A Reporter System for Assessment of Transcription from Divergently Oriented Promoters in Pseudomonas putida

Pseudomonas putida is a metabolically versatile bacterium widely used in industrial biotechnology and synthetic biology. However, the lack of rapid, sensitive, and noninvasive tools for monitoring gene expression in P. putida limits the opportunities to study its gene regulation. We developed a plasmid-based dual-reporter system optimized for P. putida, which enables simultaneous monitoring of gene expression from promoter areas that contain divergently orientated promoters. Two fluorescent proteins (SYFP2 and Scarlet-I3) were selected for a reporter based on their compatibility with the intrinsic autofluorescence of P. putida and their detectability in LB medium. We engineered plasmid backbones containing the BBR1 and RK2 origins of replication and incorporated the toxin-antitoxin module hok-sok to ensure plasmid maintenance without antibiotic selection, making it possible to use this system to quantify gene expression in both planktonic and sessile (biofilm) states. Additionally, we created reporter systems with fused reporter genes with protein half-life decreasing tags, allowing dynamic assessment of transcriptional activity. Using confocal microscopy, we demonstrated spatially distinct expression patterns of biofilm-related genes (e.g., lapF) within mature biofilms. We also tested excludon-based transcriptional repression of a reporter gene in P. putida using this system, but observed limited efficiency under the tested conditions.