Journal: Scientific Reports

Article Title: A previously uncharacterized Fur-family metalloregulator integrates iron- and manganese-sensing to control virulence gene regulatory networks in Brucella

doi: 10.1038/s41598-025-29103-1

Figure Lengend Snippet: Determination of Fur4-, Mur- and VjbR-binding sites by DNAse I footprinting. 6-FAM-labeled DNA probes corresponding to the promoter regions of virB1 ( A and B ), bab2_0131 ( B and C ) or fur4 ( bab1_0393 ) ( E and F ) were incubated with or without recombinant Fur4 ( A , C and E ), Mur ( B and F ) or VjbR ( D ) at the indicated nanomolar concentrations. The binding reactions were then subjected to DNase I digestion. Figures show DNA footprinting electrophoretograms along with Sanger sequencing reactions performed with the same 6-FAM-labeled primers used to construct the different DNA probes. Dashed rectangles indicate the regions protected from DNAse I by the corresponding TFs. Solid grey line rectangles enclose the observed central core regions fully protected from DNase I digestion by Mur or Fur4. Solid yellow rectangles enclose sequences that match the Fur-like binding site motif determined in this work. The solid blue rectangle encloses the sequence that matches the Mur-box motif previously determined by Rodionov et al . (2006) .

Article Snippet: Digested products were extracted by phenol-chloroform, ethanol precipitated and resuspended in H 2 O. DNA sequencing reactions were carried out using the Thermo Sequenase Cycle Sequencing Kit (Thermo Fisher Scientific), primer T7-FAM and the corresponding plasmids pGEM-T-P virB , pGEM-T-P bab2_0131 , or pGEM-T-P fur4 as template.

Techniques: Binding Assay, Footprinting, Labeling, Incubation, Recombinant, DNA Footprinting, Sequencing, Construct