Abstract
Azurin is a copper-containing redox protein. Previous studies have demonstrated that Azurin is involved in denitrification pathway. Recently, it was discovered to be capable of entering mammalian cells causing cell death. Nevertheless,how the biochemical properties correlates with the physiological properties of Azurin in Pseudomonas aeruginosa PAO1 is still unknown. The purpose of this study is to investigate the biological role of Azurin and its gene regulation. This study constructed an azu deletion mutant and examined its biological properteis. Significant effects on pyocyanin, pyoverdin, and biofilm production, and cytotoxicity to Int407 cells could be observed on the mutant. However, resazurin and phenol red redox assay did not reveal significant difference between PAO1 and Δazu mutant. Using β-glucuronidase reporter gene assay and electrophoretic mobility shift assay, it was revealed that transcriptional regulator PsrA does not regulate azu directly. On the other hand, RpoS, a stationary-phase sigma factor, could bind the azu promoter specifically.Based on a previous study reporting that PsrA regulates rpoS gene expression, we propose that PsrA regulates azu gene expression through RpoS. This notion was supported by the fact that the phenotypic trait of twitching motility and AHL production are consistent in ΔrpoS and Δazu. Western blot analysis illustrated that Azurin could be secreted to extracellular and ΔpsrA decreased Azurin production but not ΔrpoS. Though the function of Azurin has not been clarified, this study has established a strong basis for future analysis of the Azurin and azu regulation.