ABSTRACTInducible gene expression systems are powerful genetic tools to study bacterial physiology, probing essential and toxic gene functions, gene dosage effects, and overexpression phenotypes. For the opportunistic human pathogenPseudomonas aeruginosa, dedicated inducible gene expression systems are scarce. In the current study, we developed a minimal synthetic 4-isopropylbenzoic acid (cumate)-inducible promoter, called PQJ, that is tunable over several orders of magnitude. This was achieved by combining semirandomized housekeeping promoter libraries and control elements from thePseudomonas putidastrain F1cym/cmtsystem with powerful fluorescence-activated cell sorting (FACS) to select functionally optimized variants. Using flow cytometry and live-cell fluorescence microscopy, we demonstrate that PQJresponds rapidly and homogenously to the inducer cumate in a graded manner at the single-cell level. PQJand cumate are orthogonal to the frequently used isopropyl β-d-thiogalactopyranoside (IPTG)-regulatedlacIq-Ptacexpression system. The modular design of the cumate-inducible expression cassette together with the FACS-based enrichment strategy presented here facilitates portability, thus serving as a blueprint for the development of tailored gene expression systems for a wide range of bacteria.IMPORTANCEReverse genetics is a powerful approach to study bacterial physiology and behavior by relying on well-developed genetic tools, such as inducible promoters. For the human pathogenPseudomonas aeruginosa, well-characterized inducible promoters are scarce. In the current work, we used a synthetic biology-based approach to develop a cumate-inducible promoter forP. aeruginosa, termed PQJ, that shows excellent induction properties at the single-cell level. This genetic tool provides the means for qualitative and quantitative gene function studies describingP. aeruginosa’s physiology and virulencein vitroandin vivo. Because this synthetic approach to constructing species-specific inducible promoters is portable, it can serve as a blueprint for similar tailored gene expression systems in bacteria largely lacking such tools, including, for example, representatives of the human microbiota.