Resumen de Oenococcus oeni response to wine stress: role of glutathione and thioredoxin systems
Oenococcus oeni has been widely studied for its main role in malolactic fermentation (MLF) in red wine. One of the most relevant topics is its adaptation to wine conditions after the inoculation. When the alcoholic fermentation is finishing, in order to promote O. oeni development, in some cases starters are inoculated. Unfortunately, due to the wine harsh conditions (low pH, high ethanol content, presence of SO2 and temperature) the loss of viability is a fact.
During years the characterization of new isolates and the response to the stress suffered by O. oeni has been studied in order to understand the differences between strains. Recently, there has been an increase of global studies with the intention to reveal key proteins and genes involved in this response. Different parameters of acclimation and adaptation to wine conditions have been studied from an “omic” approach.
The aim of this thesis was to perform a general study of O. oeni genes and proteins involved in this crucial period of acclimation to wine conditions of the first hours before MLF start. Moreover, the two main redox systems glutathione (GSH) and thioredoxin (Trx), had not yet been studied in this species and they could have an important role in this bacterium response.
Starting from a genomic approach of O. oeni pan-genome, seven plasticity regions in the genome were identified using in silico tools like BLASTN analysis between genes related to stress and all the genomes of O. oeni available in database. Moreover, some of these regions were new and harboured potential candidate genes affected under wine-like conditions. Another region was identified as a possible genomic transfer from another Lactobacillus and the known genomic island harbouring one thioredoxin was detected in isolates from Priorat using PCR amplification.
For the first time in O. oeni, two complementary proteomic techniques (2D-DIGE and iTRAQ labelling) together with a DNA microarray analysis were used to unveil the crucial first hours of adaptation before MLF starts. Results revealed the up regulation of a great number of genes and some corresponding proteins related to translation. On the other hand, carbohydrate metabolism was repressed leaving the activation of malate and citrate metabolism as the possible source of energy. Cell envelope genes and proteins were activated as a protective response and well known stress targets were identified in the analysis.
The redox systems GSH and Trx were identified in the “omic” analysis and were deepen studied for the first time in O. oeni. The uptake of GSH in O. oeni was determined by a fluorescent assay, and defined as strain-dependent. Also, after different growths with the addition of GSH and stress factors like low pH and ethanol presence, the beneficial effect of this antioxidant during growth was demonstrated. The changes in the membrane reported were identified using anisotropy fluorescence technique and fatty acid analysis by gas chromatography. Also, the transcriptional analysis using RT-qPCR of the complete set of the seven GSH genes was accomplished under wine conditions.
Regarding Trx system, the transcriptional analysis identified this system activated during MLF, being the trxA3 the one with a higher over-expression in most strains tested. Using phylogenetic tree analysis and in silico BLAST tools, Trx genes were determined conserved among the different sequenced strains of O. oeni, except for one gene enclosed in a genetic horizontal transfer from Lactobacillus. However, this gene is still over-expressed in the strains which harboured it under MLF process.
These results contribute to a better global view of the response of O. oeni during adaptation to wine conditions and the possible future use of those redox systems as targets for strain selection and differentiation.
