Proteotoxic Stress Bioreporter Enables Mechanism-Informed Antibiotic Discovery.

Antibiotic resistance poses a major global health challenge, which is intensified by the decline in antibiotic discovery efforts. This highlights the urgent need for innovative antibacterial agents. Natural products remain a key source of antibiotics, yet early mechanistic insights are often lacking. Classical whole-cell screening identifies growth inhibition but provides no mechanistic information. Bioreporter strains combine bioactivity detection with preliminary mechanistic profiling; however, they remain unavailable for several promising antibiotic target areas. We developed a sensitive bioreporter that signals the induction of the heat-shock Clp-ATPase gene clpE in Bacillus subtilis. ClpE serves as a specific biomarker for proteotoxic stress, resulting from the accumulation of damaged or misfolded proteins. The self-sustained luminescence of the PclpE-lux bioreporter enables monitoring of proteotoxic stress in both solid and liquid assay formats and supports high-throughput screening. Consolidated through extensive validation with reference antibiotics, the bioreporter also identified antibacterial agents that were not previously associated with proteotoxic stress. Coupling the bioreporter with a microfractionation-based metabolomics workflow accelerated the identification of hydrophilic antibacterial metabolites from complex natural product extracts, thereby uncovering an extensive group of streptothricin derivatives, including putatively novel analogues. This study demonstrates that the PclpE-lux bioreporter is a versatile tool for mechanism-informed antibiotic discovery, enhancing the detection sensitivity and dereplication efficiency.