Eladio Barrio

A Comparative Study of Different Methods of Yeast Strain Characterization

Summary An extensive survey of different methods of yeast strain identification (classical microbiological tests, whole-cell protein electrophoresis, chromosomal patterns, DNA hybridization and mitochondrial DNA restriction analysis) has been carried out in order to differentiate, with industrial purposes, strains present in the Alicante wine ecosystem. Only chromosomal patterns and mitochondrial DNA (mtDNA) restriction analysis show differences between strains. Both techniques are very complex to be used in bio technological industries. For this reason, we have developed a new, simple, unexpensive and rapid method based on mtDNA restriction analysis.

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region

Abstract In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.

Endosymbiotic bacteria: groEL buffers against deleterious mutations.

GroEL, a heat-shock protein that acts as a molecular chaperone, is overproduced in endosymbiotic but not in free-living bacteria, presumably to assist in the folding of conformationally damaged proteins. Here we show that the overproduction of GroEL in Escherichia coli masks the effects of harmful mutations that have accumulated during a simulated process of vertical transmission. This molecular mechanism, which may be an adaptation to the bacteriums intracellular lifestyle, is able to rescue lineages from a progressive fitness decline resulting from the fixation of deleterious mutations under strong genetic drift.

Fermentative stress adaptation of hybrids within the Saccharomyces sensu stricto complex

Along the fermentation process yeasts are affected by a succession of stress conditions that affect their viability and fermentation efficiency. Among the stress conditions the most relevant are high sugar concentration and low pH in musts, temperature and, as fermentation progresses, ethanol accumulation. Nowadays, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing search for particular wine yeast strains possessing a wide range of optimized, improved or novel enological characteristics. Traditionally, the species S. cerevisiae and S. bayanus within the Saccharomyces sensu stricto species are considered some of the most important yeast species involved in fermentation processes. However, in the last years, hybrid strains between the species S. cerevisiae, S. bayanus and S. kudriavzevii have been described as yeasts conducting the alcoholic fermentations and some of them are commercially available. Our results indicate that yeasts in the Saccharomyces sensu stricto complex were not affected by low pH or high glucose content in the media; however temperature and ethanol concentration variables appreciably affected their growth. The strains pertaining to S. cerevisiae were able to tolerate high temperature stress, whereas strains within S. bayanus and S. kudriavzevii were better adapted to growth at lower temperatures. Regarding to alcohol tolerance, S. cerevisiae is tolerating alcohol better than S. bayanus or S. kudriavzevii. Surprisingly, the natural hybrids between these species have adapted to growth under ethanol and temperature stress by inheriting competitive traits from one or another parental species. These results open new perspectives in the construction of new hybrid strains with biotechnological interest, as the characteristics of the parents may result in interesting combinations in the hybrids.

RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces : a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus × S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.

Effects of temperature, pH and sugar concentration on the growth parameters of Saccharomyces cerevisiae, S. kudriavzevii and their interspecific hybrid

The effects of temperature, pH and sugar concentration (50% glucose+50% fructose) on the growth parameters of Saccharomyces cerevisiae T73, S. kudriavzevii IFO 1802(T) and the hybrid strain S. cerevisiae x S. kudriavzevii W27 were studied by means of response surface methodology based in a central composite circumscribed design. Lag phase could not be properly modelled in the wine model system, where yeasts started the fermentation in few hours after inoculation. In the case of the maximum specific growth rate (micro(max)), the temperature was the most important variable for three yeasts, although the effects of sugar concentration (in T73 and W27) and pH (W27 and 1802) were also significant (p<0.05). The only retained interaction was between the variables temperature and pH for yeast 1802. The polynomial equations built for micro(max) were used both to assess the behaviour of yeasts as a function of the factors and to predict their growth. In the case of temperature, the profiles obtained by the equations showed that response of the hybrid W27 was similar to T73 and different to 1802. When pH was the factor under study, the response of the hybrid W27 was closer to 1802 than yeast T73. For sugar concentration, the response of the hybrid W27 was similar to T73 but different to 1802. To the best of our knowledge, this is the first time that predictive models are used to assess and compare the response of a hybrid strain with respect to its parental species. The information obtained could also be useful to estimate the possible effect of climatic change on yeast growth.

