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. 2011 Jul 28;7(7):e1002205. doi: 10.1371/journal.pgen.1002205

Figure 3. Fur binds to the EAEC sci1 T6SS promoter in vivo and in vitro.

Figure 3

(A) Fur Titration assay (FURTA). H1717 reporter cells (fhuF-lacZ) carrying the empty vector or the vector bearing the sci1, sci2, or cir promoters, or the fur1 or fur2 sequences were spotted on MacConkey plates (upper panel) or on MacConkey plates supplemented with FeSO4 (30 µM; lower panel). A lacZ+ phenotype reports a derepression of the fhuF-lacZ reporter fusion by titration of the Fur protein bound to the fhuF promoter. (B) Electrophoretic mobility shift assay of the sci1 promoter (upper panel) or of the fur1 (middle panel) or fur2 (lower panel) sequences using purified Fur (lane 1, no protein; lane 2, 0.5 nM; lane 3, 2 nM, lane 4, 5 nM, lane 5, 20 nM) in presence of FeCl3 or in presence of EDTA (lane 6, Fur at 20 nM) or using purified NtrC transcriptional activator (lane 7, 50 nM). Controls include Fur shift assays of the Fur-dependent cir promoter (lane 8, no protein; lane 9, Fur at 5 nM) or of the Fur-independent sci2 promoter (lane 10, 20 nM). (C) Competition experiments for Fur binding (lane 1, no protein; lanes 2–6, Fur at 20 nM) with duplex consensus Fur- (lane 3, molecular ratio sci1:fur box 1∶2; lane 4, molecular ratio 1∶10) or σ54-binding sequence (lane 5, molecular ratio sci1:σ54-box 1∶2; lane 6, molecular ratio 1∶10). (D) Binding of the Eσ70 RNA polymerase holoenzyme (RNAP) (lanes 1, 4, 7 and 9, no RNAP; lanes 2 and 5, RNAP 0.5 unit; lanes 3, 6, 8 and 10, RNAP 2 units) on the sci1 or control cir promoter pre-incubated (+) or not (−) with Fur (20 nM). Fur-DNA, (Fur)2-DNA, and RNAP-DNA complexes are indicated by *, **, and ǂ respectively.