A LexA-independent redox-based transcriptional regulation to DNA damage response system in mycobacteria

The bacterial DNA damage response (DDR) system promotes survival under stressful conditions and facilitates the emergence of drug-resistant mutations. The DDR system in Mycobacterium tuberculosis is primarily governed by LexA-mediated transcriptional repression. Although some other regulators, such as PafBC and McdR have been reported, whether additional regulatory inputs contribute to DDR remains to be characterized.

By performing a bacterial one-hybrid screen, we identified several transcriptional regulators, including Rv2282c, Rv1985c and Rv1990c, that activate DDR gene expression and inhibit cell growth. Promoter truncation analysis revealed that these regulators act through sequences distinct from the canonical LexA-binding motif. No specific interaction between these regulators and DDR gene promoters was detected by EMSAs, indicating an indirect regulatory mechanism. RNA-seq analysis revealed that approximately 25% genes were differentially expressed upon overexpression of each of these regulators. Among these, tpx, which is responsible for oxidative stress defense, was consistently repressed by all of the three DDR-activating regulators but not by control regulators. Complementation of Tpx alleviated ROS accumulation and restored cell growth in strains overexpressing these regulators, suggesting that the DDR activation is linked to redox imbalance caused by Tpx repression.

Our data demonstrate that Rv2282c, Rv1985c, and Rv1990c activate the DDR through perturbation of redox homeostasis, revealing a previously unrecognized, LexA-independent regulation pathway in mycobacteria.