industrial interest. Many traditional breeding and selection, as well as genetic engineering, approaches have been used to achieve specific targets. These approaches can now be complemented with powerful systems biology tools which provide new insights and opportunities for the generation of new yeast strains while holistically improving our knowledge of wine microbial ecology.
Saccharomyces cerevisiae is the main actor of wine fermentation but at present, still little is known about the factors impacting its distribution in the vineyards. In this study, 23 vineyards and 7 cellars were sampled over 2 consecutive years in the Bordeaux and Bergerac regions. The impact of geography and farming system and the relation between grape and vat populations were evaluated using a collection of 1374 S. cerevisiae merlot grape isolates and 289 vat isolates analyzed at 17 microsatellites loci. A very high genetic diversity of S. cerevisiae strains was obtained from grape samples, higher in conventional farming system than in organic one. The geographic appellation and the wine estate significantly impact the S. cerevisiae population structure, whereas the type of farming system has a weak global effect. When comparing cellar and vineyard populations, we evidenced the tight connection between the two compartments, based on the high proportion of grape isolates (25%) related to the commercial starters used in the cellar and on the estimation of bidirectional geneflows between the vineyard and the cellar compartments.
Saccharomyces cerevisiae est l’acteur principal de la fermentation du moût de raisin, mais l’influence de facteurs sur sa distribution dans les vignobles est peu connue. La région bordelaise, par son histoire et ses appellations, est une région d’intérêt pour étudier la diversité de S.cerevisiae. Au total, 2422 isolats de S.cerevisiae provenant de prélèvements de raisins et de cuves en fermentation spontanées sur deux années consécutives ont été analysés par 15 à 17 marqueurs microsatellites. Une très grande diversité génétique est mise en évidence, supérieure en mode de conduite conventionnel par rapport au mode biologique. Le mode de conduite influence faiblement la structure de la population de S.cerevisiae au vignoble. L’appellation et le domaine impactent significativement la structure de population, sans que des gradients de diversité n‘apparaissent, mais nos analyses révèlent des connections importantes dans le sens Pessac-Léognan vers les autres appellations du Bordelais, en particulier le Médoc. Des flux importants bidirectionnels sont mis en évidence entre les compartiments vigne et chai, illustrés par la présence de 25% de souches apparentées à des levures commerciales au vignoble, retour des souches du chai au vignoble jusqu’alors sous-estimé, alors qu’un flux d’importance similaire est observé entre le vignoble et le chai. La présence de populations ancestrales communes dans des prélèvements anciens (plus de 20 ans) et récents révèle la stabilité des populations sur le long terme à l’échelle d’une appellation. Une succession temporelle des populations du chai pourrait être favorisée par la mise en œuvre de pied de cuve avec repiquages successifs.
Yeast have a fundamental role in winemaking. They carry out alcoholic fermentation and they contribute to the quality of the wine, although they can also cause spoilage during grape must transformation and in the final product. To detect and identify wine yeast and control their activities, a plethora of different methods can be utilized. As reported in the present chapter, these methods have different degrees of complexity and vary in terms of cost, rapidity and sensitivity. Those based on yeast isolation, namely culture-dependent methods, are widely utilized to define the composition of the microflora associated with wine-related environments and for yeast identification at the strain level, besides providing a means for ex-situ preservation of wine yeast biodiversity. Culture-independent methods bypass microorganisms cultivation, thus avoiding any bias introduced by their isolation and uncovering cell populations undetected by culture-dependent methods. These methods can be utilized to evaluate the impact of all of the components of the wine microbiota on the quality of the final product, to implement a quality control system based on real-time detection and quantification of specific targets, such as the inoculated starter (s) or the spoilage yeast, or to provide further insights into the composition of the microbial communities involved in the grape must transformation.
This study was carried out to evaluate the effect of cold stress on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) cluster analysis of Saccharomyces cerevisiae. Seven pair strains including one bread yeast strain and one or two wild type strains of S. cerevisiae were selected from yeast strain stocks at the Aichi Center for Industry and Science Technology. By using MALDI-TOF MS cluster analysis, those pair strains were shown as being not separated into single strain clusters. When those colonies were re-incubated at low temperatures before MALDI-TOF MS preparation, all pair strains were found to have separated into single strain clusters. These distinctions show that cold stress proteins could be used as biomarkers for MALDI-TOF MS identification of S. cerevisiae strains.
Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeast, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically-modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotoleran ce, increased resistances towards fermentation inhibitors, and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially-available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared twenty-one industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency, and ability to ferment lignocellulosic sugars. These data may be useful for selection of a parental strain for specific biotechnological applications of engineered yeast.
In order to discern sake yeast strains, a specific polymerase chain reaction (PCR) which targeted 100 long terminal repeat (LTR) regions was tested. We found the difference in DNA fragments amplified by using five pair primers for designed LTR regions of YDRWdelta25, YELWdelta5, YLRWdelta20, YGRWdelta21, and YPLWdelta7. Amplified DNA fragments did not differ in their fragment lengths or their presence. K-6 and No28 of sake yeast strains were characterized, and the others were classified into three classes by the presence of amplified DNA fragments.
Yeasts from the Saccharomyces complex have been used for millennia for the production of fermented food and alcoholic beverages . The availability of large genomic datasets during the past decade has provided new insights into the genetic and phenotypic diversity, population structure and evolutionary history of these yeasts. Studies of these datasets have shown that man-made environments have led to several distinct domesticated variants. Comparative genomics approaches have revealed domestication fingerprints and indicated divergent regions that may explain the adaptation of strains to different ecological niches. In addition, the genetic basis of several technological traits of S. cerevisiae has been elucidated through QTL mapping, and strains improved for various industrial traits have been developed through hybridization or evolutionary engineering. The expansion of large-scale genomic and high-throughput phenotypic data on these strains will provide a unique resource for understanding their adaptation to their ecological niches and for elucidating the missing links between genotype and phenotype, paving the way for strain improvement.
Osmotic stress represents one of the major environmental challenges experienced by yeast during industrial 15 fermentations. This stress is particularly associated with high gravity processes which utilise concentrated 16 substrates to yield products with elevated concentrations of ethanol. The aims of this work were to quantitatively 17 measure factors affecting extracellular osmotic pressure (osmolality) during brewing fermentations, and to 18 determine their effects on yeast at the physiological and molecular level. Osmolality was observed to increase 19 during fermentation due predominantly to ethanol production, indicating a strong relationship between these 20 environmental parameters. High osmolality was shown to have a negative impact on yeast physiology, viability 21 and vitality and although genome integrity was unaffected, cell membrane fluidity became altered. This data not 22 only demonstrates the occurrence of an increase in osmotic pressure during fermentation, but provides 23 explanation for the decrease in yeast quality typically observed under high gravity conditions. The results 24 presented here are directly relevant to all brewery fermentations worldwide and have applications within 25 associated industries where microorganisms are used for ethanol production, including food products, alcoholic 26 beverages and biofuels.
Significance and impact of the study:
Genotyping is routinely used for assessing the diversity of a large number of isolates/strains of a single species, e.g. a collection of wine yeasts. We tested the efficiency of interdelta genotyping on a collection of Saccharomyces wine yeasts from four wine regions of Hungary that was previously characterized physiologically. Interdelta fingerprinting recovered neither physiological nor geographical similarities, and in addition, the two different primer pairs widely used for this method showed conflicting and barely comparable results. Thus, this method does not necessarily represent the true diversity of a strain collection, but detailed clustering may be achieved by the combined use of primer sets.
