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Oenococcus oeni, the lactic acid bacterium (LAB) mainly responsible for malolactic fermentation, has been repeatedly isolated from wines, but hardly ever from grapes. In this study, a large survey of autochthonous LAB from the Catalan wine region of Priorat was made. A total of 761 LAB isolates, 254 from Grenache and Carignan grape berries and 507 from wines, were identified and typed. Around 70% of the isolates were O. oeni, mostly from wines, but remarkably, 53 of them were isolated from grapes. A minimum spanning tree of O. oeni strains constructed from the multilocus variable number tandem repeat analysis showed considerable phylogenetic diversity. Other non-Oenococcus species were also identified and typed, Lactobacillus plantarum being predominant in grapes. Other LAB isolates were Pediococcus pentosaceus, Fructobacillus tropaeoli, L. mali, L. lindneri and L. sanfranciscensis. High-throughput sequencing (HTS) analysis was also carried out with grape samples, and Oenococcus and Lactobacillus were detected in significant quantities, which corroborates the culturing results from the same samples.

The thioredoxin system protects against oxidative stress through the reversible oxidation of the thioredoxin active center dithiol to a disulphide. The genome of Oenococcus oeni PSU-1 contains three thioredoxin genes (trxA1, trxA2, trxA3), one thioredoxin reductase (trxB) and one ferredoxin reductase (fdr) which, until recently, was annotated as a second thioredoxin reductase. For the first time, the entire thioredoxin system in several O. oeni strains isolated from wine has been analysed. Comparisons at the DNA and protein levels have been undertaken between sequences from O. oeni and other genera and species, and the genera Leuconostoc and Lactobacillus were found to present the highest similarities. The gene most frequently absent from a collection of 34 strains and the sequences annotated in the NCBI database was trxA1. Moreover, phylogenetic analysis suggested that this gene was horizontally transferred from Lactobacillus to O. oeni. Strain-dependent expression profiles were determined in rich and in wine-like media. General over-expression was detected after inoculation into wine-like medium, with trxA3 being the most highly expressed gene. The increased transcriptional levels of the thioredoxin genes are indicative of the crucial role of this system in the O. oeni response to wine harsh conditions.

FIliació URV: SIInclòs a la memòria: SI Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH 4, pH 3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures.

The differential expression of five genes related to adaptation to wine conditions in Oenococcus oeni was evaluated by studying transcriptional changes in wine-like media and in real wine during malolactic fermentation. Two strains isolated from high ethanol wine and another, less tolerant, strain were used. Expression of the selected genes (mleA, citE, hsp18, clpP and gsh) was studied by means of RT-qPCR. A higher level of gene expression was found for the more ethanol-tolerant strains, especially for citE, hsp18 and clpP. Thus, these three genes might be good markers for the more stress-resistant strains of O. oeni. In most cases, the results of expression in wine-like medium with 14 % ethanol had a similar trend to those in real wine, suggesting that the wine-like medium can be a good model for studying the stress response in O. oeni. Different expression between strains was well correlated with their ability to perform malolactic fermentation. In this way, the identified gene markers can be used in the selection of new strains as starters for malolactic fermentation.

Filiació URV: SIInclòs a la memòria: SI Although Oenococcus oeni is the main species that is responsible for malolactic fermentation (MLF), harsh wine conditions can limit its performance. Although several mechanisms underlying the response to stress have been studied in this species, little is known regarding the cellular systems that protect against oxidative stress in other bacteria, such as glutathione (GSH). O. oeni cannot synthesize GSH but contains several genes related to its utilization. In this study, the relative expression (RE) of the seven genes involved in the GSH redox system found in O. oeni PSU-1 (gshR, gpo, three glutaredoxin-like genes and two subunits of an hypothetical transporter) has been measured. The study was performed using three strains, with each exhibiting a different GSH uptake capacity. The strains were grown in a stress-adaptation medium supplemented with 5 mM GSH and under different adaptation stress conditions (pH 4 and 6% ethanol). The RE showed that only some of these genes, including one for a possible glutaredoxin (OEOE_RS04215) and cydC for a subunit of a putative GSH transporter (OEOE_RS1995), responded to the addition of GSH. The presence of ethanol had a relevant effect on gene expression. Among the studied genes, the one for a NrdH-redoxin (OEOE_RS00645) showed a common response to ethanol in the strains, being over-expressed when grown with GSH. In most cases, the transcriptional changes were more evident for the strain with a higher capacity of GSH uptake. Malolactic performance of the three strains after pre-adaptation was evaluated in wine-like media (12% ethanol and pH 3.4). It was observed that the addition of GSH during pre-adaptation growth had a protective role in the cells exposed to low pH and ethanol, resulting in a