Electrophoretic Mobility Shift Assay of Bacterial Proteins

Binding of proteins onto DNA probes was determined with the electrophoretic mobility shift assay (EMSA) as described previously (Lv et al., 2018 (link)). PhcA and PhcR proteins were purified from the pET-32a based protein expression system in E. coli BL21 (DE3) and subjected for the binding reaction with biotinylated DNA probes using the LightShift Chemiluminescent EMSA Kit following the manufacturer’s protocol (Thermo, United States). Promoter regions of the rmyA operon (rmyA upstream region of ATG site about 375 bp) and impH gene (424 bp of its coding sequence) were PCR amplified and labeled by biotin using the Biotin 3′ End DNA Labeling Kit (Thermo, United States). Note that promoter DNA of impH gene, which encodes one of the type VI secretion proteins in R. solanacearum, was selected as a system control. The gels were stained by SYBR GOLD dye and subjected to screening on a phosphor screen (LAS600, GE Healthcare).