ABSTRACTOenococcus oeniis a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine,O. oenigrows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive,O. oeniis known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by thehspgenes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization ofO. oenigenes is limited, and little is known about thein vivorole of Lo18. Due to the lack of genetic tools forO. oeni, an efficient expression vector inO. oeniis still lacking, and deletion or inactivation of thehsp18gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of theO. oenihsp18genein vivo, we have developed an expression vector to produce antisense RNA targeting ofhsp18mRNA. Recombinant strains were exposed to multiple stresses inducinghsp18gene expression: heat shock and acid shock. We showed that antisense attenuation ofhsp18affectsO. oenisurvival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance ofO. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression inO. oeni.
The Antisense RNA Approach: a New Application forIn VivoInvestigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium