Abstract
Background: The current rise in multidrug-resistant pathogens highlights the urgent need for the discovery of novel antibacterial agents with potential clinical applications. A considerable proportion of these developed resistances may be attributable to the intrinsic response of bacteria to antibiotic-induced stress conditions in the environment. Consequently, the identification and characterization of genetic alterations in physiological processes in response to antibiotics represent promising strategies for the discovery and characterization of naturally produced novel antibacterial agents. This study investigated the antimicrobial activity of an antimicrobial active isolate Actinomadura coerulea derived from a meerkat fecal sample. Methods: The production of secondary metabolites that potentially compromise bacterial cell wall integrity was confirmed by the induction of promoter activity in whole-cell biosensors in which an antibiotic-inducible promoter was fused to the luciferase cassette. During plate-based biosensor assays, we identified naturally resistant Bacillus subtilis colonies growing in the zone of inhibition around A. coerulea colonies. After these successive rounds of selection, highly resistant spontaneous B. subtilis mutants had evolved that were subjected to whole-genome sequencing. Results: Non-silent mutations were identified in pssA, which encodes a phosphatidylserine synthase; mdtR, as a gene for the repressor of multidrug resistance proteins, and yhbD, whose function is still unknown. A new cinnamycin-like molecule, coerumycin, was discovered based on the physiological role of PssA and comprehensive genomic analysis of A. coerulea. Additional experiments with cell extracts containing coerumycin as well as the cinnamycin-like compound duramycin confirmed that the interaction between coerumycin and the bacterial cell envelope is inhibited by a loss-of-function mutation in pssA. Conclusion: Our approach demonstrates that combining the exploration of niche habitats for actinomycetes with whole-cell biosensor screening and characterization of natural resistance development provides a promising strategy for identifying novel antibiotics.