The long-time studies on wine yeasts have determined a wide diffusion of inoculated fermentations by commercial starters, mainly of Saccharomyces. Although the use of starter cultures has improved the reproducibility of wine quality, the main drawback to this practice is the lack of the typical traits of wines produced by spontaneous fermentation. These findings have stimulated wine-researchers and wine-makers towards the selection of autochthonous strains as starter cultures. The objective of this study was to investigate the biodiversity of 167 S. cerevisiae yeasts, isolated from spontaneous fermentation of grapes. The genetic variability of isolates was evaluated by PCR amplification of inter-δ region with primer pair δ2/δ12. The same isolates were investigated for characteristics of oenological interest, such as resistance to sulphur dioxide, ethanol and copper and hydrogen sulphide production. On the basis of technological and molecular results, 20 strains were chosen and tested into inoculated fermentations at laboratory scale. The experimental wines were analyzed for the content of some by-products correlated to wine aroma, such as higher alcohols, acetaldehyde, ethyl acetate and acetic acid. One selected strain was used as starter culture to perform fermentation at cellar level. The selection program followed during this research project represents an optimal combination between two different trends in modern winemaking: the use of S. cerevisiae as starter cultures and the starter culture selection for specific fermentations.
The general objective and unifying thread of this Thesis is the development of easy, rapid and affordable molecular methods for the detection and strain typing of the spoiling or/and industrial interesting yeasts
species as well to analyze their performance by applying them to a study case ofnorganicnyogurt spoilage.
The yeasts species included in this work are interesting from several points of view (food spoilage, starters,
biocontrol and clinic).
L'analyse des profils de restriction de l'ADN mitochondrial des levures permet une caractérisation fine des souches de Saccharomyces cerevisiae. Cette analyse a été appliquée à deux souches de levures sèches actives et à une vingtaine de souches indigènes, isolées de différents moûts lors de la fermentation spontanée.
Les profils de restriction de l'ADN mt des souches étudiées présentent une grande diversité.
La méthode mise en oeuvre est décrite de façon détaillée et les applications pratiques discutées.
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The analysis of restriction patterns from yeast's mitochondrial DNA leads to a fine characterization of different strains of Saccharomyces cerevisiae. This analysis has been applied to two commercial strains and to twenty wild yeasts, isolated from different musts in case of natural fermentations.
The DNA restriction patterns of the strains studied present a wide diversity.
The method we used is minutely described and the practical applications are discussed.
The polymerase chain reaction using only a single ‘consensus’ tRNA gene primer, or a pair of primers facing outward from tRNA
genes, amplifies a set of DNA fragments in bacterial, plant and animal genomlc DNAs. Presumably, these PCR fingerprints are
mainly derived from the regions between closely linked tRNA genes. The pattern of the PCR products is determined by which
genomes and which primer(s) are used. Genomic fingerprints are largely conserved within a species and, in bacteria, most products
in the fingerprint are conserved between closely related species. Thus, PCR with tRNA gene consensus primers helps to identify
specles and genera.
Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase
chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two
cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by
comparing polymorphisms in genomic fingerprints. The generality of the method Is demonstrated by application to twenty four
strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).
A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
Fermented mineral medium in which a pure culture yeast, Saccharomyces cerevisiae had been grown, was fractionated to find which components were responsible for foaminess. Very little of the foaminess was due to the yeast cells themselves. More than 80% of the foaminess of the supernatant was retained by an ultrafilter of nominal cut-off 10,000 daltons. The foaminess of this was found on chromatography to be due to three peaks of proteinaceous nature with molecular weights around 145×103, 125×103 and 100×103. Ethanol was found to affect the foaminess of these proteins: at concentrations above 8% it reduced foaminess, but at lower concentrations it enhanced foaminess with maximum enhancement at 5%.
