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Alicyclobacillus acidoterrestris is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of A. acidoterrestris to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of A. acidoterrestris under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene ctsR showed that dnaG and dnaN were the optimal multiple RGs for normalization at pH 4.0; ytvI , dnaG , and 16s rRNA at pH 3.5; icd and dnaG at pH 3.0; and ytv I, dnaG , and spoVE at pH 2.5. This study revealed for the first time that A. acidoterrestris had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhao, Xu, Jiao, Qiu, Zhang, Wei and Fan.)

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 .