Developing Biosensors for Specific Assessment ofTrans-translation inPseudomonas aeruginosa

Trans -translation is a crucial bacterial process and a target for new antibiotics. We developed two Pseudomonas aeruginosa biosensor strains that detect trans -translation inhibitors by exploiting the bacterium’s natural red fluorescence, linked to protoporphyrin IX accumulation. The first biosensor monitors tmRNA-SmpB-mediated tagging, while the second serves as control for biosensor 1 by keeping track of ClpP1-related proteolysis and porphyrin biosynthesis. Validation through gene deletions and complementation confirmed their specificity. These biosensors were effective in screening antibiotics and designed inhibitors, demonstrating their potential for high-throughput identification of trans -translation inhibitors in drug-resistant P. aeruginosa .