Raphaëlle Tourdot-Maréchal

Yeast–yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts

There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the volatile profiles of several mono-culture and co-cultures. Candida zemplinina, Torulaspora delbrueckii and Metschnikowia pulcherrima were used to conduct fermentations either in mono-culture or in co-culture with S. cerevisiae. Up to 48 volatile compounds belonging to different chemical families were quantified. For the first time, we show that C. zemplinina is a strong producer of terpenes and lactones. We demonstrate by means of multivariate analysis that different interactions exist between the co-cultures studied. We observed a synergistic effect on aromatic compound production when M. pulcherrima was in co-culture with S. cerevisiae. However a negative interaction was observed between C. zemplinina and S. cerevisiae, which resulted in a decrease in terpene and lactone content. These interactions are independent of biomass production. The aromatic profiles of T. delbrueckii and S. cerevisiae in mono-culture and in co-culture are very close, and are biomass-dependent, reflecting a neutral interaction. This study reveals that a whole family of compounds could be altered by such interactions. These results suggest that the entire metabolic pathway is affected by these interactions.

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase.

Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The expression of these genes as a function of growth is significantly different. Surprisingly, the clpX gene, which is induced by heat shock, was highly expressed in the early phase of growth. In addition to stress protein synthesis, adaptation to the acid pH of wine requires efficient cellular systems to extrude protons. Using inhibitors specific for different types of ATPases, we demonstrated the existence of H+-ATPase and P-type ATPase.

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation

Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.

Membrane fluidity of stressed cells of Oenococcus oeni.

The determination of membrane fluidity in whole cells of Oenococcus oeni was achieved by membrane probe 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy measurements. The results demonstrated instantaneous fluidity variations with cells directly stressed during the measure. Heat (42 degrees C) or acid (pH 3.2) shocks decreased the anisotropy values (fluidising effects), whereas an ethanol shock (10% ethanol, v/v) increased the membrane rigidity. The velocities of fluidity variation with non-adapted or adapted cells (incubation in inhibitory growth conditions) were compared. The adaptation of the cells to acid conditions had no effect on the membrane fluidity variation after an acid shock. In contrast, the rates of membrane fluidity variation of adapted cells were 5- and 3-fold lower after a heat shock and an ethanol shock, respectively. These results suggest the positive effect of an adaptation on the membrane response and can help to explain the mechanisms of stress tolerance in Oenococcus oeni.

High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.

Induction of Oenococcus oeni H+-ATPase activity and mRNA transcription under acidic conditions

The profiles of Oenococcus oeni IOB84.13 H(+)-ATPase activity under various conditions of growth were studied. Cells growing at low pH 3.5 had a 1.6-fold higher H(+)-ATPase activity compared to control cells grown at pH 5.3. While the pH of the growth medium was shown to be stable in the presence of malic acid, a drastic decrease in pH from 5.3 down to 3.9 during growth in the absence of malic acid induced an increase in H(+)-ATPase activity by 1.5-fold. This induction was even greater when the initial pH was 3.5. Partial cloning of the genes encoding the beta-subunit and the epsilon-subunit of the H(+)-ATPase suggested a typical F(1)F(0)-ATPase genetic organization in O. oeni. The atp mRNA was detected by slot blots. Cells shocked at acidic pH were shown to contain higher levels of atp mRNA compared to the control cells grown at pH 5.3. Taken together, these results indicate that the H(+)-ATPase of O. oeni is induced at low pH and that regulation seems to occur at the level of transcription. This agrees with the role of this enzyme in the regulation of the cytoplasmic pH and in the acid tolerance of O. oeni.

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

ABSTRACT Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Wine microbiome: A dynamic world of microbial interactions

ABSTRACT Most fermented products are generated by a mixture of microbes. These microbial consortia perform various biological activities responsible for the nutritional, hygienic, and aromatic qualities of the product. Wine is no exception. Substantial yeast and bacterial biodiversity is observed on grapes, and in both must and wine. The diverse microorganisms present interact throughout the winemaking process. The interactions modulate the hygienic and sensorial properties of the wine. Many studies have been conducted to elucidate the nature of these interactions, with the aim of establishing better control of the two fermentations occurring during wine processing. However, wine is a very complex medium making such studies difficult. In this review, we present the current state of research on microbial interactions in wines. We consider the different kinds of interactions between different microorganisms together with the consequences of these interactions. We underline the major challenges to obtaining a better understanding of how microbes interact. Finally, strategies and methodologies that may help unravel microbe interactions in wine are suggested.

Absence of Malolactic Activity Is a Characteristic of H+-ATPase-Deficient Mutants of the Lactic Acid Bacterium Oenococcus oeni

ABSTRACT The lack of malolactic activity in H+-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni. Results obtained in this study show that the mleR transcript was not detected in the mutants by RT-PCR. No mutation in the nucleotide sequences of the mleR gene and the malolactic operon was found. The effect of a reduction in H+-ATPase activity on l-malate metabolism was then investigated by using other malolactic bacteria. Spontaneous H+-ATPase-deficient mutant strains of Lactococcus lactis and Leuconostoc mesenteroides were isolated by using neomycin resistance. Two mutants were selected. These mutants exhibited ATPase activities that were reduced to 54 and 70% of the activities obtained for the L. lactis and L. mesenteroides parental strains, respectively. These mutants were also acid sensitive. However, in contrast to the ATPase-deficient mutants of O. oeni, activation of l-malate metabolism was observed with the L. lactis and L. mesenteroides mutants under optimal or acidic growth conditions. These data support the suggestion that expression of the genes encoding malolactic enzymes in O. oeni is regulated by the mleR product, as it is in L. lactis. Nevertheless, our results strongly suggest that there is a difference between the regulation of expression of the malolactic locus in O. oeni and the regulation of expression of this locus in less acidophilic lactic acid bacteria.

Involvement of osmotic cell shrinkage on the proton extrusion rate in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been subjected to hyperosmotic shocks by using permeating (sorbitol, xylitol, glycerol, NaCl) and nonpermeating (PEG 600) solutes. The proton extrusion rate decreased as the osmotic pressure increased, whichever solute was used. However, the total inhibition of the cellular H+ extrusion depended on the solute used. A total inhibition was observed at about 20 MPa with glycerol, xylitol and sorbitol. With PEG 600, a total inhibition of extracellular acidification was obtained at 8.5 MPa. NaCl, with an extracellular pressure of 37.8 MPa (near saturation), did not completely inhibit the extracellular acidification. These results showed that the total inhibition of proton extrusion, involving probably the membrane H+-ATPase. was not correlated to the hydric state of the external medium but was strictly linked to the degree of permeation of solutes across the plasma membrane. The extracellular acidification was totally inhibited by a critical final cell volume reached after the osmotic shrinkage, whichever solute was used. This critical final cell volume represented 50% of the initial cell volume. This result suggests that the final cell volume reached after an osmotic stress represents a key thermodynamic parameter for cell osmoregulation in which H+-ATPase would be implicated.