College of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China.
College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, Henan 466001, China.
Biochem J. 2019 Nov 15;476(21):3141-3159. doi: 10.1042/BCJ20190320.
MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)-uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882-ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR-uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.
MarR（多重抗生素耐药调节剂）蛋白是一种在谷氨酸棒杆菌中普遍存在的转录调节剂家族。了解谷氨酸棒杆菌中 MarR 同源物的生理和生化功能集中在基于半胱氨酸氧化的氧化还原感应和涉及底物代谢的调节剂上。在这项研究中，我们表征了谷氨酸棒杆菌 MarR 型调节剂 CarR（谷氨酸棒杆菌抗生素反应调节剂）的与应激相关的配体结合功能。我们证明 CarR 负调控 carR（ncgl2886）-uspA（ncgl2887）操纵子和相邻的、反向取向的基因 ncgl2885 的表达，该基因编码假定的脱酰酶 DecE。我们还表明，CarR 直接激活位于 carR 上游的反向排列的 ncgl2882-ncgl2884 操纵子的转录，该操纵子编码肽聚糖合成操纵子（PSO）。应激相关配体如青霉素和链霉素的添加诱导了体内 carR、uspA、decE 和 PSO 的表达，并减弱了 CarR 与体外操纵子 DNA 的结合。重要的是，应激反应诱导的 carR、uspA 和 PSO 基因表达的上调与细胞对β-内酰胺抗生素和芳香族化合物的抗性相关。CarR 中的六个高度保守残基被发现强烈影响其配体结合和转录调控特性。总的来说，结果表明 CarR 的配体结合诱导其从 carR-uspA 启动子解离，以解除 carR 和 uspA 的转录抑制。无配体的 CarR 还激活 PSO 的表达，这反过来又有助于谷氨酸棒杆菌的应激抵抗。结果表明，CarR 在谷氨酸棒杆菌中的应激反应机制是通过蛋白质的配体诱导构象变化发生的，而不是通过基于半胱氨酸氧化的硫醇修饰。
