Brigitte Cambon

Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations.

Effects of GPD1 Overexpression in Saccharomyces cerevisiae Commercial Wine Yeast Strains Lacking ALD6 Genes

ABSTRACT The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.

EXPRESSION OF SPECIFIC WHITE ADIPOSE TISSUE GENES IN DENERVATION-INDUCED SKELETAL MUSCLE FATTY DEGENERATION

Denervation of skeletal muscle results in rapid atrophy with loss of contractile mass and/or progressive degeneration of muscle fibers which are replaced to a greater or lesser degree by connective and fatty tissues. In this study, we show that denervated rabbit muscles are transformed into a white adipose tissue, depending on their fiber types. This tissue does express LPL, G3PDH and particularly the ob gene, a white adipose tissue‐specific marker, and does not express the brown adipose tissue molecular marker UCP1 mRNA.

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway

Glycerol 3‐phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1—the kinase involved in the response to osmotic stress—in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast‐derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1Δ mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild‐type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1Δ mutant was not as severely affected as the gpd1Δ mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1Δ mutant was able to increase glycerol production in response to high sugar concentration (15–28% glucose), to almost the same extent as the wild‐type, whereas this response was totally abolished in the gpd1Δ mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation. Copyright

Genetic characterization of commercial Saccharomyces cerevisiae isolates recovered from vineyard environments.

One hundred isolates of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 were recovered from spontaneous fermentations carried out with grapes collected from vineyards located close to wineries in the Vinho Verde wine region of Portugal. Isolates were differentiated based on their mitochondrial DNA restriction patterns and the evaluation of genetic polymorphisms was carried out by microsatellite analysis, interdelta sequence typing and pulsed‐field gel electrophoresis (PFGE). Genetic patterns were compared to those obtained for 30 isolates of the original commercialized Zymaflore VL1 strain. Among the 100 recovered isolates we found a high percentage of chromosomal size variations, most evident for the smaller chromosomes III and VI. Complete loss of heterozygosity was observed for two isolates that had also lost chromosomal heteromorphism; their growth and fermentative capacity in a synthetic must medium was also affected. A considerably higher number of variant patterns for interdelta sequence amplifications was obtained for grape‐derived strains compared to the original VL1 isolates. Our data show that the long‐term presence of strain VL1 in natural grapevine environments induced genetic changes that can be detected using different fingerprinting methods. The observed genetic changes may reflect adaptive mechanisms to changed environmental conditions that yeast cells encounter during their existence in nature. Copyright

Expression of c-erbAα, c-erbAβ and Rev-erbAα mRNA during the conversion of brown adipose tissue into white adipose tissue

The levels of mRNA encoding uncoupling protein (UCP), thyroid hormone receptors (c-erbA alpha, c-erbA beta) and a related protein Rev-erbA alpha have been studied in brown (pericervical) and white (perirenal) rabbit adipose tissues from birth to 180 days. The c-erbA alpha and c-erbA beta genes are expressed at similar levels in the two tissues. The alpha 1, alpha 2 and beta 1 transcripts do not change notably during development or during the conversion from brown to white phenotype which occurs in pericervical during postnatal life. Rev-erbA alpha mRNA is barely detectable at birth and dramatically increases between 7 and 30 days. However, this increase is not tissue-specific and is also observed in liver and heart. In conclusion, our results show that the decline in UCP expression during the transition from brown to white phenotype cannot be related to changes in the profiles of thyroid hormone receptors or Rev-erbA alpha mRNA expression. These profiles are not different between adipose tissue sites which are brown or white at birth.

In vitro induction of UCP1 mRNA in preadipocytes from rabbit considered as a model of large mammals brown adipose tissue development: importance of PPARγ agonists for cells isolated in the postnatal period

UNLABELLED In mammals with a lower mass-specific metabolic rate than small laboratory rodents, the brown adipose tissue (BAT) loses its thermogenic activity after birth and undergoes a transformation into white adipose tissue (WAT). Rabbit is a model of these mammals of larger body mass. Preadipocytes from cervical BAT of foetal or newborn rabbits differentiated in a chemically-defined medium and expressed low levels of uncoupled protein-1 (UCP1) mRNA, greatly increased by beta3-adrenergic or retinoic acid stimulations. On the contrary, preadipocytes from 1-month-old animals differentiated in the same conditions with no detectable,expression of UCP1. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists were necessary to induce UCP1 in these cells from older animals, a synergistic increase being noted in the presence of beta3-adrenergic agonists. In contrast to these results, preadipocytes from perirenal WAT stimulated by PPARgamma agonists never expressed UCPI. CONCLUSION preadipocytes in the postnatal period are determined as brown or white preadipocytes. PPARgamma agonists induce UCP1 expression in brown postnatal preadipocytes, but they are unable to trigger the gene in white preadipocytes.