Selection favors cost-ordered adaptive mutational paths in a stress-magnitude-dependent manner.

Chronic exposure to stress requires adaptive strategies beyond canonical regulatory mechanisms. Stress varies, both qualitatively and quantitatively, across physiological niches and exerts distinct selection pressures on colonizing bacteria. Bacteria employ diverse defense strategies to withstand various stressful conditions, yet how they tailor their responses to different magnitudes of the same stressor remains poorly understood. We used multiple adaptive laboratory evolution experiments of Escherichia coli across varying paraquat concentrations and genetic backgrounds to dissect adaptive strategies at different levels of stress. Integrating multi-omic analyses with a tailored genome-scale metabolic model-based parametrized cost calculations, we identify two fundamentally distinct tolerance mechanisms. Under low-paraquat stress, blocking the polyamine transporter that is reported to be hijacked for paraquat influx suffices to maintain optimal growth. In contrast, higher stress levels activate an energetically demanding program involving enhanced detoxification and efflux. The transport flux regulation establishes a primary defense layer, upon which metabolic repair systems provide additional fitness advantages. The stress magnitude-dependent differential engagement of previously reported paraquat tolerance approaches offers insights into the principles governing dynamic bacterial adaptation.