Qiao Jiaxin, Zhu Weiwei, Du Dongdong, Morigen Morigen
Inner Mongolia Key Laboratory for Molecular Regulation of the Cell, School of Life Sciences, Inner Mongolia University, Hohhot 010070, China.
State Key Laboratory of Vaccines for Infectious Diseases, Xiang-An Biomedicine Laboratory, Department of Laboratory Medicine, School of Public Health, Xiamen University, Xiamen 361102, China.
Int J Mol Sci. 2025 Mar 25;26(7):2968. doi: 10.3390/ijms26072968.
Oxidative stress is prevalent in organisms, and excessive oxidative damage can trigger cell death. Bacteria have evolved multiple pathways to cope with adverse stress, including the regulation of the cell cycle. Previous studies show that non-lethal exposure to HO and mutations in antioxidant enzymes suppress replication initiation in . The existence of common regulatory factors governing replication initiation across diverse causes-induced oxidative stress remains unclear. In this study, we utilized flow cytometry to determine the replication pattern of , and found that oxidative stress also participated in the inhibition of replication initiation by a defective iron regulation (-- deletion). Adding a certain level of ATP promoted replication initiation in various antioxidant enzyme-deficient mutants and the ΔΔΔ mutant, suggesting that low ATP levels could be a common factor in the inhibition of replication initiation by different causes-induced oxidative stress. More potential common factors were screened using proteomics, followed by genetic validation with HO stress. We found that oxidative stress might mediate the inhibition of replication initiation by interfering with the metabolism of glycine, glutamate, ornithine, and aspartate. Blocking CcmA-dependent cytochrome biosynthesis, deleting the efflux pump proteins MdtABCD and TolC, or the arabinose transporter AraFHG eliminated the replication initiation inhibition by HO. In conclusion, this study uncovers a common multifactorial pathway of different causes-induced oxidative stress inhibiting replication initiation. Dormant and persistent bacteria exhibit an arrested or slow cell cycle, and non-lethal oxidative stress promotes their formation. Our findings contribute to exploring strategies to limit dormant and persistent bacterial formation by maintaining faster DNA replication initiation (cell cycle progression).
氧化应激在生物体中普遍存在，过度的氧化损伤会触发细胞死亡。细菌已经进化出多种途径来应对逆境胁迫，包括细胞周期调控。先前的研究表明，非致死性暴露于羟基自由基（HO）和抗氧化酶突变会抑制大肠杆菌（E. coli）的复制起始。目前尚不清楚在多种原因诱导的氧化应激中，是否存在共同的调控因子来控制复制起始。在本研究中，我们利用流式细胞术来确定大肠杆菌的复制模式，发现氧化应激也参与了铁调控缺陷型（ΔfurΔfhuAΔtonB缺失）对复制起始的抑制作用。添加一定水平的ATP可促进各种抗氧化酶缺陷型突变体和ΔfurΔfhuAΔtonB突变体的复制起始，这表明低ATP水平可能是不同原因诱导的氧化应激抑制复制起始的一个共同因素。我们使用蛋白质组学筛选了更多潜在的共同因素，随后用HO胁迫进行了基因验证。我们发现氧化应激可能通过干扰甘氨酸、谷氨酸、鸟氨酸和天冬氨酸的代谢来介导对复制起始的抑制。阻断CcmA依赖的细胞色素生物合成、删除外排泵蛋白MdtABCD和TolC或阿拉伯糖转运蛋白AraFHG可消除HO对复制起始的抑制作用。总之，本研究揭示了不同原因诱导的氧化应激抑制复制起始的一个共同的多因素途径。休眠和持留菌表现出细胞周期停滞或缓慢，非致死性氧化应激促进它们的形成。我们的研究结果有助于探索通过维持更快的DNA复制起始（细胞周期进程）来限制休眠和持留菌形成的策略。
