Genetic characterization of commercial Saccharomyces cerevisiae isolates recovered from vineyard environments using comparative genome hybridization on array (aCGH)

The use of commercial S. cerevisiae wine strains as fermentation starters has been extensively generalized over the past two decades. Within our previous work we showed that such strains are disseminated from the winery and their permanence in nature induced genetic changes in comparison to the commercial “mother” strain. The objective of the present study was to evaluate genome variations among four isolates of the commercial strain Zymaflore VL1 that were re-isolated from vineyards close the wineries, in comparison to the commercial “mother” strain by the use of comparative genome hybridization on array (aCGH). Hierarchical cluster analysis showed some genome variability between both groups. Using one-way ANOVA we identified more than 50 genes with significant differences. Multi-class significance analysis revealed a group of 130 genes with reduced fold changes in the recovered strains, being the most relevant the PAU family, involved in the adaptation of yeast cells to stress during alcoholic fermentations. Our studies showed that genetic changes occur among isogenic strains of the commercial yeast Zymaflore VL1 that were re-isolated from environments. The depletion of several genes may reflect adaptive mechanisms that strains undergo as response to the conditions of vineyards that are very distinct from must fermentation environments.Fundação para a Ciência e a Tecnologia (FCT

Dissemination and survival of commercial wine yeast in the vineyard: a large-scale, three years study

The use of commercial wine yeast strains as starters has been extensively generalised over the past two decades. In this study, a large-scale sampling plan was devised over a period of three years in six different vineyards to evaluate the dynamics and survival of industrial yeast strains in the vineyard. A total of 198 grape samples were collected at various distances from the wineries, before and after harvest, and yeast strains isolated after spontaneous fermentation were subsequently identified by molecular methods. Among 3780 yeast strains identified, 296 isolates had a genetic profile identical to that of commercial yeast strains. For a large majority (94%), these strains were recovered at very close proximity to the winery (10-200m). Commercial strains were mostly found in the post harvest samples, reflecting immediate dissemination. Analysis of population variations from year to year indicated that permanent implantation of commercial strains in the vineyard did not occur, but instead that these strains were subject to natural fluctuations of periodical appearance/disappearance like autochthonous strains. Our data show that dissemination of commercial yeast in the vineyard is restricted to short distances and limited periods of times and is largely favoured by the presence of water runoff.ENOSAFE (Nº 762, Programa AGRO, medida 8.1) and the grant nº 657 C2 from the cooperation agreement between the Portuguese Institute for International Scientific and Technological Cooperation (ICCTI) and the French Embassy in Lisbon and the Marie Curie Fellowship of the European Community programme of Quality of Life under Contract QLK4-CT-2001-51873

Comparative genomics of commercial Saccharomyces cerevisiae isolates recovered from vineyard environments

Apresentação efetuada nas 36.ª Jornadas Portuguesas de Genética, Coimbra, 2011The use of commercial Saccharomyces cerevisiae wine yeast strains as fermentation starters has been extensively generalised over the past two decades. These strains are used by wineries every year at the harvest time in large quantities. Within our previous work we showed that such strains are disseminated from the winery and can be recovered from locations in close proximity (10-200m) (Valero et al., 2005). Their permanence in natural environments induced genetic changes such as loss of heterozygosity, microsatellite expansion and size variations of chromosomes III and/or VI. These changes were not found among a group of isolates that were used as a control and that derived from the commercial “mother” strain (Schuller et al., 2007). The objective of the present study was to evaluate genome variations among four isolates of the commercial strain S. cerevisiae Zymaflore VL1 that were re-isolated from vineyards surrounding the wineries where this strain was applied, in comparison to the commercial “mother” strain by the use of comparative genome hybridization on array (aCGH). These approaches were carried out as previously described (Carreto, et al., 2008). Hierarchical cluster analysis showed some genome variability between isolates recovered from the environment and the commercial VL1 reference strain. Using one-way ANOVA we identified more than 50 genes with significant differences between the environmental isolates and the original VL1 strain. Multi-class significance analysis (SAM) for two-class unpaired comparisons revealed a group of 130 genes with significantly reduced fold changes in the recovered strains. The most relevant genes (PAU2, PAU3 and PAU5) belonged to the seripauperin family. They are located in subtelomeric chromosomal regions, where gene families are maintained that serve for adaptive purposes. These genes are involved in the adaptation of yeast cells to stress during alcoholic fermentations and are highly upregulated during early stages of wine fermentation. Our studies showed that genetic changes occur among isogenic strains of the commercial yeast S. cerevisiae Zymaflore VL1 that were re-isolated from environments close to vineyards where this strain is used as starter yeast. The depletion of several genes may reflect adaptive mechanisms that strains undergo as response to the ecological conditions of vineyards that are very distinct from must fermentation environments.Fundação para a Ciência e a Tecnologia (FCT

