Dose-dependent duality: Extracellular vesicles shift from metabolic facilitators to cytotoxic effectors during anaerobic denitrification.

Extracellular vesicles (EVs) mediate critical intercellular interactions, yet their special functions in anaerobic denitrification process remain poorly understood. Utilizing the model denitrifier Pseudomonas aeruginosa PAO1, we investigated how environmental oxygen and nitrate gradients reprogram vesicular cargo and functionality. Proteomic profiling revealed a clear condition-specific cargo: aerobically generated EVs selectively packaged virulence factors and nutrient acquisition proteins. In contrast, imposing severe bioenergetic stress via anaerobic denitrification forced a radical compositional inversion, driving a targeted accumulation of with peptidoglycan-degrading enzymes. Functionally, these EVs exhibited a dose-dependent duality. At physiological EV-to-cell ratios, they facilitated transient denitrification activity and early growth in recipient cohorts. Crossing a critical density threshold, however, they transitioned to cytotoxic effectors, suppressing culture density through a cytotoxic activity that emerged post-denitrification. Notably, despite carrying a full suite of denitrification enzymes, purified EVs displayed no intrinsic activity, indicating their primary roles is that of effector carriers rather than independent catalytic units. Furthermore, a substantial fraction of these bioactive nanoparticulates survived sequential multi-barrier municipal drinking water purification processes. Together, these findings redefine EVs not merely as condition-regulated ecological regulators during severe bioenergetic stress, but also as structurally recalcitrant colloidal entities within engineered aquatic environments.