Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus

Genetic bases of nitrogen requirement in wine yeast assessed trhrough QTL analysis

In grape must, nitrogen content is ofteninsufficient for the completion of alcoholic fermentation by yeast. For Saccharomyces cerevisiae, response to nitrogen deficiency is strain-dependent, some strains being able to complete fermentation despite nitrogen deficiency whereas others are not and result in sluggish or stuck fermentation. Thus, it is of high interest to study the mechanisms behind those different responses and exploit them to improve yeast strain for wine fermentation when nitrogen content is low. Previous study highlighted different genomic regions involved in nitrogen requirement through BSA (Bulk Segregant Analysis), and the contributions of three genes: MDS3, GCN1, and ARG81 have been shown (1). However, many other large genomic regions were also defined for which we could not find evident candidate genes. In addition, BSA did not provide any information on possible interactions between loci. In order to explore further the genetic bases of nitrogen requirement, we applied a QTL analysis to the fermentation rate in nitrogen deficient medium, on a population of 131 individually genotyped segregants obtained from the same cross as (1). The dense genetic map available for the segregant population (3727 markers) enabled us to perform single and multiple map QTL and thus define genomic regions which could be implied in low nitrogen requirement. In order to further validate the impact of candidate genes on the phenotype, alleles were “swapped” by CRISPR-Cas9 technique and phenotype was evaluated in comparison with haploid parent strains.Several regions with high LOD scores were identified, some above the significance threshold, and others below, among which the regions containing the genes identified by (1), probably in relation with the multigenic character of the trait. In the region with the highest LOD score, two candidate genes in relation with nitrogen metabolism (namely, Target of Rapamycin (TOR) pathwayand lifespan regulation) were identified. In addition, in order to reveal possible interaction between genes, strains carrying different combinations of GCN1 and MDS3 parental alleles (implied in TOR pathway) have been evaluated. These constructions confirm their role on the fermentation rate in low-nitrogen conditions and indicate dependence on the genetic background. These results confirm the complexity of mechanisms involved in nitrogen requirement during alcoholic fermentation and will permit to optimise wine yeast strain selection in response to winemaking industry demands

How different anthropogenic environments have shaped the genome of S. cerevisiae ?

The yeast Saccharomyces cerevisiae plays an important role in food and beverage fermentations. In order to known how environmental constraints imposed by anthropogenic niches have shaped S. cerevisiae genomes and phenotypes, we sequenced the genome of 82 S. cerevisiae strains from various ecological origins. Using these genomic data, we found additional genetic elements acquired by introgression or by horizontal transfer. Here, we present two remarkable examples of divergent adaptation associated to yeast domestication for wine and milk fermentation. Firstly, we demonstrated the role of oligopeptide transporters encoded by FOT genes, which are recently acquired by wine yeasts from Torulaspora microellipsoides. These transporters with a broader specificity than S. cerevisiae dipeptides transporters, confer a strong competitive advantage during grape must fermentation and thus play a key role in the adaptation of wine yeasts to the nitrogen-limited wine fermentation environment. The genome of cheese strains, secondly, present some particular features. Genes of the GAL locus were replaced by their orthologues from a species apparently basal to the Saccharomyces clade. Allelic exchange of this locus in a wine strain enables improves growth speed in a media containing the two hexoses such as when released from the hydrolysis of lactose. In addition, a highly divergent high affinity transporter GAL2 and a specific allele of the regulator GAL80 were found. This work highlights the remarkable plasticity of yeast genomes as a mechanism of their adaptation to their environment

Evolutionary Advantage Conferred by an Eukaryote-to-Eukaryote Gene Transfer Event in Wine Yeasts

Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding oligopeptide transporters. We show that FOT genes confer a strong competitive advantage during grape must fermentation by increasing the number and diversity of oligopeptides that yeast can utilize as a source of nitrogen, thereby improving biomass formation, fermentation efficiency, and cell viability. Thus, the acquisition of FOT genes has favored yeast adaptation to the nitrogen-limited wine fermentation environment. This finding indicates that anthropic environments offer substantial ecological opportunity for evolutionary diversification through gene exchange between distant yeast species

SSU1 Checkup, a Rapid Tool for Detecting Chromosomal Rearrangements Related to the SSU1 Promoter in Saccharomyces cerevisiae: An Ecological and Technological Study on Wine Yeast