Microevolutionary changes of commercial Saccharomyces cerevisiae strains recovered from vineyard environments identified by comparative genome hybridization on array

The use of commercial Saccharomyces cerevisiae wine yeast strains as fermentation starters has been extensively generalised over the past two decades. These strains are used by wineries every harvest time in large quantities. Within our previous work we showed that such strains are disseminated from the winery [1] and their permanence in nature induced genetic changes that were not found among a control group of isolates that derived from clonal expansion of the commercial “mother” strain [2]. The objective of the present study was to evaluate genome variations among four isolates of the commercial strain S. cerevisiae Zymaflore VL1 that were re-isolated from vineyards surrounding the wineries where this strain was applied, in comparison to the commercial “mother” strain, by the use of comparative genome hybridization on array. These approaches were carried out as described [3]. Data analysis showed genetic differences among the recovered isolates in comparison with the “mother” strain. Amplification (between 1 and 2 fold changes) of 14 genes were detected, related with mitosis (SHE1) or meiosis (HFM1), lysine biosynthesis (LYS14), galactose (GAL1) and asparagine catabolism (ASP3-2). ASP3-2 amplification is in agreement with the previously shown increased expression during nitrogen starvation. This might occur as an adaptation to natural environments with poor yeast-utilizable nitrogen sources. Eight Ty elements were also amplified, whereas each of the recovered strains had a unique pattern of amplifications. Phenotypic screening was performed considering 28 physiological tests. Seven phenotypic traits distinguished recovered strains from the “mother” strain which was unable to grow at 18ºC, but evidenced some growth in the presence of CuSO4 5mM and SDS 0.01%. Variable growth patterns were found for NaCl 1.5M, KHSO3 (300 mg/l) and wine supplemented with glucose (0.5% and 1% w/v). We hypothesize that the transition from nutrient-rich musts to nutritionally scarce natural environments induces adaptive responses and microevolutionary changes promoted by Ty elements. These changes (and possibly others as well) may contribute to intra-strain phenotypic variability.Fundação para a Ciência e a Tecnologia (FCT

Intra-strain phenotypic and genomic variability of the commercial Saccharomyces cerevisiae strains Zymaflore VL1 recovered from vineyard environments

The use of commercial Saccharomyces cerevisiae wine strains as fermentation starters has been extensively generalised over the past two decades. These strains are used by wineries every harvest in large quantities. We showed that such strains are disseminated from the winery and their permanence in nature induced genetic changes, not found among a control group of isolates that derived from clonal expansion of the commercial “mother” strain. The objective of the present study was to evaluate by comparative genome hybridization on array (aCGH) the genome variations among four isolates of the commercial strain S. cerevisiae Zymaflore VL1, re-isolated from vineyards surrounding the wineries where this strain was applied, in comparison to the commercial “mother” strain. Data analysis showed genetic differences among the recovered isolates in comparison with the “mother” strain. Amplification (1 to 2 fold changes) of 14 genes were detected, related with mitosis (SHE1), meiosis (HFM1), lysine biosynthesis (LYS14), galactose (GAL1) and asparagine catabolism (ASP3-2). ASP3-2 amplification is in agreement with the previously shown increased expression during nitrogen starvation. This might occur as adaptation to natural environments with poor yeast-utilizable nitrogen sources. Eight Ty elements were also amplified, whereas each of the recovered strains had a unique amplification pattern. SNP analysis of the four isolates strengthened the hypothesis of microevolutionary changes, as complement to the aCGH approaches. A phenotypic screening was performed considering 28 physiological tests. Seven phenotypic traits distinguished the recovered strains from the “mother” strain which was unable to grow at 18ºC, but evidenced some growth in the presence of CuSO4(5mM) and SDS 0.01%(v/v). Variable growth patterns were found for NaCl(1.5M), KHSO3(300mg/L) and wine supplemented with glucose (0.5% and 1%w/v). Sequencing and genome comparison of the five isolates is currently underway. We hypothesize that the transition from nutrient-rich musts to nutritionally scarce natural environments induces adaptive responses and microevolutionary changes promoted by Ty elements. These changes (and possibly others as well) may contribute to intra-strain phenotypic variability.Fundação para a Ciência e a Tecnologia (FCT

Circulating adrenomedullin estimates survival and reversibility of organ failure in sepsis: the prospective observational multinational Adrenomedullin and Outcome in Sepsis and Septic Shock-1 (AdrenOSS-1) study

