Loss of PA0619 Enhances Crude Rhamnolipid Extract Yield and Attenuates Virulence in P. aeruginosa

Rhamnolipids are versatile biosurfactants with immense industrial potential, yet their scalable production in P. aeruginosa is constrained by limited yields. To identify novel regulators, we constructed a rhlAB-luxCDABE reporter strain to screen a genome-wide transposon insertion library containing 30,000 mutant colonies and identified 53 candidate genes whose disruption resulted in more than two-fold changes in the normalized rhlAB reporter activity. Among the four selected candidates with 15-fold variation in rhlAB reporter activity, PA0619 disruption consistently enhanced PrhlAB promoter activity and shifted P. aeruginosa toward a stronger extracellular biosurfactant-associated output. Furthermore, ΔPA0619 also increased crude rhamnolipid extract yield and elevated rhlAB promoter activity. In parallel, ΔPA0619 showed reduced pyocyanin production, biofilm formation, and tissue damage in a Chinese cabbage infection model. These findings identify PA0619 as a genetic locus connecting rhlAB expression, extracellular biosurfactant-associated phenotypes, and selected virulence-related traits in P. aeruginosa.