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological Saccharomyces cerevisiae strains, CR involving the promoter region of the gene SSU1 lead to a higher sulfite tolerance by enhancing the SO2 efflux. To date, three different SSU1 associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method (SSU1 checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 S. cerevisiae strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the SSU1 promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged SSU1 promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of SSU1 promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO2 concentrations. The SSU1 checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers.Fil: Marullo, Philippe. Universite de Bordeaux; FranciaFil: Claisse, Olivier. Universite de Bordeaux; FranciaFil: Raymond Eder, María Laura. Universidad Católica de Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J.; ArgentinaFil: Börlin, Marine. Universite de Bordeaux; FranciaFil: Feghali, Nadine. Lebanese University; LíbanoFil: Bernard, Margaux. Universite de Bordeaux; FranciaFil: Legras, Jean Luc. Université Montpellier II; FranciaFil: Albertin, Warren. Universite de Bordeaux; FranciaFil: Rosa, Alberto Luis. Universidad Católica de Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J.; ArgentinaFil: Masneuf Pomarede, Isabelle. Universite de Bordeaux; Franci

A influência do teatro no turismo em Curitiba

Orientador : Sandro Campos NevesMonografia (graduação) - Universidade Federal do Paraná, Setor de Ciências Humanas, Curso de Graduação em TurismoInclui referênciasResumo : O presente trabalho analisou o atual cenário de teatro em Curitiba e a influência exercida pelo Festival de Teatro de Curitiba no turismo da cidade. Para cumprir com o objetivo central, realizou-se pesquisa exploratória e bibliográfica, que fundamentaram o referencial teórico, dividido nos seguintes tópicos: turismo e cultura; eventos e turismo; teatro e festivais; festival de teatro de Curitiba; Espaço Positivo. Em seguida, foi aplicada a técnica de estudo de caso para o público presente na 27° edição do Festival, sendo a coleta de dados realizada por meio de questionários previamente estruturados, além de um roteiro de entrevista aplicado a gestora do Teatro Positivo, para compreender melhor a oferta da cidade. A análise dos dados coletados foi realizada por análise de conteúdo, onde as informações foram apresentadas através de gráficos e quadros, que auxiliaram na interpretação dos dados. As informações obtidas possibilitaram uma melhor interpretação da cena teatral de Curitiba, com foco no Festival de Teatro de Curitiba e no Teatro Positivo, objetos de estudo desse trabalho. Foi possível identificar que o teatro como influência para atrair a visita de turistas não é satisfatória, sendo a maior parte do público presente de Curitiba e região. Em resposta, propôs-se um projeto que utiliza uma estratégia de marketing online, através de mídias sociais, que consiste na elaboração de postagens em plataformas acessadas pelo público alvo do evento, com o objetivo de melhorar a divulgação, trazendo um número maior de visitantes.Abstract : The present paper analyzed the current theater scene in Curitiba and the influence exerted by the Festival de Teatro de Curitiba in the city tourism. In order to fulfill the central objective, an exploratory and bibliographical research was carried out, which based the theoretical reference, divided in the following topics: tourism and culture; events and tourism; theater and festivals; Festival de Teatro de Curitiba; Espaço Positivo. Then, the case study technique was applied to the audience present at the 27th Festival de Teatro de Curitiba, and the data collection was carried out through previously structured questionnaires, as well as an interview script applied to the Teatro Positivo manager, to understand better the offer of the city. The analysis of the collected data was performed by content analysis, where the information was presented through graphs and tables, which helped in the interpretation of the data. The information obtained allowed a better interpretation of the theatrical scene of Curitiba, focusing on the Festival de Teatro de Curitiba and Teatro Positivo, objects of study of this paper. It was possible to identify that the theater as an influence to attract the visit of tourists is not satisfactory, being the majority of the present public of Curitiba and region. In response, a project was proposed that uses an online marketing strategy, through social media, which consists in the elaboration of postings on platforms accessed by the target audience of the event, with the aim of improving the publicity, bringing a greater number of visitors

Front Microbiol

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological strains, CR involving the promoter region of the gene lead to a higher sulfite tolerance by enhancing the SO efflux. To date, three different associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method ( checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO concentrations. The checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers

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