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Ohsr Acts As an Organic Peroxide-Sensing Transcriptional Si et al. Microb Cell Fact (2018) 17:200 https://doi.org/10.1186/s12934-018-1048-y Microbial Cell Factories RESEARCH Open Access OhsR acts as an organic peroxide‑sensing transcriptional activator using an S‑mycothiolation mechanism in Corynebacterium glutamicum Meiru Si1*† , Tao Su1†, Can Chen2*†, Jinfeng Liu1, Zhijin Gong1, Chengchuan Che1, GuiZhi Li1 and Ge Yang1 Abstract Background: Corynebacterium glutamicum is a well-known producer of various L-amino acids in industry. During the fermenting process, C. glutamicum unavoidably encounters oxidative stress due to a specifc reactive oxygen species (ROS) produced by consistent adverse conditions. To combat the ROS, C. glutamicum has developed many common disulfde bond-based regulatory devices to control a specifc set of antioxidant genes. However, nothing is known about the mixed disulfde between the protein thiol groups and the mycothiol (MSH) (S-mycothiolation)-based sen- sor. In addition, no OhrR (organic hydroperoxide resistance regulator) homologs and none of the organic hydroper- oxide reductase (Ohr) sensors have been described in the alkyl hydroperoxide reductase CF-missing C. glutamicum, while organic hydroperoxides (OHPs)-specifc Ohr was a core detoxifcation system. Results: In this study, we showed that the C. glutamicum OhsR acted as an OHPs sensor that activated ohr expres- sion. OhsR conferred resistance to cumene hydroperoxide (CHP) and t-butyl hydroperoxide but not H­ 2O2, hypochlor- ous acid, and diamide; this outcome was substantiated by the fact that the ohsR-defcient mutant was sensitive to OHPs but not inorganic peroxides. The DNA binding activity of OhsR was specifcally activated by CHP. Mutational analysis of the two cysteines (Cys125 and Cys261) showed that Cys125 was primarily responsible for the activation of DNA binding. The oxidation of Cys125 produced a sulfenic acid (C125-SOH) that subsequently reacted with MSH to generate S-mycothiolation that was required to activate the ohr expression. Therefore, OhsR regulated the ohr expres- sion using an S-mycothiolation mechanism in vivo. Conclusion: This is the frst report demonstrating that the regulatory OhsR specifcally sensed OHPs stress and responded to it by activating a specifc ohr gene under its control using an S-mycothiolated mechanism. Keywords: MarR, Organic peroxide stress, Transcription regulation, C. glutamicum, S-mycothiolation *Correspondence: [email protected]; [email protected] †Meiru Si, Tao Su and Can Chen contributed equally to this work 1 College of Life Sciences, Qufu Normal University, Qufu 273165, Shandong, China 2 College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, Henan, China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Si et al. Microb Cell Fact (2018) 17:200 Page 2 of 15 Background S-thiolation-based regulation model (S-glutathionylation, During normal cellular functions or under unfavora- S-mycothiolation and S-bacillithiolation) in bacteria has ble external environmental stimuli, bacteria unavoid- so far only been shown for a few selected sensors, such as ably produce signifcant levels of reactive oxygen species the Escherichia coli OxyR (the thiol-based redox sensor (ROS), including hydrogen peroxide (H 2O2), superox- for peroxides) and B. subtilis OhrR (organic hydroperox- ·− ide radical (O2 ), and organic peroxides (OHPs) [1]. ide resistance regulator) [16, 17]. In addition, researchers Te accumulation of excessive concentrations of ROS found that each of the many regulators sensed a specifc is toxic to biological systems, leading to DNA damage, oxidant and responded to it by activating or depressing lipid peroxidation, protein oxidation, and eventually a specifc set of genes under its control. For example, causing various diseases, such as cancer, vascular and in bacteria, OxyR and OhrR respond to peroxide and neurodegenerative disorders, and aging [2]. Among the organic hydroperoxide stress, respectively, while SoxRS external environmental stimuli, OHPs cause the most (a sensor of superoxide and nitric-oxide stress) specif- serious damage to the cells due to their ability to gener- cally responds to superoxide stress [4, 18, 19]. However, ate reactive organic radicals through the reaction with the exact molecular mechanism in diferent stimulus- membranes, free fatty acids, or other macromolecules segregating regulators or specifc stimulus-sensing regu- [3]. Terefore, OHPs detoxifcation is critical for bacte- lators remains to be