Developing Biosensors for Specific Assessment of Trans-Translation in Pseudomonas aeruginosa

We developed two Pseudomonas aeruginosa biosensors to detect trans-translation inhibitors in this medically relevant pathogen. These biosensors leverage the red fluorescence produced by the accumulation of protoporphyrin IX, the substrate of ferrochelatase. The first biosensor monitors tmRNA-SmpB-mediated tagging and degradation of ferrochelatase, while the second serves as a control by tracking ClpP1-mediated proteolysis and porphyrin biosynthesis. Both biosensors were tested in wild-type and mutant backgrounds, and red fluorescence was measured relative to absorbance at 600 nm in microtiter plates. The results confirmed a link between fluorescence and trans-translation or proteolysis activity. These biosensors offer a promising tool for high-throughput screening of trans-translation inhibitors in P. aeruginosa.