The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

Abstract

Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni, an efficient expression vector in O. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. 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 in O. oeni. Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for wine malolactic fermentation (MLF) (1). In wine medium, O. oeni evolves under extreme physicochemical conditions: low pH (3.2 to 3.5), low temperature (18°C), and the presence of biological competitors such as yeast, producing inhibitory compounds, i.e., fatty acids, ethanol, 2-phenylethanol, and sulfites. The winemaking process itself may also constitute a hostile factor for bacterial growth. All these hostile environmental factors can cause a delay in MLF initiation. Thus, this fermentation step is currently not yet fully managed. However, the gradual increase in the ethanol level in wine during the alcoholic fermentation performed by yeasts and the production of substances inhibiting growth of lactic acid bacteria (dodecanoic acid or decanoic acid) led to the selection of the species most suited and adapted to wine conditions: O. oeni (1, 2). A better understanding of the mechanisms involved in stress-adaptive responses is essential to improve O. oeni development in wine and to perfect industrial processes of preparing wine malolactic starter. Because of its acidophilic properties, O. oeni is an interesting model for investigating stress response mechanisms in lactic acid bacteria (LAB). Like any organism undergoing an environmental stress, O. oeni tries to restore a metabolic profile beneficial for survival. Over the past decades, several mechanisms involved in adaptation to wine were described, inclu...

Oenococcus oeni: Queen of the cellar, nightmare of geneticists

“…An improved method for electroporation of O. oeni, using ethanol as a membrane fluidifier, was developed in 2008, allowing effective and reproducible DNA transfer. More recently, a major advance has been achieved by the application of anti-sense RNA technology to modulate gene expression [5]. A new stable and effective expression vector was developed and antisense attenuation was successfully carried out to confirm the involvement of Lo18 in stress tolerance.…”

Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile

“…The O. oeni ATCC BAA-1163 estA2 and estA7 genes were cloned under the control of a synthetic constitutive promoter using the E. coli/LAB shuttle vector pSIPSYN (Darsonval et al, 2015). After verification of both cloning nucleotide sequences of estA2 and estA7 genes by sequencing, E. coli recombinant strains carrying native pSIPSYN vector (Ecsyn, control) or recombinant vector pSIPSYNestA2 and pSIPSYNestA7 (EcestA2 and EcestA7) were used to test the functionality of cloned esterase genes.…”

“…T4 DNA ligase and other restriction endonucleases were purchased from New England Biolabs Inc. (New England Biolabs, Ipswich, MA, USA) and were used as recommended by the manufacturer for the cloning experiments. Plasmids pSIPSYNestA2 and pSIPSYNestA7 were constructed by inserting the amplified estA2 and estA7 esterase ORFs into the multiple cloning sites of the E. coli/LAB shuttle vector pSIPSYN (Darsonval et al, 2015) under the control of a synthetic constitutive promotor. The ORFs of estA2 and estA7 were amplified by PCR by using primer couples estA2N/estA2E and estA7N/estA7E respectively (Table 2).…”

“…At this point, no investigation of the ability of O. oeni esterases in enological conditions has been reported due to the obvious lack of genetic expression tools. Recently, we provided a new efficient expression tool, the pSIPSYN vector (Darsonval et al, 2015). The aim of the present study is to use this new genetic tool to investigate the impact of O. oeni esterases EstA2 and EstA7 activity on the ester substrates naturally present in wine.…”

Small RNAs in major foodborne pathogens: from novel regulatory activities to future applications

“…Additionally, sRNAs may also be considered a great biotechnological toolbox in synthetic biology. For instance, base pairing antisense RNAs have successfully been employed to knockdown selected genes in organisms such as the wine-associated lactic acid bacterium Oenocuccus oeni [51] and the butanolproducing C. acetobutylicum [52], which are difficult to genetically engineer. Under some conditions, it might even be advantageous to knockdown, rather than knockout, gene expression; for example, a double knockdown strategy was used to screen 18 E. coli strains for the influence of genetic background on phenol production and tolerance [53], a task that would have been very time-…”