Pseudomonas aeruginosa PAO1 was shown to have three acyl homoserine lactone acylases encoded by pvdQ, quiP and pa0305. One additional gene, pa1893, still remains to be proved to be another penicillin acylase protein. Initial studies on PAO1 pa0305 deletion strains suggested that triple mutant (ΔpvdQ, ΔquiP, Δpa0305)showed a higher 3-oxo-C12-HSL accumulation compared to the double mutant PAO1 (ΔpvdQ, ΔquiP). When PA0305 was checked for its activities, the activity towards 3- oxo-C12-HSL was recorded with kcat/Km 7.8x10-4 M-1s-1. However, when the pa0305 was over expressed in PAO1, it did not show quenching activity as high as when it was done in vitro. mRNA quantification showed that pa0305 was expressed at low amounts during the late logarithmic phase from the growth curve of PAO1 and Δpa0305. It suggested the existence of a regulation controlling the pa0305 expression in cells. In order to check when the pa0305 was expressed, a plasmid containing the interregional sequence (between pa0305 and pa0306) was introduced into PAO1 and Δpa0305. This interregional sequence was cloned upstream the lacZ gene reporter. Every two hours along the growth each culture was analyzed for the β-galactosidase and LasA elastase activities and the pyocyanin accumulation. The results showed that the interregional activated during the early logarithmic phase, followed by the de novo production of pyocyanin and de novo activity of LasA elastase. Compared to the wild type, the absence of pa0305 in the PAO1 chromosome led to a greater accumulation of pyocyanin and to a stronger activity of LasA elastase. The increase was occurred during the log phase where the increase of virulence factors at late stationary phase was doubled compared to the wild type. These results showed that the activation of putative promoter of pa0305 in AO1chromosome lead to the decrease of virulence factors.