H2O2 concentration-dependent kinetics of gene expression: linking the intensity of oxidative stress and mycobacterial physiological adaptation

Defence against oxidative stress is crucial for Mycobacterium tuberculosis to survive and replicate within macrophages. Mycobacteria have evolved multilayer antioxidant systems, including scavenging enzymes, iron homeostasis, repair pathways, and metabolic adaptation, for coping with oxidative stress. How these systems are coordinated to enable the physiological adaptation to different intensities of oxidative stress, however, remains unclear. To address this, we investigated the expression kinetics of the well-characterized antioxidant genes at bacteriostatic H2O2 concentrations ranging from 1 mM to 10 mM employing Mycolicibacterium smegmatis as a model. Our results showed that most of the selected genes were expressed in a H2O2 concentration-dependent manner, whereas a subset exhibited sustained induction or repression without dose-effect, reflecting H2O2 concentration-dependent physiological adaptations. Through analyzing the dynamics of the coordinated gene expression, we demonstrated that the expressions of the H2O2 scavenging enzymes, DNA damage response, and Fe-S cluster repair function were strikingly correlated to the intensity of oxidative stress. The sustained induction of mbtB, irtA, and dnaE2 indicated that mycobacteria might deploy increased iron acquisition and error-prone lesion bypass function as fundamental strategies to counteract oxidative damages, which are distinct from the defence tactics of Escherichia coli characterized by shrinking the iron pool and delaying the DNA repair. Moreover, the distinct gene expression kinetics among the tricarboxylic acid cycle, glyoxylate shunt, and methylcitrate cycle suggested that mycobacteria could dynamically redirect its metabolic fluxes according to the intensity of oxidative stress. This work defines the H2O2 concentration-dependent gene expression kinetics and provides unique insights into mycobacterial antioxidant defence strategies.