No abstract

Oenococcus oeni is a lactic acid bacterium responsible for malolactic fermentation of wine. While many stress response mechanisms implemented by O. oeni during wine adaptation have been described, little is known about their regulation. CtsR is the only regulator of stress response genes identified to date in O. oeni. Extensively characterized in Bacillus subtilis, the CtsR repressor is active as a dimer at 37°C and degraded at higher temperatures by a proteolytic mechanism involving two adapter proteins, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not encode orthologs of these adapter proteins and the regulation of CtsR activity remains unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of CtsR activity was studied using a heterologous expression system to demonstrate that O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni CtsR acts as a temperature sensor and is inactivated at growth temperatures above 33°C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni.

<p style="text-align: justify;"><em>Brettanomyces bruxellensis</em> is well adapted to high ethanol concentrations and low pH which allows it to develop in difficult environments, such as wine. <em>B. bruxellensis</em> is mainly found in red wine and is regarded as a spoilage yeast due to its production of ethylphenols and other compounds responsible for organoleptic defects. The detection and quantification of this yeast is essential to preventing wine spoilage. Several specific detection and quantification kits based on real time quantitative PCR are commercially available. Although these kits are frequently used by private enological and research laboratories, no scientific report on the reliability and performance of these kits, including inter-laboratory and inter-assay comparisons have been published. The aim of this work was to compare available kits to quantify <em>B</em>. <em>bruxellensis</em> in red wine to classical method (plate counting on selective medium) in an interlaboratory study. Three different commercial kits were tested on three different wines from Bordeaux, Côtes du Rhône, and Burgundy inoculated with <em>B</em>. <em>bruxellensis </em>at four different concentrations. Five naturally contaminated wines from different French wine regions were also tested. Our results suggest that all the kits tested probably over or underestimate the quantity of <em>B</em>. <em>bruxellensis</em> in red wine and, under specific conditions, give false positives. Quantification may be very heterogeneous depending on the wine, laboratory, or population level. Underestimations or false negative results may have serious consequences for winemakers. Overestimation may be partly due to the quantification of dead cells qPCR.</p><p style="text-align: justify;">This study highlights that quantification of<em> B</em>. <em>bruxellensis</em> in red wine using commercial kits requires a high level of expertise in molecular biology. We recommend that all users use a microbiological internal control to validate DNA extraction yield.</p>