Figure 4

ChIP-Seq validation for HrcA and PhoB. (a) Binding of His₆-HrcA on promoters identified by ChIP-Seq (P_groES1, P_groES3, P_hrcA, P_phoH and P_{wcw_1080}). The promoters were amplified by PCR using specific primers coupled to Cy5. DNA fragments were incubated in the absence (−) or in presence of an increasing concentration of His₆-HrcA (50, 250 and 1250 nm) and analysed by EMSA. (b) EMSA analysis showing the binding of His₆-HrcA to a synthetic fragment harbouring the HrcA-triple consensus. As a negative control, we used an analogous triple-repeat fragment harbouring the predicted NrdR consensus motif. (c, d) In vivo binding of HrcA on the triple consensus and on its own promoter (P_hrcA) using lacZ reporter gene in E. coli. HrcA was expressed from an arabinose-inducible promoter on pBAD22 (Guzman et al., 1995). As a negative control, the expression empty vector was used. Data are means±standard deviation of three biological triplicates. Panel (c) absolute values, panels (d) and (e) normalized value according to the empty plasmid (set as 100%). When HrcA was expressed, a decrease of 40% of the LacZ activity was observed, suggesting that HrcA is able to bind and to repress the expression of lacZ. (e) In vivo binding of PhoB (expressed from pBAD22) on three promoters (P_{wcw_0193}, P_{wcw_1016} and P_{wcw_1714}) identified by ChIP-Seq and on the PhoB-triple consensus using lacZ reporter gene in E. coli. A decrease of the LacZ activity was observed for the four lacZ promoter-probe plasmids.