Fig. 5.
WalK-activated WalR bound to agr promoter region to control the expression of agr in S. aureus. (A) β-galactosidase assay. The pOS1-agrP reporter plasmid was transformed into S. aureus XN108 and XN108-R. The LacZ activity was detected and represented as mean ± SD (n = 3). Statistical significance was calculated by Student’s t-test. ***P < 0.001. (B) Predicted WalR-binding site in the promoter regions of agr P2. (C) Mutation of the predicted WalR-binding site in agr promoter regions for EMSA experiment. Interaction between agr promoter region (agr-P2) and (D) WalK-activated WalR proteins or (E) WalK(S221P)-activated WalR proteins was detected. The unlabeled DNA fragment of mecA promoter region (50 pM) was used for non-specific competition of the interaction. (F) EMSA with agr promoter mutant (agr-P2M), the sle1 promoter DNA fragment (10 pM) was used as positive control. (G) Evaluation of gray value of the bound probe in each lane using ImageJ software. The value of the free probe in the first lane (0 pM WalR protein) was used as loading control (LC), and the relative gray values of the bound probe to LC in other lanes were calculated and indicated.