H-031. Structural and Functional Analysis of Vibrio vulnificus Quorum Sensing Regulator, SmcR

B. Kim1, Y. Park1, M. Kim2, S. Choi1;
1Seoul Natl. Univ., Seoul, REPUBLIC OF KOREA, 2Systems Microbiology Res. Ctr., KRIBB, Daejeon, REPUBLIC OF KOREA.

Vibrio vulnificus SmcR is a homologue of Vibrio harveyi LuxR which plays a role in quorum sensing II system. Recently, SmcR has been reported as a novel global regulator controlling numerous genes contributing to pathogenesis. Previous chemical cross-linking study give the impression that SmcR exists as a dimer. Recently identified crystallographic structure is consistent with this presumption. In this study, a set of mutants of SmcR was constructed and then dimerizing activity as well as DNA binding activity was analyzed using bacterial two hybrid system and EMSA. Among 11 mutations in C-terminal domain, mutations with Y171A substitution or L165A and Y193A double substitutions resulted in most detrimental effects on dimerization. Since these residues are located in the putative dimeric region of α-helix 9 or α-helix 10, deletion mutants that lack α-helix 10 or both α-helix 9 and α-helix 10 were constructed and examined. The result reveals that dimerizing activities of the deletion mutants were 5~20% level of that of WT. The in vivo activities of WT SmcR and dimerization defective SmcR were compared upon SmcR target promoter, PvvpE using PvvpE::lacZ fusion. The result indicates that dimerizing activity is also important to activate target promoter. To analyze DNA binding domain proposed on crystallographic structure, 8 substitution mutations and one deletion mutation were introduced into N-terminal DNA binding domain of SmcR, and the resulting mutant SmcRs were purified. EMSA with the vvpE upstream region shows that mutant with the deletion of 2nd~11th residues and mutant with R11A were completely defective for DNA binding. In addition, H39A or R60A substitution mutant proteins showed less reduction of the electrophoretic mobility. These results suggest that N-terminal domain is essential for DNA binding while C-terminal domain is mainly involved in its dimerization.