Cost adjusted hierarchical defense strategies enables stratified oxidative stress tolerance

Abstract Organisms employ diverse adaptive strategies to withstand environmental stresses, yet how bacteria tailor their responses to different magnitudes of the same stressor remains poorly understood. This question is particularly salient for oxidative stress, which varies substantially across physiological niches and exerts distinct selection pressures on colonizing bacterial populations. Here, we used four separate adaptive laboratory evolutions of Escherichia coli across multiple paraquat concentrations and genetic backgrounds to dissect how cells adapt to varying levels of superoxide stress. Integrating multi-omic analyses with tailored genome-scale metabolic modeling, we identify two fundamentally distinct tolerance strategies based on flux control and cellular resource optimization. Under low superoxide stress, blocking polyamine transporter that is hijacked for paraquat influx suffices to maintain redox balance. In contrast, higher stress levels activate an energetically demanding program involving enhanced detoxification of reactive radicals and active efflux that allows cells to maintain higher aerotype. We further demonstrate that flux regulation establishes a primary defense layer, upon which metabolic repair systems act to provide additional fitness advantages. Together, our findings reveal how E. coli differentially engages modular stress-response programs depending on stress magnitude, offering generalizable insights into the principles governing dynamic bacterial adaptation.