Disinfection-driven rewiring of bacterial regulatory networks through c-di-GMP signaling in engineered water systems

Disinfection is indispensable for drinking water safety, yet its long-term evolutionary consequences for surviving microorganisms remain poorly resolved. Here, integrating macro-transcriptomics with experimental evolution, we demonstrate that sustained sublethal chlorination drives systematic rewiring of bacterial regulatory architecture, rather than simple selection for resistance. Chronic chlorine exposure consistently depleted intracellular cyclic di-guanosine monophosphate (c-di-GMP) and reprogrammed chemotaxis, motility, and host-interaction-associated phenotypes across independently evolved lineages, despite divergent genetic trajectories. Comparative genomic and transcriptomic analyses revealed that this regulatory shift is underpinned by extensive plasmid remodeling and expansion of toxin-antitoxin systems. Importantly, chemical restoration of intracellular c-di-GMP partially reversed chlorine-associated behavioral changes, establishing a causal link between disinfectant-driven signaling reprogramming and functional adaptation. Together, our findings identify regulatory and signaling rewiring as a central evolutionary outcome of long-term chlorination and highlight microbial signaling networks as potential targets for mitigating unintended adaptive responses in engineered water systems.