Molecular tools for the analysis of the microbiota involved in malolactic fermentation: from microbial diversity to selection of lactic acid bacteria of enological interest.

Winemaking is a complex process involving two successive fermentations: alcoholic fermentation, by yeasts, and malolactic fermentation (MLF), by lactic acid bacteria (LAB). During MLF, LAB can contribute positively to wine flavor through decarboxylation of malic acid with acidity reduction and other numerous enzymatic reactions. However, some microorganisms can have a negative impact on the quality of the wine through processes such as biogenic amine production. For these reasons, monitoring the bacterial community profiles during MLF can predict and control the quality of the final product. In addition, the selection of LAB from a wine-producing area is necessary for the formulation of native malolactic starter cultures well adapted to local winemaking practices and able to enhance the regional wine typicality. In this sense, molecular biology techniques are fundamental tools to decipher the native microbiome involved in MLF and to select bacterial strains with potential to function as starter cultures, given their enological and technological characteristics. In this context, this work reviews the different molecular tools (both culture-dependent and -independent) that can be applied to the study of MLF, either in bacterial isolates or in the microbial community of wine, and of its dynamics during the process.

Relative expression of stress-related genes during acclimation at low temperature of psychrotrophic Oenococcus oeni strains from Patagonian wine.

In the present study, we evaluated the transcriptional response of four stress-related genes in three Oenococcus oeni strains after acclimation at two different temperatures. Gene expression was analyzed at time zero and after 48 h acclimation at 18 and 21 °C. After the acclimation period cells were inoculated into sterile Pinot noir wine and MLF was followed for 25 days to investigate if different acclimation temperatures could influence cell survival and MLF performance. L-malic acid consumption, population survival, and transcriptional behavior were different upon the acclimation temperature. rmlB and hsp20 genes presented a considerable increase in their expression level when strains were acclimated at 18 °C particularly in the psychrotrophic strains UNQOe19 and UNQOe4 isolated from Patagonian Pinot noir wine in comparison with the control strain (ATCC 27310). The increase in rmlB and hsp20 expression could account for the better survival of these strains in Pinot noir in comparison with the control strain. In addition, Patagonian populations acclimated at 18 °C were able to consume a higher percentage of L-malic acid in comparison with cells acclimated at 21 °C. Our results suggest that gene expression analysis of cells acclimated at sub-optimal temperatures could benefit the selection of psychrotrophic strains aimed as starter cultures.

Genome Sequence of Oenococcus oeni UNQOe19, the First Fully Assembled Genome Sequence of a Patagonian Psychrotrophic Oenological Strain.

Oenococcus oeni UNQOe19 is a native strain isolated from a Patagonian pinot noir wine undergoing spontaneous malolactic fermentation. Here, we present the 1.83-Mb genome sequence of O. oeni UNQOe19, the first fully assembled genome sequence of a psychrotrophic strain from an Argentinean wine.

Neurotransmitter amines and antioxidant agents in neuronal protection against methylmercury-induced cytotoxicity in primary cultures of mice cortical neurons.

Methylmercury (MeHg) is an environmental toxicant with detrimental effects on the developing brain and adult nervous system. The main mechanisms identified include oxidative stress, changes in intracellular calcium, mitochondrial changes, inhibition of glutamate uptake, of protein synthesis and disruption of microtubules. However, little is known about mechanisms of protection against MeHg neurotoxicity. We found that resveratrol (10 µM) and ascorbic acid (200 µM) protected MeHg-induced cell death in primary cultures of cortical neurons. In this work, we aimed at finding additional targets that may be related to MeHg mode of action in cell toxicity with special emphasis in cell protection. We wonder whether neurotransmitters may affect the MeHg effects on neuronal death. Our findings show that neurons exposed to low MeHg concentrations exhibit less mortality if co-exposed to 10 µM dopamine (DA). However, DA metabolites, HVA (homovanillic acid) and DOPAC (3,4-dihydroxyphenylacetic acid) are not responsible for such protection. Furthermore, both DA D1 and D2 receptors agonists showed a protective effect against MeHg toxicity. It is striking though that DA receptor antagonists SKF83566 (10 µM) and haloperidol (10 µM) did not inhibit DA protection against MeHg. In addition, the protective effect of 10 µM DA against MeHg-induced toxicity was not affected by additional organochlorine pollutants exposure. Our results also demonstrate that cells exposed to MeHg in presence of 100 µM acetylcholine (ACh), show an increase in cell mortality at the "threshold value" of 100 nM MeHg. Finally, norepinephrine (10 µM) and serotonin (20 µM) also had an effect on cell protection. Altogether, we propose to further investigate the additional mechanisms that may be playing an important role in MeHg-induced cytotoxicity.