Temperature Adaptation Markedly Determines Evolution within the Genus Saccharomyces

ABSTRACT The present study uses a mathematical-empirical approach to estimate the cardinal growth temperature parameters (T min, the temperature below which growth is no longer observed; T opt, the temperature at which the μmax equals its optimal value; μopt, the optimal value of μmax; and T max, the temperature above which no growth occurs) of 27 yeast strains belonging to different Saccharomyces and non-Saccharomyces species. S. cerevisiae was the yeast best adapted to grow at high temperatures within the Saccharomyces genus, with the highest optimum (32.3°C) and maximum (45.4°C) growth temperatures. On the other hand, S. kudriavzevii and S. bayanus var. uvarum showed the lowest optimum (23.6 and 26.2°C) and maximum (36.8 and 38.4°C) growth temperatures, respectively, confirming that both species are more psychrophilic than S. cerevisiae. The remaining Saccharomyces species (S. paradoxus, S. mikatae, S. arboricolus, and S. cariocanus) showed intermediate responses. With respect to the minimum temperature which supported growth, this parameter ranged from 1.3 (S. cariocanus) to 4.3°C (S. kudriavzevii). We also tested whether these physiological traits were correlated with the phylogeny, which was accomplished by means of a statistical orthogram method. The analysis suggested that the most important shift in the adaptation to grow at higher temperatures occurred in the Saccharomyces genus after the divergence of the S. arboricolus, S. mikatae, S. cariocanus, S. paradoxus, and S. cerevisiae lineages from the S. kudriavzevii and S. bayanus var. uvarum lineages. Finally, our mathematical models suggest that temperature may also play an important role in the imposition of S. cerevisiae versus non-Saccharomyces species during wine fermentation.

Molecular Characterization of New Natural Hybrids of Saccharomyces cerevisiae and S. kudriavzevii in Brewing

ABSTRACT We analyzed 24 beer strains from different origins by using PCR-restriction fragment length polymorphism analysis of different gene regions, and six new Saccharomyces cerevisiae × Saccharomyces kudriavzevii hybrid strains were found. This is the first time that the presence in brewing of this new type of hybrid has been demonstrated. From the comparative molecular analysis of these natural hybrids with respect to those described in wines, it can be concluded that these originated from at least two hybridization events and that some brewing hybrids share a common origin with wine hybrids. Finally, a reduction of the S. kudriavzevii fraction of the hybrid genomes was observed, but this reduction was found to vary among hybrids regardless of the source of isolation. The fact that 25% of the strains analyzed were discovered to be S. cerevisiae × S. kudriavzevii hybrids suggests that an important fraction of brewing strains classified as S. cerevisiae may correspond to hybrids, contributing to the complexity of Saccharomyces diversity in brewing environments. The present study raises new questions about the prevalence of these new hybrids in brewing as well as their contribution to the properties of the final product.

Population dynamics of natural Saccharomyces strains during wine fermentation

Using mitochondrial DNA restriction endonuclease analysis, the dynamics of the natural Saccharomyces cerevisiae strains present in spontaneous wine fermentations have been studied. We observed a sequential substitution of Sacch. cerevisiae strains along fermentation agreeing with different fermentation phases. When the restriction patterns similarity (measured as the fraction of shared restriction fragments) was high, a clear sequential substitution of the strains was seen. However, when the similarity was low, although a sequential substitution could be also observed between secondary strains, a clearly predominant strain was present along the whole fermentation process.

Mitotic Recombination and Genetic Changes in Saccharomyces cerevisiae during Wine Fermentation

ABSTRACT Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3homozygotes were detected at a rate of 1 × 10−5 to 3 × 10−5 per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.