Fig 4. Electrophoretic mobility shift assays showing the ability of FurA to bind in vitro the promoter regions of novel direct target genes.

DNA fragments free (1) or mixed with recombinant FurA protein at concentration of 300 nM (2), 500 nM (3) and 700 nM (4) in the presence of Mn²⁺ and DTT were separated on a 4% PAGE. The impact of the metal co-regulator (removing Mn²⁺/adding EDTA) and reducing conditions (removing DTT) on the in vitro affinity of FurA (700 nM) to each target are also showed. The promoter region of nifJ gene was used as non-specific competitor DNA in all assays. Binding of FurA (700 nM) to its own promoter was included as positive controls, while promoter regions of superoxide dismutases genes sodA and sodB were used as negative controls.