elucidated to a large degree. rial survival. Bacteria have evolved diverse defense strat- Corynebacterium glutamicum, a well-known various egies against OHPs toxicity, including enzymes and low l-amino acid producer in industry and a model organism molecular weight antioxidants [4–11]. Te OHPs detoxi- for systems biology, unavoidably generates or encounters fcation enzymes alkyl hydroperoxide reductase subu- a series of unfavorable circumstances during the cul- nit CF (AhpCF) and organic hydroperoxide resistance ture process [20]. However, C. glutamicum robustly sur- (Ohr) are the two core detoxifcation systems in bacte- vives the various adverse stresses of the culture process ria [6, 7]. Tey can reduce OHPs into their correspond- using diverse defense strategies, including thiol-based ing alcohols. In addition to enzymes, another unique regulator-control of a variety of antioxidants and MSH feature of the antioxidant system is the presence of low [11, 21–24]. However, surprising observations in previ- molecular weight thiols, such as tripeptide glutathione ous reports showed that although MSH-defcient C. glu- (GSH; γ-l-glutamyl-l-cysteinylglycine), 1-d-myo-inosityl tamicum mutants were highly sensitive to OHPs, MSH 2-(N-acetyl-l-cysteinyl) amido-2-deoxy-d-glucopyra- did not directly react with peroxides to protect the cells noside (mycothiol or MSH) and bacillithiol (BSH) [6, 7, [11, 25, 26]. More recently, the ohr expression in a C. glu- 11, 12]. Te low molecular weight thiols have a role in tamicum strain lacking AhpC was found to be specifcally OHPs detoxifcation in vivo, confrmed by the increased induced by the OHPs and not regulated by the reactive sensitivity to cumene hydroperoxide (CHP), menadione electrophilic species (RES)-sensing zinc-associated anti (MD), and tert-butyl hydroperoxide (t-BHP) in MSH- sigma factors SigH, and ohr mutants were hypersensitive defcient Mycobacterium smegmatis, M. tuberculosis and to the OHPs and were not sensitive to other ROS-gener- Corynebacterium glutamicum mutant strains [8, 9, 11]. ating agents [21]. In addition, in vitro biochemical studies Several studies have demonstrated that the diverse showed that the thiol-dependent, Cys-based peroxidase defense systems against oxidant toxicity are coordinated Ohr could detoxify OHPs more efectively than H2O2 by thiol-based redox transcription sensors that sense [21]. Tese recent discoveries raised questions about the exclusive ROS, modulate the appropriate regulon tran- existence of a specifc Ohr-regulating and MSH-depend- scription, and fnally confer resistance to oxidative stress ent response OHPs regulator. Although many H2O2-, and restore the redox balance [13–15]. Direct sensing quinone-, diamide-, or NaOCl-responding disulfde and the rapid responses of thiol-based transcription fac- bond-based redox regulators, such as OxyR, RosR (reg- tors are considered to be an efcient way to enhance the ulator of oxidative stress response), QorR (quinone oxi- survival of the organism under oxidation stress. Tiol- doreductase regulator), and SigH (the stress-responsive based redox regulators control the antioxidant gene extracytoplasmic function-sigma (ECF-σ) factor), have expression by versatile posttranslational thiol-modifca- been investigated extensively in C. glutamicum, the study tions mechanisms, including the disulfdes-switch model of transcriptional regulators specifcally responding to (sulfenic acid, disulfde bond, and S-thiolation), Cys- organic oxidation and controlling ohr expression in C. phosphorylation and Cys-alkylation. Although many of glutamicum remains enigmatic, and very little is known the common disulfde bond model-based regulators in about transcriptional regulators that use an S-mycothi- bacteria have been well described, and S-thiolations in olated regulation model [27–30]. eukaryotes have emerged as the major redox-regulatory In this study, we demonstrated that the regulator OhsR mechanism of redox sensing transcription factors, the from C. glutamicum specifcally sensed OHPs stress Si et al. Microb Cell Fact (2018) 17:200 Page 3 of 15 using a new thiol oxidation-based mechanism. Te mL for C. glutamicum; chloramphenicol, 20 µg/mL for E. reduced OhsR did not bind to the Ohr promoters. Te coli and 10 µg/mL for C. glutamicum. oxidative modifcation of Cys125 produced an intermedi- ate Cys-SOH and reacted further with MSH to the mixed Plasmid construction disulfdes formation. S-mycothiolation caused the struc- To obtain an expression plasmid, the OOhsR-F/OOhsR-
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