Figure 5

ArcA(-P) Binding to selected promoters by EMSA. (A). Overproduced and purified recombinant S. oneidensis His₆-ArcA from E. coli BL21 cells. (B). Interaction of so1661 promoter DNA with S. oneidensis His₆-ArcA. The probe was prepared by PCR with SO1661-EMSA-F (³³P end-labeled) and SO1661-EMSA-R primers (Table S4 in additional file 5). The EMS assay was performed with 2 nM ³³P end-labeled probes and various amounts of ArcA (left panel) or ArcA-P proteins (right panel). The protein concentrations for lanes 1–9 are 0, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 4.0, 4.0 μM, respectively. Non-specific competitor DNA, (2 μg poly dI·dC), was added (lane 8) and specific competitor (10 μM unlabeled SO1661 probe) was added (lane 9). (C). The binding assay was performed in the presence of 0, 1, or 2 μM ArcA-P and 2–5 nM radiolabeled promoter DNA 0.2 μg/μl poly(dI·dC) was used in all these binding reactions to block non-specific interactions. Promoter region of so0011 (gyrB) was included as negative control. The phosphorylation of the ArcA protein was done with carbamoyl phosphate.