The genotypes of the Carlsberg and Tuborg bottom fermenting (lager) Saccharomyces strains as well as those of 13 other bottom fermenting yeasts and 9 top fermenting yeasts from the collection of the Wissenschaftliche Station fur Brauerei in Munich were analysed fro restriction fragment length differences in theRDN1 ribosomal RNA gene cluster on chromosome XII as well as in the chromosome III regions containing theHIS4 (histidine 4) andLEU2 (leucine 2) genes. All brewing strains had the same form IIRDN1 gene cluster. The top fermenting strains yielded identical Sall restriction endonuclease fragment patterns (I) when probed with theHIS4 gene probe of plasmid pC503. The pattern corresponds to that found in genetic standard strain S288C. The bottom fermenting strains were all heterozygous for theHIS4 patterns I and II known to originate from homeologous regions of two chromosomes. The transposable elementTy1 from a genetic standard strain distinguishes the genotypes of various lager strains as well as those of top fermenting yeasts. Hybridisation ofTy1 to EcoRI restricted DNA of Carlsberg lager strain BK 2246 yields four diagnostic fragments, while the DNA of Tuborg lager strain BK 2224 is characterized by an additional 5.5 kb fragment. Conversely Sall restricted DNA of the Carlsberg strain contains a 10 kb fragment absent from the genome of the Tuborg strain. Three different restriction fragment patterns are found after probing with theTy1 element among the Bavarian bottom fermenting yeasts. More complex patterns with at least 10 restriction fragments hybridising to theTy1 element are found among the top fermenting strains from the Munich collection. Five different genotypes have been identified among 9 strains tested.Ty1 transposing elements will be helpful markers in the characterisation of improved brewing strains.
Chromosomal DNA patterns using the transverse alternating field electrophoresis technique and mitochondrial DNA restriction profiles have been achieved for 22 enological strains of Saccharomyces cerevisiae. Both methods have evidenced a marked polymorphism of these strains. Twenty different karyotypes and 17 mitochondrial DNA banding patterns have been observed. Only three strains originating from the same vineyard could not be differentiated by either of the two methods. The polymorphism observed at the chromosomal and mitochondrial levels makes the techniques investigated powerful tools for identification and control of industrial strains.
30 bottom fermenting strains, 13 hybrid lager strains, 11 top fermenting strains. In addition, 24 other yeast species and
strains have been analysed in this study. Four DNA regions have been monitored by restriction endonuclease fragment pattern
polymorphisms. The regions analysed are:RDN1 (encoding cytosolic ribosomal RNA molecules) located on chromosome XII in S. cerevisiae,HIS4 (histidine 4) andLEU2 (leucine 2) both located on chromosome III and theTy elements which are distributed at different positions in the genome of S. cerevisiae. SevenTy fragment patterns have been detected in the bottom fermenting strains. Patterns I, II, IIa, IIb, and IIc are present in lager
strains while pattern III and IV are found in S. carlsbergensis bottom fermenting strain No. I and in S. monacensis, respectively.
Hybridization of theTy1 p14 probe from S. cerevisiae to OFAGE separated chromosomes from bottom fermenting strains show that at least fiveTy elements are present in the genomes of the lager strains, at least five in S. carlsbergensis bottom fermenting strain No.
I and at least three in S. monacensis. From the restriction fragment patterns and the karyotypes of the bottom fermenting
strains it is suggested that these strains are closely related and different from the S. bayanus, S. pastorianus, and S. uvarum
group as well as from the S. cerevisiae strains. In spite of their differences inTy element patterns the large strains are so homogenous that most likely they all originate from a single strain which is related
to S. carlsbergensis and S. monacensis type strains. In contrast to the bottom fermenting strains there is a large variability
among the top fermenting strains, especially with regard to theLEU2 gene andTy elements. It seems that S. bayanus, S. inusitatus, S. pastorianus, S. validus, and S. uvarum are closely related, and none
of the studied strains from these taxons contained DNA which hybridized to theTy1 probe from S. cerevisiae. This is a clear difference from the DNA of bottom fermenting strains which consistently hybridized
strongly to theTy1 probe.
Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.
Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms
(RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random
DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which
amplify from one parent but not the other, are inherited in a Mendellan fashion and can be used to construct genetic maps
in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme,
isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed
at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments
up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule
present only once in a sample of 10(5) cells.
In this paper we present a systematic approach to gene mapping and genotyping based on the simultaneous analysis of multiple amplified sequence polymorphisms (ASPs). These genetic markers measure variation in DNA sequences which have been amplified by a polymerase and/or a ligase. The amplified sequence lengths are determined by appropriate choice of oligonucleotides used in the amplification reaction. We describe three classes of ASPs: restriction site polymorphisms, sequence length polymorphisms, and DNA base pair changes not associated with restriction sites. Simultaneous analysis of multiple ASPs using a modified automated DNA sequencing apparatus should be possible because amplification with oligonucleotides provides control over the fragment lengths generated. Development of an automated ASP technology is therefore the next logical step for efficient gene mapping and genotyping of individuals. With this technology, one gel would be sufficient to indicate the most probable locations of a gene and a second gel would permit the selection of the correct location while simultaneously providing a fine structure map.
Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
We studied the NAM2 genes of Saccharomyces douglasii and Saccharomyces cerevisiae, and showed that they are interchangeable for all the known functions of these genes, both mitochondrial protein synthesis and mitochondrial mRNA splicing. This confirms the prediction that the S. douglasii NAM2D gene encodes the mitochondrial leucyl tRNA synthetase (EC 6.1.1.4.). The observation that these enzymes are interchangeable for their mRNA splicing functions, even though there are significant differences in the intron/exon structure of their mitochondrial genome, suggests that they may have a general role in yeast mitochondrial RNA splicing. A short open reading frame (ORF) precedes the synthetase-encoding ORF, and we showed that at least in S. cerevisiae this is not essential for the expression of the gene; however, it may be involved in a more subtle type of regulation. Sequence comparisons of S. douglasii and S. cerevisiae revealed a particularly interesting situation from the evolutionary point of view. It appears that the two yeasts have diverged relatively recently: there is remarkable nucleotide sequence conservation, with no deletions or insertions, but numerous (albeit non-saturating) silent substitutions resulting from transitions. This applies not only to the NAM2 coding regions, but also to two other ORFs flanking the NAM2 ORF. The regions between the ORFs (believed to be intergenic regions) are much less conserved, with several deletions and insertions. Thus S. douglasii and S. cerevisiae provide an ideal system for the study of molecular evolution, being two yeasts "caught in the act" of speciation.
Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stable in vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 degrees C. At pH 4.0, closer to the in vitro pH optimum of the K2 killer toxin, the K1 strain again predominated at 18 degrees C, but at 30 degrees C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1-K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1-K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.
Chromosomal DNAs from various yeast species were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). To this end we developed a spheroplasting and lysis method to obtain intact DNA from both ascomycetous and basidiomycetous yeasts. The OFAGE banding patterns of 22 ascomycetous and four basidiomycetous yeast strains were compared. The strains represented species from the genera: Brettanomyces, Candida, Cryptococcus, Filobasidiella, Geotrichum, Hansenula, Kluyveromyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccharomyces and Zygosaccharomyces. Variations occurred in the number of bands and their positions in the gel, not only among strains of different genera but also among species from the same genus and even between varieties of the same species. The ascomycetous yeasts, with the exception of Saccharomyces cerevisiae, only showed one to five bands of DNA larger than 1000 kilobase pairs (kb) in general none smaller. The patterns of the four basidiomycetous yeasts revealed also a few large DNA bands but in addition one to six bands ranging in size from 500 to 1000 kb, with the exception of a single smaller chromosome in Rhodotorula mucilaginosa. From the OFAGE banding patterns of strains studied here it appears that in Sacch. cerevisiae the partitioning of DNA over chromosomes is unique. But rather than the large number of chromosomes, the presence of four chromosomes with less than 500 kb of DNA is characteristic for Sacch. cerevisiae.
The chromosomal DNA molecules of a standard laboratory strain of Saccharomyces cerevisiae have been separated into 12 well-resolved bands by orthogonal-field-alternation gel electrophoresis. DNA X DNA hybridization probes derived from cloned genes have been used to correlate this banding pattern with yeast's genetically defined chromosomes. The 12 bands are shown to represent 9 singlets and 3 comigrating doublets, thereby accounting for 15 chromosomes that were identified as I-XI and XIII-XVI. Because the three comigrating doublets could be readily resolved in certain laboratory yeast strains that contain chromosome-length polymorphisms relative to our standard strain, all 15 of these chromosomes could be displayed as a single band in at least one of four strains that were studied. A 16th chromosome (number XII), which is known to contain the genes for rRNA, does not reproducibly enter the gels. By making use of the band identifications, the previously unmapped fragment F8 was assigned to chromosome XIII. With the possible exception of chromosomes that differ greatly in size or electrophoretic behavior from all the known chromosomes, the results appear to define a complete "electrophoretic karyotype" for yeast.
sigma is a DNA element of about 340 base pairs (bp) that is repeated many times in the yeast genome. The element has 8-bp inverted repeats at its ends and is flanked by 5-bp direct repeats. The 5-bp repeats are different for each sigma and have no homology with the ends of the sigma sequence. sigma is located 16 or 18 bp from the 5' end of several tRNA genes. Southern analysis of different yeast strains shows that the pattern of hybridization is different even for closely related strains.
