College of Life Sciences, Qufu Normal University, Qufu, Shandong, 273165, China.
Key Laboratory of Plant Genetics and Molecular Breeding, College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, Henan, 466001, China.
Microb Cell Fact. 2022 Jun 21;21(1):123. doi: 10.1186/s12934-022-01850-0.
The TetR (tetracycline repressor) family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. NCgl0886 protein, designated as AtsR, is a member of the TetR family identified in Corynebacterium glutamicum, which is conserved in several species of the genera Corynebacterium, also including the well-known pathogen C. diphtheriae. AtsR is located at no far upstream of the identically oriented ncgl0884 gene, encoding a putative multidrug efflux pump protein, and in the same operon with ncgl0887, encoding a resistance, nodulation and cell division (RND) superfamily drug exporter. However, the role of AtsR is not clearly understood.
Here we showed that dimeric AtsR directly repressed the expression of the ncgl0887-atsR operon, as well as indirectly controlled the ncgl0884 transcription. Antibiotics and toxic compounds induced the expression of ncgl0887-atsR operon. A perfect palindromic motif (5΄-TGCAA-N-TTGCA-3΄; 12 bp) was identified in the upstream region of ncgl0887-atsR operon. Electrophoretic mobility shift assays (EMSAs) demonstrated specific binding of AtsR to this motif, and hydrogen peroxide (HO) blocked binding. HO oxidized cysteine residues to form Cys123-Cys187 intermolecular disulfide bonds between two subunits in AtsR dimer, which altered its DNA-binding characteristics and caused its dissociation, thereby leading to derepression of the drug efflux protein. Deletion of ncgl0884 and ncgl0887 increased the susceptibilities of C. glutamicum for several toxic compounds, but overexpression of atsR decreased the drug tolerance of C. glutamicum.
Our study revealed that AtsR was a redox regulator that sensed oxidative stress via thiol modification. The results obtained here will contribute to our understanding of the drug response mechanism not only in C. glutamicum but also in the related bacteria C. diphtheriae.
TetR（四环素阻遏物）家族是调控细菌抗菌系统中基因表达的主要转录因子家族之一。NCgl0886 蛋白，被命名为 AtsR，是谷氨酸棒杆菌中鉴定出的 TetR 家族的成员，在棒杆菌属的几个种中保守，包括著名的病原体白喉棒状杆菌。AtsR 位于方向相同的 ncgl0884 基因的上游不远处，该基因编码一种假定的多药外排泵蛋白，与 ncgl0887 基因（编码抗性、结节和细胞分裂（RND）超家族药物外排泵）在同一个操纵子中。然而，AtsR 的作用尚不清楚。
我们发现二聚体 AtsR 直接抑制 ncgl0887-atsR 操纵子的表达，并间接控制 ncgl0884 的转录。抗生素和有毒化合物诱导 ncgl0887-atsR 操纵子的表达。在 ncgl0887-atsR 操纵子的上游区域鉴定出一个完美的回文基序（5' - TGCAA-N-TTGCA-3'；12 个碱基）。电泳迁移率变动分析（EMSA）表明 AtsR 特异性结合该基序，而过氧化氢（HO）阻断结合。HO 将半胱氨酸残基氧化形成二聚体 AtsR 中两个亚基之间的 Cys123-Cys187 分子间二硫键，改变其 DNA 结合特性并导致其解离，从而导致外排蛋白去阻遏。ncgl0884 和 ncgl0887 的缺失增加了谷氨酸棒杆菌对几种有毒化合物的敏感性，但 atsR 的过表达降低了谷氨酸棒杆菌的药物耐受性。
我们的研究表明，AtsR 是一种通过巯基修饰感应氧化应激的氧化还原调节剂。本研究结果将有助于我们不仅在谷氨酸棒杆菌，而且在相关细菌白喉棒状杆菌中理解药物反应机制。