Methylmercury-induced developmental toxicity is associated with oxidative stress and cofilin phosphorylation. Cellular and human studies.

Environmental exposure to methylmercury (MeHg) during development is of concern because it is easily incorporated in children's body both pre- and post-natal, it acts at several levels of neural pathways (mitochondria, cytoskeleton, neurotransmission) and it causes behavioral impairment in child. We evaluated the effects of prolonged exposure to 10-600nM MeHg on primary cultures of mouse cortical (CCN) and of cerebellar granule cells (CGC) during their differentiation period. In addition, it was studied if prenatal MeHg exposure correlated with altered antioxidant defenses and cofilin phosphorylation in human placentas (n=12) from the INMA cohort (Spain). Exposure to MeHg for 9days in vitro (DIV) resulted in protein carbonylation and in cell death at concentrations ≥200nM and ≥300nM, respectively. Exposure of CCN and CGC to non-cytotoxic MeHg concentrations for 5 DIV induced an early concentration-dependent decrease in cofilin phosphorylation. Furthermore, in both cell types actin was translocated from the cytosol to the mitochondria whereas cofilin translocation was found only in CGC. Translocation of cofilin and actin to mitochondria in CGC occurred from 30nM MeHg onwards. We also found an increased expression of cortactin and LIMK1 mRNA in CGC but not in CCN. All these effects were prevented by the antioxidant probucol. Cofilin phosphorylation was significantly decreased and a trend for decreased activity of glutathione reductase and glutathione peroxidase was found in the fetal side of human placental samples from the highest (20-40µg/L) MeHg-exposed group when compared with the low (<7µg/L) MeHg-exposed group. In summary, cofilin dephosphorylation and oxidative stress are hallmarks of MeHg exposure in both experimental and human systems.

Transcriptomic and proteomic analysis of Oenococcus oeni PSU-1 response to ethanol shock.

The correct development of malolactic fermentation depends on the capacity of Oenococcus oeni to survive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on O. oeni PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were over-expressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of O. oeni to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue.

Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons.

Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce. In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse cortical neurons. Exposure to 0.1-100µM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca(2+)]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo.

Multigenic expression analysis as an approach to understanding the behaviour of Oenococcus oeni in wine-like conditions.

The correct performance of wine malolactic fermentation (MLF) depends on the metabolic characteristics of the Oenococcus oeni strain/s responsible for this process. This study characterizes four O. oeni strains, which behave differently in terms of malolactic performance, the citric acid use related to acetic acid production, and stress adaptation. Metabolic evolution and its associated enzymatic activities were studied and the transcriptional response of the genes related to MLF, citrate metabolism and stress response was compared among strains. A higher initial expression of both the malolactic enzyme and the encoding gene mleA may be related to faster MLF. The initial transcriptional levels of citrate lyase (citE) proved indicative of early citrate consumption. Moreover, the strains that performed best in wine-like conditions presented a much higher relative expression of several stress responsive genes, particularly hsp18, clpP, ctsR and rmlB. Finally, an inter-strain comparison of the transcriptional levels of selected genes at different times during MLF proved a useful tool in characterizing strains based on their metabolic behaviour.

Influence of ethanol and pH on the gene expression of the citrate pathway in Oenococcus oeni.

The consumption of citrate by the malolactic bacterium Oenococcus oeni changes the aromatic profile of wines due to the production of volatile compounds such as diacetyl and acetic acid. In this study, the expression of genes related to citrate utilization in the O. oeni strain PSU-1 was investigated to further understand the role of this metabolic pathway in the adaptation to wine environment and its impact on organoleptic qualities. Different conditions of ethanol content (0% and 10%) and pH (3.5 and 4.0) were assayed to evaluate the transcriptional response to both these stress factors. In the presence of ethanol, metabolic and transcriptional behavior was different than the observed when ethanol was absent. The expression of citrate pathway genes was mainly affected by ethanol, while pH showed a lower effect. Among the studied genes, citE, ackA and alsD were the genes revealing a distinctive transcriptional response. The differences observed in gene expression were in correlation with the different content of end products such as acetic acid and diacetyl. The increment of gene expression observed in the presence of ethanol at low pH suggests the participation of citrate metabolism in the response to stress conditions.