A comparative analysis of a number of yeast DNA-pBR322 recombinant plasmids carrying repetitive sequence elements has revealed that Ty1 or delta elements occur in the vicinity of several tRNA genes. Four examples have been characterized in detail: three glutamate tRNA genes and a serine tRNA gene. The tRNAGlu3 genes occupy different chromosomal locations; two of these genes are found adjacent to Ty1 elements, and the third is found adjacent to an independent delta element. A delta unit is also found adjacent to a tRNASer2 gene. Next to one of the tRNAGlu3 genes, the delta element is joined to a truncated sigma element. Junctions between different delta units were characterized by the sequence analysis of two DNA segments that carry no tRNA genes.
We have found a 371-base-pair (bp) repeated DNA element, tau, in Saccharomyces cerevisiae. The ends of tau are composed of a 5-bp inverted repeat, similar in sequence to those reported for the Ty, sigma, copia, and spleen necrosis virus elements. These inverted repeats are flanked by 5-bp direct repeats of a target sequence that occurs only once in an allele that lacks the tau element. This overall structure is characteristic of transposable elements. Like sigma, tau elements have been found (in both orientations) closely associated with tRNA genes (409 and 198 bp from the 5' end, respectively). It is noteworthy that one representative of tau was isolated in a concentric insertion of tau, delta, and sigma.
A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.
Two so far undetected tRNA genes were found close to delta (delta) sequences at the sup4 locus on chromosome X in the genome of Saccharomyces cerevisiae. The two genes were identified from their abundant transcription products in frog oocytes. Hybridisation experiments allowed the mapping of the transcripts in cloned DNA and DNA sequence analysis revealed the presence of one AGGtRNA and one GACtRNA gene. tRNA genes with sequences similar or identical to GACtRNA exist in 14-16 copies per haploid yeast genome, whereas only one copy was detected for AGGtRNA. In vivo labelling of total yeast tRNA with P followed by hybridisation revealed that the unique AGGtRNA gene is transcribed in S. cerevisiae. delta sequences are present 120 bp upstream from the first coding nucleotide in the case of AGGtRNA, 80 bp in the case of GACtRNA and 405 bp in the case of the known UACtRNA (sup4) gene. delta sequences, as part of Ty elements or alone, were also found by other investigators at similar distances upstream of the mRNA start in mutant alleles of protein-coding yeast genes. Although protein-coding genes are transcribed by RNA polymerase II and tRNA genes by RNA polymerase III, the 5' non-coding region of both types of genes could conceivably have a peculiar DNA or chromatin structure used as preferred landing sites by transposable elements.
The macromolecular compounds excreted during growth by yeasts as exocellular fractions were studied and found to contain a high proportion of carbohydrates. On the basis of the specificity of these fractions, an attempt was made to distinguish between nine strains of a single species of yeast-Saccharomyces cerevisiae. Each strain came from a different source. The electrophoretic patterns of the exocellular fractions tested made it possible to distinguish between the nine strains, on the basis of Lodder's criteria for the morphological and physiological identification of yeasts. We considered that strains giving exocellular fractions distinguishable by electrophoresis belonged to distinct clones. Consequently, this technique enables us to go further than diagnosis of the species.
BouixM1990Application de l'immunofluorescence à la différentiation fine des souches de levures
- S A Lallemand
- Toulouse
Implantation de levures sélectionnées, étude en site industriel de vinification
- Rozières C
A rapid identification technique to differentiate between in vitro
- Tredoux HG