e In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.T he spread of antibiotic resistance genes among pathogenic bacteria is leading to a gradual decrease in the efficiency of known antibiotics. Substantial efforts have been invested in platforms for new antibiotic development (1). High-throughput screening (HTS) is a major method for the discovery of new chemical scaffolds for drug discovery. However, in the search for new antibiotics, HTS demonstrated low efficiency (for a discussion, see references 2 and 3). Acceleration of the antibiotic development pipeline demands increased efficiency in the identification of mechanisms of action with both HTS of chemical libraries and screening of natural compounds. Ideally, the mechanism of action should be determined while screening for antibacterial activity. One of the major challenges in high-throughput screening is the development of a cost-effective procedure that could maximize information output while concomitantly minimizing the number of pipetting steps and the reagent costs.The most efficient way to reveal the mechanism of action is the application of reporter strains (for a recent review, see reference 4). However, the majority of the reporter strains developed thus far aim to identify a narrow group of chemically related compounds, such as tetracyclines (5), macrolides (6, 7), or ␤-lactams (8). Broader-spectrum reporters based on stress response promoters are also available (9-11). A combination of several reporter strains could help to classify more mechanisms of action, but that would require multiple experiments for a single substance being tested.The majority of antibiotics currently in clinical use target the cell wall, DNA, or protein biosynthesis. For the latter two mechanisms ...