Technological Advances in Bioreporter-Based Screening for Mechanism-Informed Antibiotic Discovery in Bacillus subtilis

Abstract

The discovery of new antibacterial agents with potent and well-defined mechanisms of action has become essential for addressing the growing burden of antibiotic resistance. However, traditional screening pipelines remain constrained by low throughput, limited specificity, and labor-intensive dereplication. These limitations highlight the need for new screening strategies that accelerate antibacterial discovery and enable early mechanistic assessment. This dissertation describes the advancement of a bioreporter-based screening platform in Bacillus subtilis that combines mechanism-informed whole-cell screening with an efficient compound discovery and dereplication workflow. For this purpose, a new generation of bioreporters based on the bacterial luciferase system was developed, including a novel bioreporter specific for proteotoxic stress. In parallel, a customized workflow for the discovery and rapid dereplication of antibacterial agents was established, enabling the seamless integration of the bioreporter technology. The first study implemented the compound-resolved bioactivity-based metabolomics pipeline, which combines the bioreporter panel with high-frequency microfractionation onto microfluidic paper-analytical devices and non-targeted LC-MS/MS. This approach enabled high-throughput antibiotic screening and the identification of bioactive compounds from pure compounds, crude extracts, and producer strains, while providing early insights into their mechanisms of action. The second study expanded the mechanistic coverage of the bioreporter panel by developing a sensitive bioreporter that signals proteotoxic stress caused by the accumulation of damaged or misfolded proteins. Validation with an extensive set of antibacterial reference compounds confirmed the high specificity of the bioreporter for antibacterial agents that induce proteotoxic stress and enabled the discovery of several compounds not previously associated with this mechanism. Integration of the bioreporter with the microfractionation workflow facilitated the identification of an extensive molecular network of streptothricin derivatives from the Tübingen collection of actinomycete producer strains, including multiple putatively novel analogues. Overall, this work establishes a versatile, bioreporter-based screening platform for mechanism-informed antibiotic discovery and dereplication. By directly linking bioactivity to compound identity at early stages of screening, this approach enables efficient exploration of natural products and the discovery of new antibacterial agents.