College of Life Sciences, Qufu Normal University, Qufu, 273165, Shandong, China.
College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, 466001, Henan, China.
Microb Cell Fact. 2018 Dec 26;17(1):200. doi: 10.1186/s12934-018-1048-y.
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 specific reactive oxygen species (ROS) produced by consistent adverse conditions. To combat the ROS, C. glutamicum has developed many common disulfide bond-based regulatory devices to control a specific set of antioxidant genes. However, nothing is known about the mixed disulfide between the protein thiol groups and the mycothiol (MSH) (S-mycothiolation)-based sensor. In addition, no OhrR (organic hydroperoxide resistance regulator) homologs and none of the organic hydroperoxide reductase (Ohr) sensors have been described in the alkyl hydroperoxide reductase CF-missing C. glutamicum, while organic hydroperoxides (OHPs)-specific Ohr was a core detoxification system.
In this study, we showed that the C. glutamicum OhsR acted as an OHPs sensor that activated ohr expression. OhsR conferred resistance to cumene hydroperoxide (CHP) and t-butyl hydroperoxide but not HO, hypochlorous acid, and diamide; this outcome was substantiated by the fact that the ohsR-deficient mutant was sensitive to OHPs but not inorganic peroxides. The DNA binding activity of OhsR was specifically 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 expression using an S-mycothiolation mechanism in vivo.
This is the first report demonstrating that the regulatory OhsR specifically sensed OHPs stress and responded to it by activating a specific ohr gene under its control using an S-mycothiolated mechanism.
谷氨酸棒杆菌是工业上生产各种 L-氨基酸的知名生产者。在发酵过程中，由于持续的不利条件产生的特定活性氧物种 (ROS)，谷氨酸棒杆菌不可避免地会遇到氧化应激。为了应对 ROS，谷氨酸棒杆菌已经开发了许多常见的基于二硫键的调节装置来控制一组特定的抗氧化基因。然而，对于蛋白质巯基与麦硫醇 (MSH) 之间的混合二硫键（S-麦硫醇化）-基于传感器，我们一无所知。此外，在烷基过氧化氢酶 CF 缺失的谷氨酸棒杆菌中，没有描述过 OhrR（有机过氧化物抗性调节剂）同源物和任何有机过氧化物还原酶（Ohr）传感器，而有机过氧化物 (OHPs)-特异性 Ohr 是核心解毒系统。
在这项研究中，我们表明谷氨酸棒杆菌 OhsR 作为 OHPs 传感器发挥作用，激活 ohr 表达。OhsR 赋予对 cumene hydroperoxide (CHP) 和 t-butyl hydroperoxide 的抗性，但对 HO、次氯酸和联氨没有抗性；ohsR 缺陷突变体对 OHPs 敏感但对无机过氧化物不敏感，这一结果得到了证实。OhsR 的 DNA 结合活性被 CHP 特异性激活。对两个半胱氨酸 (Cys125 和 Cys261) 的突变分析表明，Cys125 主要负责 DNA 结合的激活。Cys125 的氧化产生了亚磺酸 (C125-SOH)，随后与 MSH 反应生成 S-麦硫醇化，这是激活 ohr 表达所必需的。因此，OhsR 利用体内 S-麦硫醇化机制调节 ohr 表达。
这是第一项表明调节 OhsR 特异性感知 OHPs 应激并通过其控制下的特定 oh 基因的激活来响应的报告，该机制使用 S-麦硫醇化机制。
