Аннотация:Due to dramatic increase of the resistance to well-known antibi-otics, discovery of new antibacterial agents becomes one of theprincipal goal of the modern biochemistry. For acceleration ofdrug discovery process, we created a simple, reliable and cost-effective system for high-throughput identification of a potentialantibiotic mechanism of action. Two fluorescent proteins withinducible expression were used for double reporter system, whichallows not only antimicrobial activity detecting, but also forsimultaneous sorting of potential antimicrobials into those thatcause ribosome stalling, and others that induce SOS response dueto DNA damage. The mechanism of action determination is nec-essary for rational improvement of the found compound; rapididentification of drug target allows significantly decrease time and coast of the molecule optimization process. By means of this sys-tem, more than 100 thousands of different synthetic and naturalcompounds were tested, and several new antibacterial agentswere found. This work was supported by the RSF grant 17-74-30012.