Population Size, Sex and Purifying Selection: Comparative Genomics of Two Sister Taxa of the Wild Yeast Saccharomyces paradoxus.

This study uses population genomic data to estimate demographic and selection parameters in two sister lineages of the wild yeast Saccharomyces paradoxus and compare their evolution. We first estimate nucleotide and recombinational diversities in each of the two lineages to infer their population size and frequency of sex and then analyze the rate of mutation accumulation since divergence from their inferred common ancestor to estimate the generation time and efficacy of selection. We find that one of the lineages has significantly higher silent nucleotide diversity and lower linkage disequilibrium, indicating a larger population with more frequent sexual generations. The same lineage also shows shorter generation time and higher efficacy of purifying selection, the latter consistent with the finding of larger population size and more frequent sex. Similar analyses are also performed on the ancestries of individual strains within lineages and we find significant differences between strains implying variation in rates of mitotic cell divisions. Our sample includes some strains originating in the Chernobyl nuclear-accident exclusion zone, which has been subjected to high levels of radiation for nearly 30 years now. We find no evidence, however, for increased rates of mutation. Finally, there is a positive correlation between rates of mutation accumulation and length of growing period, as measured by latitude of the place of origin of strains. Our study illustrates the power of genomic analyses in estimating population and life history parameters and testing predictions based on population genetic theory.

Induced expression of the alcohol acetyltransferase gene ATF1 in industrial yeast Saccharomyces pastorianus TUM 34/70.

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.

Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

Exploring the potential of Saccharomyces eubayanus as a parent for new interspecies hybrid strains in winemaking.

Yeast cryotolerance brings some advantages for wine fermentations, including the improved aromatic complexity of white wines. Naturally cold-tolerant strains are generally less adept at wine fermentation but fermentative fitness can potentially be improved through hybridization. Here we studied the potential of using hybrids involving Saccharomyces eubayanus and a S. cerevisiae wine strain for low-temperature winemaking. Through screening the performance in response to variable concentrations of sugar, nitrogen and temperature, we isolated one hybrid strain that exhibited the superior performance. This hybrid strain was propagated and dried in pilot scale and tested for the fermentation of Macabeu and Sauvignon blanc grape musts. We obtained highly viable active dry yeast, which was able to efficiently ferment the grape musts with superior production of aroma active volatiles, in particular, 2-phenylethanol. The genome sequences of the hybrid strains revealed variable chromosome inheritance among hybrids, particularly within the S. cerevisiae subgenome. With the present paper, we expand the knowledge on the potentialities of using S. eubayanus hybrids in industrial fermentation at beverages other than lager beer.

Induced gene expression in industrial Saccharomyces pastorianus var. carlsbergensis TUM 34/70: evaluation of temperature and ethanol inducible native promoters.

Induced gene expression is an important trait in yeast metabolic engineering, but current regulations prevent the use of conventional expression systems, such as galactose and copper, in food and beverage fermentations. This article examines the suitability of temperature-inducible native promoters for use in the industrial yeast strain Saccharomyces pastorianus var. carlsbergensis TUM 34/70 under brewing conditions. Ten different promoters were cloned and characterized under varying temperature shifts and ethanol concentrations using a green fluorescent protein reporter. The activities of these promoters varied depending upon the stress conditions applied. A temperature shift to 4°C led to the highest fold changes of PSSA3, PUBI4 and PHSP104 by 5.4, 4.5 and 5.0, respectively. Ethanol shock at 24°C showed marked, concentration-dependent induction of the promoters. Here, PHSP104 showed its highest induction at ethanol concentrations between 4% (v/v) and 6% (v/v). The highest fold changes of PSSA3 and PUBI4 were found at 10% (v/v) ethanol. In comparison, the ethanol shock at a typical fermentation temperature (12°C) leads to lower induction patterns of these promoters. Taken together, the data show that three promoters (PHSP104, PUBI4 and PSSA3) have high potential for targeted gene expression in self-cloning brewing yeast using temperature shifts.

