Elucidating the Mechanism of Temporal Adaptation to Hydrogen Peroxide-Induced Oxidative Stress in Corynebacterium...

Corynebacterium glutamicum serves as a pivotal industrial chassis for biomanufacturing and an ideal model for studying the phylogenetically related pathogen Mycobacterium tuberculosis. Oxidative stress poses a critical challenge to microorganisms during aerobic industrial processes and immune cell-mediated antibacterial killing by perturbing cellular redox homeostasis, affecting central metabolism, and damaging the integrity of biomacromolecules. However, the intricate mechanisms underlying the dynamic defence of C. glutamicum, despite previous transcriptomic studies on acute and adaptive responses to oxidative stresses, remain largely unclear, hindering strain engineering for industrial applications and the development of effective antimicrobial treatments. In this study, the susceptibility of C. glutamicum to hydrogen peroxide (H₂O₂) was evaluated, and the inhibitory dynamics of H₂O₂ were characterised through viable cell counting. RNA sequencing (RNA-seq) was employed to analyse gene expression changes after exposure to 720 mM H₂O₂. The treatment induced differential expression of 966 and 787 genes at 2 and 6 h, respectively, reflecting perturbations across a broad array of pathways, including (i) enhanced H₂O₂ and peroxide scavenging, mycothiol biosynthesis, and iron chelation; (ii) repressed central metabolism and enhanced anaplerosis; (iii) elevated sulphur assimilation; (iv) altered amino acid biosynthesis; and (v) altered transcriptional regulation in response to oxidative stress. Further validation by overexpression of ahpD, cysN, and exogenous supplementation with l-methionine and l-cysteine significantly enhanced bacterial tolerance to H₂O₂. Overall, this study provides the most comprehensive analysis to date of temporal cellular adaptation to H₂O₂ stress in C. glutamicum, establishing a foundation for future applications in both biomanufacturing and antimicrobial research.