Abstract: Objective To study the regulatory effect of arginine regulatory protein ArgR on the gene expression of type Ⅵ secretion system (T6SS) in P. aeruginosa (Pseudomonas aeruginosa). Methods The ArgR protein expression vector with SUMO tag was constructed, and the ArgR protein of P. aeruginosa was expressed heterologously by using E. coli DH5α. The gel electrophoretic mobility shift assay (EMSA) was used to explore the interaction between ArgR and the promoter of T6SS-related gene hsiA2. Hcp2-Flag expression vector with FLAG tag was constructed. Western blotting and qRT-PCR assays were used to detect the regulatory effect of argR gene on the T6SS structural gene hcp2. Results The soluble ArgR protein was obtained by cutting off the label by restriction enzymes. EMSA analysis showed that there was an interaction between ArgR protein and the hsiA2 promoter. The results of Western blotting and qRT-PCR showed that the expression level of hcp2 was significantly increased in the mutants lacking argR. Moreover, the expression level of hcp2 in the mutants could be returned to an equivalent level as in the wild-type bacteria after argR expression level was supplemented. Conclusion This study shows that ArgR can bind to the promoter of hsiA2 and negatively regulate the expression of T6SS-related genes. 

Key words: Pseudomonas aeruginosa, ArgR, type Ⅵ secretion system, transcriptional regulation, gene expression