S. cerevisiae × S. eubayanus interspecific hybrid, the best of both worlds and beyond.

Saccharomyces pastorianus lager-brewing yeasts have descended from natural hybrids of S. cerevisiae and S. eubayanus. Their alloploidy has undoubtedly contributed to successful domestication and industrial exploitation. To understand the early events that have led to the predominance of S. pastorianus as lager-brewing yeast, an interspecific hybrid between S. cerevisiae and S. eubayanus was experimentally constructed. Alloploidy substantially improved the performance of the S. cerevisiae × S. eubayanus hybrid as compared to either parent regarding two cardinal features of brewing yeasts: tolerance to low temperature and oligosaccharide utilization. The hybrid's S. eubayanus subgenome conferred better growth rates and biomass yields at low temperature, both on glucose and on maltose. Conversely, the ability of the hybrid to consume maltotriose, which was absent in the S. eubayanus CBS12357 type strain, was inherited from its S. cerevisiae parent. The S. cerevisiae × S. eubayanus hybrid even outperformed its parents, a phenomenon known as transgression, suggesting that fast growth at low temperature and oligosaccharide utilization may have been key selective advantages of the natural hybrids in brewing environments. To enable sequence comparisons of the parental and hybrid strains, the genome of S. eubayanus CBS12357 type strain (Patagonian isolate) was resequenced, resulting in an improved publicly available sequence assembly.

Three Agt1 transporters from brewer's yeasts exhibit different temperature dependencies for maltose transport over the range of brewery temperatures (0­20 °C).

Zero-trans rates of maltose transport by brewer's yeasts exert strong control over fermentation rates and are strongly temperature-dependent over the temperature range (20­0 °C) of brewery fermentations. Three α-glucoside transporters, ScAgt1(A60) (a Saccharomyces cerevisiae version of Agt1 from an ale strain), ScAgt1-A548V (a variant of ScAgt1(A60) with a single amino acid change in a transmembrane domain), and SbAgt1 (a Saccharomyces (eu)bayanus version from a lager strain), were compared. When expressed in the same laboratory yeast, grown at 24 °C and assayed at 0, 10, and 20 °C, SbAgt1 had the lowest absolute maltose uptake activity at 20 °C but smallest temperature dependence, ScAgt1-A548V had the highest activity but greatest temperature dependence, and ScAgt1(A60) had intermediate properties. ScAgt1(A60) exhibited higher absolute rates and smaller temperature dependencies when expressed in laboratory rather than brewer's strains. Absolute rates closely reflected the amounts of GFP-tagged ScAgt1(A60) transporter in each host's plasma membrane. Growth at 15 °C instead of 24 °C decreased the absolute activities of strains expressing ScAgt1(A60) by two- to threefold. Evidently, the kinetic characteristics of at least ScAgt1(A60) depended on the nature of the host plasma membrane. However, no consistent correlation was observed between transport activities and fatty acid or ergosterol compositions.

Breeding of lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance.

Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent.

Temperature adaptation markedly determines evolution within the genus Saccharomyces.

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 µ(max) equals its optimal value; µ(opt), the optimal value of µ(max); 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.

The temperature dependence of maltose transport in ale and lager strains of brewer's yeast.

Lager beers are traditionally made at lower temperatures (6-14 degrees C) than ales (15-25 degrees C). At low temperatures, lager strains (Saccharomyces pastorianus) ferment faster than ale strains (Saccharomyces cerevisiae). Two lager and two ale strains had similar maltose transport activities at 20 degrees C, but at 0 degrees C the lager strains had fivefold greater activity. AGT1, MTT1 and MALx1 are major maltose transporter genes. In nine tested lager strains, the AGT1 genes contained premature stop codons. None of five tested ale strains had this defect. All tested lager strains, but no ale strain, contained MTT1 genes. When functional AGT1 from an ale strain was expressed in a lager strain, the resultant maltose transport activity had the high temperature dependence characteristic of ale yeasts. Lager yeast MTT1 and MALx1 genes were expressed in a maltose-negative laboratory strain of S. cerevisiae. The resultant Mtt1 transport activity had low temperature dependence and the Malx1 activity had high temperature dependence. Faster fermentation at low temperature by lager strains than ale strains may result from their different maltose transporters. The loss of Agt1 transporters during the evolution of lager strains may have provided plasma membrane space for the Mtt1 transporters that perform better at a low temperature.

[The prognosis of the portion of irreversible radiation damages after simultaneous action of hyperthermia and ionizing radiation on yeast cells].

