Ctsr, the Master Regulator of Stress-Response in Oenococcus

CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1 Maud Darsonval, Frédérique Julliat, Tarek Msadek, Hervé Alexandre, Cosette Grandvalet To cite this version: Maud Darsonval, Frédérique Julliat, Tarek Msadek, Hervé Alexandre, Cosette Grandvalet. CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1. Frontiers in Microbiology, Frontiers Media, 2018, 9, pp.1-14. 10.3389/fmicb.2018.03135. hal-01986589 HAL Id: hal-01986589 https://hal.archives-ouvertes.fr/hal-01986589 Submitted on 18 Jan 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Distributed under a Creative Commons Attribution| 4.0 International License fmicb-09-03135 December 15, 2018 Time: 15:10 # 1 ORIGINAL RESEARCH published: 18 December 2018 doi: 10.3389/fmicb.2018.03135 CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1 Maud Darsonval1†, Frédérique Julliat1†, Tarek Msadek2,3, Hervé Alexandre1,4 and Cosette Grandvalet1,5* 1 UMR A. 02.102 Procédés Alimentaires et Microbiologique, AgroSup Dijon, Université Bourgogne Franche-Comté, Dijon, France, 2 Unité de Biologie des Bactéries Pathogènes à Gram Positif, Institut Pasteur, Paris, France, 3 CNRS ERL 6002, Paris, France, 4 Institut Universitaire de la Vigne et du Vin – Jules Guyot, Dijon, France, 5 Institut National Supérieur des Sciences Agronomiques, de L0Alimentation et de L0Environnement, AgroSup Dijon, Dijon, France 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 Edited by: Teresa Zotta, regulator of stress response genes identified to date in O. oeni. Extensively characterized Italian National Research Council, Italy in Bacillus subtilis, the CtsR repressor is active as a dimer at 37◦C and degraded Reviewed by: at higher temperatures by a proteolytic mechanism involving two adapter proteins, Albert Bordons, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not Rovira i Virgili University, Spain Giuseppe Spano, encode orthologs of these adapter proteins and the regulation of CtsR activity remains University of Foggia, Italy unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA Vittorio Capozzi, University of Foggia, Italy silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by *Correspondence: asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock Cosette Grandvalet (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of [email protected] CtsR activity was studied using a heterologous expression system to demonstrate that † These authors have contributed O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when equally to this work produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni Specialty section: CtsR acts as a temperature sensor and is inactivated at growth temperatures above This article was submitted to ◦ Food Microbiology, 33 C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and a section of the journal ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni. Frontiers in Microbiology Keywords: Oenococcus oeni, stress response, CtsR, RNA silencing, heterologous expression system, two-hybrid Received: 15 October 2018 system Accepted: 04 December 2018 Published: 18 December 2018 Citation: INTRODUCTION Darsonval M, Julliat F, Msadek T, Alexandre H and Grandvalet C (2018) Oenococcus oeni is an acidophilic wine-associated lactic acid bacterium (LAB), mainly responsible CtsR, the Master Regulator of Stress-Response in Oenococcus for malolactic fermentation (MLF) of wine, usually following yeast-driven alcoholic fermentation oeni, Is a Heat Sensor Interacting (Lonvaud-Funel, 1999). Wine and the winemaking process form a harsh and challenging ◦ With ClpL1. Front. Microbiol. 9:3135. environment combining stresses such as low pH (3–3.5), low temperatures (14–18 C), the presence doi: 10.3389/fmicb.2018.03135 of ethanol, nutrient starvation and competing organisms (yeasts) generating abiotic growth Frontiers in Microbiology| www.frontiersin.org 1 December 2018| Volume 9| Article 3135 fmicb-09-03135 December 15, 2018 Time: 15:10 # 2 Darsonval et al. O. oeni CtsR Regulator inhibitors (ethanol, sulfites, decanoic, and dodecanoic acids). HrcA (Grandvalet et al., 2005). In Bacillus subtilis, CtsR is active Like most microorganisms facing stress conditions, O. oeni must as a dimer under optimal growth conditions and represses