Regulatory time-kill sequencing maps antibiotic-specific transcriptional control of Escherichia coli persistence at...

Bacterial persistence enables a phenotypically tolerant subpopulation to survive lethal antibiotic exposure, contributing to the recalcitrance of chronic infections and facilitating the evolution of heritable resistance. Despite its importance, a systems-level, quantitative understanding of how transcriptional regulation shapes persistence kinetics has remained limited, largely because conventional time-kill assays are inherently low-throughput. Here, we develop Regulatory Time-Kill Sequencing (RTK-seq), a high-throughput functional genomics strategy that combines a pooled CRISPR interference (CRISPRi) library targeting transcription factors with time-resolved antibiotic selection and deep barcode sequencing. RTK-seq simultaneously resolves multiphasic killing trajectories for 174 Escherichia coli transcriptional regulators over five orders of magnitude in survival. The resulting profiles recapture known persistence pathways and identify previously unrecognized regulators, including rcsA, narP, and csgD, that modulate distinct phases of antibiotic killing. Additionally, applying RTK-seq across multiple antibiotic classes reveals a highly condition-dependent regulatory landscape, demonstrating that persistence is governed not by a single universal program but by drug-specific metabolic and stress responses. Overall, RTK-seq offers a scalable, versatile strategy for quantitatively mapping microbial survival networks and identifies targets for anti-persister therapies.