Fig. 4

Identification of the target genes regulated by IsaJ. (A, B) Transcriptional analysis of isa genes in the isaJ overexpression strain (CPCC 204095/pL-isaJ), and isaJ gene knockout strain (JKO) and genetic complementary strain (JKO/pL-isaJ) by RT-qPCR analysis. The relative mRNA level of the target genes was normalized to the principal sigma factor gene hrdB. The relative expressional 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) ChIP-PCR analysis of Flag-tagged IsaJ. PCR using primers flanking target promoter regions were performed with immunoprecipitated DNA from CPCC 204095/JKO/pL-isaJ-Flag (lanes 4–6) and negative control DNA from CPCC 204095/JKO/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) EMSA to analyze the interaction of the promoter regions with purified IsaJ. The amounts of IsaJ in the reaction is indicated above the lanes. The “competitor” lane contains 10 µl of IsaJ with a 200-fold excess unlabeled specific competitor