Developing Biosensors for Specific Assessment of <i>Trans</i>-Translation in <i>Pseudomonas aeruginosa</i>

We developed two <i>Pseudomonas aeruginosa</i> biosensors to detect <i>trans</i>-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 <i>trans</i>-translation or proteolysis activity. These biosensors offer a promising tool for high-throughput screening of <i>trans</i>-translation inhibitors in <i>P. aeruginosa</i>.