The results of experimental research of diploid yeasts cells survival after simultaneous action of hyperthermia and ionizing radiation (60Co) have been described. It was shown that the cell ability to liquid holding recovery decreased with an increase in the temperature, at which the exposure was carried out. due to the increase in the irreversible component determining the relative part of radiation damage which cells are incapable to recover. To predict theoretically the relative part of irreversible radiation damage after combined action, the mathematical model was suggested taking into account the synergistic interaction of agents. Good correlation between experimental results and model prediction was demonstrated. The importance of the results obtained for the interpretation of the mechanism of synergistic interaction of various factors is discussed.

[Study of dose-rate effects of neutron radiation in a wild-type and a repair-deficient yeasts saccharomyces].

We report here a comparative analysis of RBE for lethality of a single pulse (duration 65 micros) of fast neutron with ultra high dose rates (up to 6 x 10(6) Gy/s) and continuous neutron radiation (3.6 x 10(3) s) of the pulse reactor BARS-6. Three diploid strains, one haploid strain and three diploid repair-deficient strains (rad52-1/rad52-1; rad54/rad54; rad2/rad2) were used. The RBE values (D(0gamma)/1D(0n)) of a single pulse and continuous neutron irradiation were equal (1.7-1.8) with maximum RBE (4.1-3.1) in region of low doses (shoulder region). Haploid cells were found to be more (3 times) sensitive to both gamma-rays and neutrons than the wild type. There was no obvious decrease in the RBE of 1.9 in highly sensitive haploid cells as compared with highly resistant diploid cells. The repair-deficient strains (rad52-1/rad52-1; rad54/rad54) were more (up to 10 fold) sensitive to both neutrons and gamma-rays as compared with their parent line. The RBE values of 1.5-1.7 of neutrons for these mutants (independent by of the mode of irradiation) were found. The repair-deficient mutant rad2/rad2 had similar sensitivity as a wild type and a RBE value was 2.0. We have concluded that biological effectiveness of the neutrons of pulse reactor BARS-6 was independent of the dose-rate, differing up to 10(8) fold. The RBE didn't vary significantly with the capacity of cells to repair DNA damages.

Survival and recovery of yeast cells after combined treatment with ionizing radiation and heat.

Cell survival, recovery kinetics and inactivation forms after successive and simultaneous treatments with gamma rays (60Co) and high temperatures were studied in diploid yeast cells capable of recovery. Both the extent and the rate of the recovery were shown to be greatly decreased with increase in the duration of heat treatment (60 degrees C) followed by radiation and with increase in exposure temperature after simultaneous treatment with heat and radiation. A quantitative approach describing the recovery process was used to estimate the probability of recovery per unit time and the irreversible component of damage after the combined treatment with heat and radiation. It was shown that the probability of recovery was independent of the conditions of the treatment with heat and radiation, while the irreversible component gradually increased as a function of the duration of heat treatment (60 degrees C) after sequential treatment with heat and radiation and as a function of the exposure temperature after simultaneous treatment with heat and radiation. The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division. It is concluded on this basis that the synergistic interaction of ionizing radiation and hyperthermia in yeast cells is not related to the impairment of the recovery capacity itself and that it may be attributed to an increased yield of irreversible damage.

Critical evaluation of sampling techniques for residual glucose determination in carbon-limited chemostat culture of Saccharomyces cerevisiae.

In this paper, three sampling techniques for rapid quenching of cellular metabolism and subsequent separation of cells from fermentation broth are compared: (i) quick freezing of fermentation broth directly in liquid nitrogen; (ii) quenching metabolism by exposing the fermentation broth to stainless steel beads (4-mm diameter) in a filter syringe precooled to -18 degrees C; and (iii) withdrawal of the filtrate through a 0.45 microm filter attached to a syringe and a needle inserted directly into the fermentor. It was concluded that use of liquid nitrogen as a quenching method to rapidly arrest cellular metabolism, for quantitative analysis of extracellular glucose, is not a very reliable method and that the filter syringe steel beads work very well.

[Recovery of yeast cells after exposure to ionizing radiation and hyperthermia]. / Vosstanovlenie drozhzhevykh kletok posle vozdeistviia ioniziruiushchego izlucheniia i gipertermii.

