Mario Polsinelli

On the origins of wine yeast

There is still a lack of agreement concerning the relative contribution of wine yeast that may originate in the vineyard compared to that which may originate in the cellar. Part of this controversy is due to the extreme difficulty of finding Saccharomyces cerevisiae on the grapes. We estimate that only about one in one-thousand grape berries carries wine yeast. However, we have found that grape berries that are damaged (i.e. the skin is broken) are very rich depositories of microorganisms including S. cerevisiae, and that one in four such berries is S. cerevisiae-positive. These positive berries have between 100,000 and 1,000,000 wine yeast cells on them, and there is evidence that these yeasts are clonal. We believe that the yeasts are brought to the berries by insects such as bees, wasps, and Drosophila and that they multiply in the rich medium of the grape interior. Even though there are many cells of S. cerevisiae on the damaged berries, they are in a definite minority. All the other organisms that are found in wine fermentations are also present on these berries, and their total numbers are in the range of 10 million to 100 million cells per berry.

Role of social wasps in Saccharomyces cerevisiae ecology and evolution

Saccharomyces cerevisiae is one of the most important model organisms and has been a valuable asset to human civilization. However, despite its extensive use in the last 9,000 y, the existence of a seasonal cycle outside human-made environments has not yet been described. We demonstrate the role of social wasps as vector and natural reservoir of S. cerevisiae during all seasons. We provide experimental evidence that queens of social wasps overwintering as adults (Vespa crabro and Polistes spp.) can harbor yeast cells from autumn to spring and transmit them to their progeny. This result is mirrored by field surveys of the genetic variability of natural strains of yeast. Microsatellites and sequences of a selected set of loci able to recapitulate the yeast strain’s evolutionary history were used to compare 17 environmental wasp isolates with a collection of strains from grapes from the same region and more than 230 strains representing worldwide yeast variation. The wasp isolates fall into subclusters representing the overall ecological and industrial yeast diversity of their geographic origin. Our findings indicate that wasps are a key environmental niche for the evolution of natural S. cerevisiae populations, the dispersion of yeast cells in the environment, and the maintenance of their diversity. The close relatedness of several wasp isolates with grape and wine isolates reflects the crucial role of human activities on yeast population structure, through clonal expansion and selection of specific strains during the biotransformation of fermented foods, followed by dispersal mediated by insects and other animals.

Evidence for S. cerevisiae Fermentation in Ancient Wine

Saccharomyces cerevisiae is the principal yeast used in modern fermentation processes, including winemaking, breadmaking, and brewing. From residue present inside one of the earliest known wine jars from Egypt, we have extracted, amplified, and sequenced ribosomal DNA from S.cerevisiae. These results indicate that this organism was probably responsible for wine fermentation by at least 3150 B.C. This inference has major implications for the evolution of bread and beer yeasts, since it suggests that S. cerevisiae yeast, which occurs naturally on the surface bloom of grapes, was also used as an inoculum to ferment cereal products.

A new phage of Bacillus subtilis with infectious DNA having separable strands

A new phage (SPP1) active on Bacillus subtilis has been isolated. The DNA extracted from the phage is infectious on competent cells of B. subtilis and shows the highest efficiency of infection so far observed for B. subtilis phage DNA, namely 5 to 6 × 10 3 phage equivalents per plaque-forming unit. The phage genome can be extracted as a single DNA molecule having a molecular weight of 2·5 × 10 7 . The molecular weight of phage DNA after denaturation is approximately 1·3 × 10 7 , thus demonstrating the absence of single-strand breaks. The buoyant density of SPP1 DNA in a neutral CsCl density-gradient is 1·703 g cm −3 ; denatured DNA has a bimodal density distribution at neutral pH, the two bands corresponding to the separated strands; their densities are 1·713 g cm −3 and 1·725 g cm −3 , respectively. The interaction of ribosomal RNA with the heavy strand strongly enhances its density and allows a large-scale preparation of the two separated strands. The isolated strands can be reannealed, yielding a good recovery of infectious activity.

Quest for wine yeasts—An old story revisited

Numerous studies have described the yeast biota of grapes, and grape must in order to understand better the succession of yeasts during fermentation of wine. The origin of the wine yeasts has been rather controversial. By using more elaborate isolation methods, classical genetic analysis and electrophoretic karyotyping of monosporic clones, with this study, credible proof now exists that the vineyard is the primary source for the wine yeasts and that strains found on the grapes can be followed through the fermentation process.

