Fig. 3

Identification of the target genes regulated by IsaF. (A, B) Transcriptional analysis of isa genes in the isaF overexpression strain (CPCC 204095/pL-isaF), and isaF knockout strain (FKO) and genetic complementary strain (FKO/pL-isaF) by RT-qPCR analysis. The relative mRNA level of the target genes was normalized to the principal sigma factor gene hrdB. The relative expression level of each sample was represented as the value related to the control strain CPCC 204095/pSET152 or CPCC 204095. Values are presented as mean ± SEM (two biological repeats for each strain). (C) Chromatin immunoprecipitation (ChIP) -PCR analysis of Flag-tagged IsaF. PCR using primers flanking target promoter regions was performed with immunoprecipitated DNA from CPCC 204095/FKO/pL-isaF-Flag (lanes 4–6) and negative control DNA from CPCC 204095/FKO/pSET152 (lanes 1–3). Total DNA prior to immunoprecipitation (lanes 1 and 4) was used as positive control for PCR. DNA treated with anti-Flag antibody (lanes 2 and 5) or with IgG antibody (lanes 3 and 6) was analyzed by PCR. (D) IsaF consensus binding sites revealed by MEME analysis. The data for this logo consist of four IsaF binding sites including the promoter regions of isaD, isaH, isaL, and isaS. The height of each letter is proportional to the frequency of the base. The grey shades and arrows indicate forward repeats. (E) Electrophoretic mobility shift assays (EMSAs) were used to analyze the interaction of the promoter regions with purified His-SUMO-tagged IsaF. The amounts of IsaF in the reaction are indicated above each lane. The “competitor” lane contains 10 µl IsaF with a 200-fold excess of unlabeled specific competitor