Background: Adrenomedullin (ADM) regulates vascular tone and endothelial permeability during sepsis. Levels of circulating biologically active ADM (bio-ADM) show an inverse relationship with blood pressure and a direct relationship with vasopressor requirement. In the present prospective observational multinational Adrenomedullin and Outcome in Sepsis and Septic Shock 1 (, AdrenOSS-1) study, we assessed relationships between circulating bio-ADM during the initial intensive care unit (ICU) stay and short-term outcome in order to eventually design a biomarker-guided randomized controlled trial. Methods: AdrenOSS-1 was a prospective observational multinational study. The primary outcome was 28-day mortality. Secondary outcomes included organ failure as defined by Sequential Organ Failure Assessment (SOFA) score, organ support with focus on vasopressor/inotropic use, and need for renal replacement therapy. AdrenOSS-1 included 583 patients admitted to the ICU with sepsis or septic shock. Results: Circulating bio-ADM levels were measured upon admission and at day 2. Median bio-ADM concentration upon admission was 80.5 pg/ml [IQR 41.5-148.1 pg/ml]. Initial SOFA score was 7 [IQR 5-10], and 28-day mortality was 22%. We found marked associations between bio-ADM upon admission and 28-day mortality (unadjusted standardized HR 2.3 [CI 1.9-2.9]; adjusted HR 1.6 [CI 1.1-2.5]) and between bio-ADM levels and SOFA score (p < 0.0001). Need of vasopressor/inotrope, renal replacement therapy, and positive fluid balance were more prevalent in patients with a bio-ADM > 70 pg/ml upon admission than in those with bio-ADM ≤ 70 pg/ml. In patients with bio-ADM > 70 pg/ml upon admission, decrease in bio-ADM below 70 pg/ml at day 2 was associated with recovery of organ function at day 7 and better 28-day outcome (9.5% mortality). By contrast, persistently elevated bio-ADM at day 2 was associated with prolonged organ dysfunction and high 28-day mortality (38.1% mortality, HR 4.9, 95% CI 2.5-9.8). Conclusions: AdrenOSS-1 shows that early levels and rapid changes in bio-ADM estimate short-term outcome in sepsis and septic shock. These data are the backbone of the design of the biomarker-guided AdrenOSS-2 trial. Trial registration: ClinicalTrials.gov, NCT02393781. Registered on March 19, 2015

A collection of indigenous saccharomyces cerevisiae strains from appellations of origin in Portugal and France

Apresentação efectuada nas "Jornadas Portuguesas de Genética, 35, Braga, 2010"The model organism Saccharomyces cerevisiae stands today at the forefront of molecular biology and functional analysis in genetics and genomics. However, understanding of the ecological, evolutionary and population genetic features that shaped the biology of this species is underscored by a wealth of knowledge on molecular and cellular biology obtained from a very limited number of laboratory strains. In this reasoning, we constituted one of the largest bio-databanks of S. cerevisiae that were obtained from winemaking environments in Portugal and France. During the harvest time of 2001 to 2009, 604 grape samples were collected in appellations of origin in Portugal (Vinho Verde, Dão, Douro, Bairrada, Estremadura, Palmela, Ribatejo, Açores) and France (Languedoc). The grape samples belonged to the varieties Alvarinho, Aragonez, Arinto, Avesso, Baga, Bical, Castelão, Carignan, Loureiro, Maria Gomes, Terrantez, Touriga Nacional and Verdelho. Yeast populations, in particular S. cerevisiae, were isolated after spontaneous fermentation of the extracted grape juice. From the final stage of 258 fermentations, 7740 yeast isolates were obtained, belonging mainly (5496 isolates) to the species S. cerevisiae. An initial genetic screen, based on mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP), electrophoretic karyotyping or interdelta sequence analysis, was followed by microsatellite analysis of strains with unique genetic profiles. Isolates were assigned to 752 different strains, based on their microsatellite allelic distribution. The resulting web-based autochthonous strain collection is one of the largest S. cerevisiae bio-databanks and is a resource for sustainable biodiversity preservation, equitable sharing of genetic data and winemaking strain selection.Fundação para a Ciência e Tecnologia (FCT)Direcção Regional da Ciência e Tecnologia (DRCT

Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits

The species Saccharomyces cerevisiae includes natural strains, clinical isolates, and a large number of strains used in human activities. The aim of this work was to investigate how the adaptation to a broad range of ecological niches may have selectively shaped the yeast metabolic network to generate specific phenotypes. Using 72 S. cerevisiae strains collected from various sources, we provide, for the first time, a population-scale picture of the fermentative metabolic traits found in the S. cerevisiae species under wine making conditions. Considerable phenotypic variation was found suggesting that this yeast employs diverse metabolic strategies to face environmental constraints. Several groups of strains can be distinguished from the entire population on the basis of specific traits. Strains accustomed to growing in the presence of high sugar concentrations, such as wine yeasts and strains obtained from fruits, were able to achieve fermentation, whereas natural yeasts isolated from “poor-sugar” environments, such as oak trees or plants, were not. Commercial wine yeasts clearly appeared as a subset of vineyard isolates, and were mainly differentiated by their fermentative performances as well as their low acetate production. Overall, the emergence of the origin-dependent properties of the strains provides evidence for a phenotypic evolution driven by environmental constraints and/or human selection within S. cerevisiae

Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations