Fig 4. Alanine substitutions in the linker peptide alter the kinase activity of SaeS.

(A) Expression of the alanine substitution mutants of SaeS analyzed by Western blot. An equal number of the cells were harvested at exponential growth phase. LC, loading control. (B) The effect of the alanine substitution mutations on the transcription of the low affinity target Pcoa and blood coagulating activity. The promoter activity of Pcoa was measured as described for Fig 2C. Coagulation activity was measured by mixing bacterial culture with rabbit plasma at 37°C. Coagulation time is indicated under each result. Error bars represent the standard error of the mean. WT, wild type; AU, arbitrary unit; ND, not detected. (C) The effect of the alanine substitution mutations on the transcription of the high affinity target Phla and the production of alpha-hemolysin (Hla). The production of Hla was examined by Western blot analysis and hemolysis assay on a rabbit blood agar. In the hemolysis assay, red zone indicates complete hemolysis. In the bar graphs, the error bars indicate the standard error of the mean. GFP expressions were compared with that of the strain carrying wild type SaeS, and statistical significance was assessed by unpaired, two-tailed student’s t-test. ***, p < 0.001.