Isolation and Characterization of Glutamate Synthase Mutants of Azospirillum brasilense

Six mutants of Azospirillum brasilense Sp6 unable to fix nitrogen have been isolated and characterized. Analysis of the enzymes involved in nitrogen metabolism has shown that the mutants are deficient in glutamate synthase activity (asm). They also have a low activity of glutamine synthetase and no or very low nitrogenase activity (assayed by acetylene reduction). In addition, the mutants were unable to grow on various sources of combined nitrogen such as nitrate, nitrite, alanine, histidine, adenine and xanthine.

Social wasps are a Saccharomyces mating nest

Significance Despite the widespread interest on Saccharomyces cerevisiae, its wild lifestyle is far from being completely understood, with one of the most resounding examples being its sexual attitude. We show that the intestine of social wasps favors the mating of Saccharomyces strains by providing a succession of environmental conditions prompting sporulation and germination. We also demonstrate that the insect intestine favors hybridization of S. cerevisiae and Saccharomyces paradoxus. Although S. paradoxus survives in wild environments and rarely mates with S. cerevisiae, we discover that two European S. paradoxus strains cannot survive the wasps intestinal environment but can be rescued through interspecific hybridization with S. cerevisiae. These findings are introducing insects as environmental alcoves in which yeast cells can meet and mate. The reproductive ecology of Saccharomyces cerevisiae is still largely unknown. Recent evidence of interspecific hybridization, high levels of strain heterozygosity, and prion transmission suggest that outbreeding occurs frequently in yeasts. Nevertheless, the place where yeasts mate and recombine in the wild has not been identified. We found that the intestine of social wasps hosts highly outbred S. cerevisiae strains as well as a rare S. cerevisiae×S. paradoxus hybrid. We show that the intestine of Polistes dominula social wasps favors the mating of S. cerevisiae strains among themselves and with S. paradoxus cells by providing a succession of environmental conditions prompting cell sporulation and spores germination. In addition, we prove that heterospecific mating is the only option for European S. paradoxus strains to survive in the gut. Taken together, these findings unveil the best hidden secret of yeast ecology, introducing the insect gut as an environmental alcove in which crosses occur, maintaining and generating the diversity of the ascomycetes.

Trifluoroleucine resistance and regulation of alpha-isopropyl malate synthase in Saccharomyces cerevisiae.

Abstract Seven spontaneous Saccharomyces cerevisiae mutants that express dominant resistance to 5,5,5-trifluoro-DL-leucine have been characterised at the molecular level. The gene responsible for the resistance was cloned from one of the mutants (FSC2.4). Determination of its nucleotide sequence showed that it was an allele of LEU4 (LEU4-1), the gene that encodes α-isopropyl malate synthase I (α-IPM synthase I), and that the mutation involved a codon deletion localised close to the 3′ end of the LEU4 ORF. Six different point mutations – four transitions and two transversions – were found in the remaining mutants. α-IPM synthase activity was found to be insensitive to feedback inhibition by leucine in five of the strains. In the other two the enzyme was resistant to Zn2+-mediated inactivation by Coenzyme A, a previously postulated control mechanism in energy metabolism; as far as we know, this represents the first direct in vivo evidence for this mechanism. The seven mutations define a region, the R-region, involved in both leucine feedback inhibition and in Zn2+-mediated inactivation by CoA. Deletion experiments involving the R-region showed that it is also necessary for enzyme activity.

Multiple strains of Saccharomyces cerevisiae on a single grape vine

M. POLSINELLI, P. ROMANO, G. SUZZI AND R. MORTIMER. 1996. On the basis of the levels of secondary product formation four different phenotypes were represented among the 28 strains of Saccharomyces cerevisiae isolated during the spontaneous fermentation of grape juice. The genetic analysis indicated that four different strains, representing each phenotypic class, were derived, one from the other, by mutation. The spontaneous fermentation of a Malvasia must was dominated by different strains of Saccharomyces cerevisiae at different stages of fermentation.

DNA fingerprinting by random amplified polymorphic DNA and restriction fragment length polymorphism is useful for yeast typing

Random amplified polymorphic DNA (RAPD) analysis was applied to genomic DNA from nineteen yeast strains belonging to the genera Saccharomyces and Zygosaccharomyces. Results obtained with five primers indicated that this technique is a powerful tool for yeast differentiation and identification. The data were consistent with those derived from restriction fragment length polymorphism (RFLP) using two S. cerevisiae DNA probes. We conclude that RAPD fingerprinting, combined with the analysis of RFLP, can provide unambiguous type assignment in yeasts.