Quantitative regularities of recovery of wild-type diploid yeast cells irradiated with gamma-rays (60Co) simultaneously with exposure to high temperatures were studied. It was shown that in conditions of such a combined action the constant of recovery did not depend on the temperature at which the irradiation was carried out. However, with an increase of acting temperature an augmentation in the portion of irreversible component was registered. The analysis of cell inactivation revealed that the augmentation of the irreversible component was accompanied by a continuous increase of cell killing without any postirradiation division after which cells are incapable of recovery. The reproductive death was mainly exerted after ionizing radiation applied alone while in conditions of simultaneous thermoradiation action the interphase killing (cell death without division) predominated. It is concluded on this base that the mechanism of synergistic interaction of ionizing radiation and hyperthermia may be related with cardinal change in mechanisms of cell killing.

Pulsed-field gel electrophoresis of chromosomal DNA of Saccharomyces pastorianus after exposure to x-rays (30-50 keV) and neutrons (14 MeV).

Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).

Adhesion of inactivated probiotic strains to intestinal mucus.

It has been suggested that probiotics should be viable in order to elicit beneficial health effects. Inactivation of probiotics has been suggested to interfere with the binding to the mucosa and thereby with the immune modulating activity of probiotics. The effect of different inactivation methods on the mucus adhesion of nine probiotic strains was studied. Inactivation by heat or gamma-irradiation generally decreased the adhesive abilities. However, heat treatment increased the adhesion of Propionibacterium freudenreichii and gamma-irradiation enhanced the adhesion of Lactobacillus casei Shirota. Inactivation by u.v. was not observed to modulate the adhesion of the tested strains and it was concluded to be the most appropriate method for studying non-viable probiotics and preparing control products.

Repair of cellular radiation damage in space under microgravity conditions.

The influence of microgravity on the repair of x-ray-induced DNA double-strand breaks was studied in the temperature-conditional repair mutant rad54-3 of diploid yeast Saccharomyces cerevisiae. Cells were exposed on the ground and kept at a low temperature until microgravity conditions were achieved. In orbit, they were incubated at the permissive temperature to allow repair. Before re-entry they were again cooled down and kept at a low temperature until final analysis. The experiment, which was flown on the shuttle Atlantis on flight STS-76 (SMM-03), showed that repair of pre-formed DNA double-strand breaks in yeast is not impaired by microgravity.

[Radiosensitivity of Saccharomyces yeasts and srm genes: effects of srm1 and srm5 mutations]. / Radiochuvstvitel'nost' drozhzhei sakharomitsetov i geny srm: éffekty mutatsii srm1 i srm5.

The presence in the cell genotype of srm1 and srm5 (cdc28-srm) mutations decreasing the spontaneous rho- mutability was shown to have no effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination. Mutation cdc28-srm exerts a marked effect on cell sensitivity to the lethal action of ionizing radiation and on the appearance of homoplasmic segregants generated from heteroplasmic diploids. Additive interactions between mutations cdc28-srm and each of the rad6 and rad52 mutations were revealed by an analysis of double mutants with respect to their sensitivity to radiation. Mutation rad9 was epistatic with mutation cdc28-srm. These data agree with the idea that the p34CDC28 gene product is a target for the RAD9-dependent feedback control operating at the cell cycle checkpoints (checkpoint control) and ensuring an additional amount of time for premitotic repair of chromosomal DNA damage.

Mutant PCNA alleles are associated with cdc phenotypes and sensitivity to DNA damage in fission yeast.

Twenty-eight site-directed mutations were introduced into the fission yeast gene (pcn1+) that encodes proliferating cell nuclear antigen (PCNA) and their in vivo effects analyzed in a strain with a null pcn1 background. Mutants defective in enhancing processivity of DNA polymerase delta have previously been identified. In this study, we assessed all of the mutants for their sensitivities to temperature, hydroxyurea, UV irradiation and methyl methanesulfonate (MMS), and specific mutants were also tested for sensitivity to gamma irradiation. One cold-sensitive allele, pcn1-3, was characterized in detail. This mutant had a recessive cold-sensitive cdc phenotype and showed sensitivity to hydroxyurea, UV, and gamma irradiation. At the non-permissive temperature pcn1-3 protein was able to form homotrimers in solution and showed increased stimulation of both synthetic activity and processivity of DNA polymerase delta relative to the wild-type Pcn1+ protein. Epistasis analyses indicated that pcn1-3 is defective in the repair pathway involving rad2+ but not defective in the classical nucleotide excision repair pathway involving rad13+. Furthermore, pcn1-3 is either synthetically or conditionally lethal in null checkpoint rad backgrounds and displays a mitotic catastrophe phenotype in these backgrounds. A model for how pcn1-3 defects may affect DNA repair and replication is presented.