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.
Highlights
Oenococcus oeni is an acidophilic wine-associated lactic acid bacterium (LAB), mainly responsible for malolactic fermentation (MLF) of wine, usually following yeast-driven alcoholic fermentation (Lonvaud-Funel, 1999)
In order to validate the efficiency of this antisense RNA (asRNA) approach, the effect of ASctsR RNA production was examined at the protein level by immunodetection of Lo18 (Figure 1)
By targeting ctsR for knockdown, we undertook an in vivo approach with the aim of broadening our knowledge on O. oeni CtsR, the only stress response regulator described to date
Summary
Oenococcus oeni is an acidophilic wine-associated lactic acid bacterium (LAB), mainly responsible for malolactic fermentation (MLF) of wine, usually following yeast-driven alcoholic fermentation (Lonvaud-Funel, 1999). In Bacilli, the two regulons are entirely distinct while in Staphylococci the HrcA regulon is completely embedded within the CtsR regulon (Chastanet et al, 2003) Both transcriptional repressors control expression of their regulon by binding to their operator sequences in the promoter region, preventing RNA polymerase recruitment. CtsR is the only regulator of stress response gene expression identified and studied in O. oeni so far (Grandvalet et al, 2005). The sequence analysis of the scaffold O. oeni ATCC BAA-1163 genome allowed the identification of the ctsR gene sequence and CtsR operator sequences, but no hrcA gene, CIRCE sequences or other genes encoding known stress response regulators have been found. Likewise in O. oeni PSU-1 (NC_008528), the only complete sequenced strain, no gene encoding known regulators of stress response have been identified, except cstR. O. oeni is the only Firmicutes where expression of dnaK and groEL is controlled exclusively by CtsR and not by
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