adapt to survive and deciphering the molecular mechanisms transcription of its regulon by binding its operator sequence involved in responding to stress is an important step to improve (Derré et al., 1999, 2000). Under stress conditions, the CtsR O. oeni MLF performance and design future malolactic starter dimer is phosphorylated by McsA and McsB and then recognized strains. Because of its acidophilic profile and its unique genome and degraded by the ClpCP proteolytic complex (Derré et al., organization, O. oeni is an intriguing and challenging model 2000; Kirstein et al., 2005, 2007; Elsholz et al., 2010, 2011). LAB to investigate stress response mechanisms in LAB (Bartowsky, are mcsAB-deficient Firmicutes and alternative mechanisms for 2017; Grandvalet, 2017). Over the past decades, several genetic regulating the CtsR activity have been described. In Lactococcus responses adopted by O. oeni during wine adaptation have been lactis, ClpE is required to restore repression by CtsR after heat described, including genes involved in general stress response, shock. Indeed, replacement of clpE by mcsA was shown to restore membrane composition and fluidity, pH homeostasis, oxidative hsp gene repression suggesting that ClpE in L. lactis has the stress response, presence of sulfites and DNA damage (Salema same function as McsA in B. subtilis by interacting with CtsR et al., 1996; Guzzo et al., 1997, 1998; Jobin et al., 1997, 1999; through its zinc finger motif (Varmanen et al., 2003). More Tourdot-Maréchal et al., 2000; Da Silveira et al., 2003; Beltramo recently, Tao and Biswas(2013) showed that the ClpCP complex et al., 2004; Chu-Ky et al., 2005; Coucheney et al., 2005b; is not required for specific degradation of CtsR in Streptococcus Grandvalet et al., 2005, 2008; Da Silveira and Abee, 2009; Maitre mutans but that ClpL displays a chaperone protective role et al., 2014; Darsonval et al., 2016; Margalef-Català et al., 2016). helping CtsR to bind its operator sequence (Tao et al., 2012; Oenococcus oeni stress response mechanisms involve Tao and Biswas, 2013). In addition, in L. lactis, Geobacillus the synthesis of Heat Shock Proteins (HSPs), a universal stearothermophilus, and B. subtilis, CtsR has been shown to act stress response with several regulatory pathways described in directly as a heat sensor with distinct species-specific thermal Firmicutes. Indeed, hsp genes can be induced by the alternative derepression temperature thresholds (Elsholz et al., 2010). The sigma factor sB or repressed by transcriptional repressors O. oeni genome does not contain clpE, mcsA, and mcsB genes, such as CtsR or HrcA (Benson and Haldenwang, 1993; Schulz however two clpL genes are present: clpL1, in an operon with and Schumann, 1996; Derré et al., 1999; Schumann, 2003). In clpP, and clpL2 (Beltramo et al., 2006; Assad-García et al., 2008). Streptococci, HrcA and CtsR control two partially overlapping This strongly suggests a likely involvement of ClpL1 and/or regulons that include most hsp genes (Chastanet et al., 2001; ClpL2 in the regulation of CtsR activity in O. oeni. O. oeni is not Chastanet and Msadek, 2003; Grandvalet et al., 2005; Spano readily genetically tractable, few genetic tools are available and and Massa, 2006; Frees et al., 2007). In contrast, in Bacilli, none for directed mutagenesis or gene deletion. Because of these the two regulons are entirely distinct while in Staphylococci technical barriers, the in vivo function of CtsR and the regulatory the HrcA regulon is completely embedded within the CtsR mechanisms controlling its activity in O. oeni remain unknown. regulon (Chastanet et al., 2003). Both transcriptional repressors In this study, we first investigated the in vivo role of CtsR in control expression of their regulon by specifically binding to O. oeni using antisense RNA silencing, a technique we recently their operator sequences in the promoter region, preventing used to show the first modulation of gene expression in O. oeni RNA polymerase recruitment. HrcA specifically recognizes the and confirm the molecular chaperone role of the small Hsp CIRCE (“Controlling inverted repeat of chaperone expression”) Lo18 (Darsonval et al., 2016). Using this approach, we inhibited palindromic

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