DNA Electrophoretic Mobility Shift Assay (EMSA)

The purified K. pneumoniae Fur protein and DNA fragments containing the putative Fur boxes were used on DNA Electrophoretic Mobility Shift Assays (EMSA). These DNA fragments were obtained by PCR amplifying the pGEM^®-T Easy vectors cloned with the putative Fur boxes. Amplifications were done with universal M13 primers and the resulting 285 base pairs long fragments were then used as probes on EMSA. The negative control consisted of a 254 base pairs DNA fragment without Fur box sequence. This fragment was obtained by PCR amplifying a pGEM^®-T Easy vector without insert (i.e., vector not cloned with the putative Fur boxes).

EMSA was performed as described elsewhere⁷³, with minor modifications. In brief, 500 ηM of His-Fur protein was initially equilibrated for 10 minutes on ice in a 10 µL reaction volume containing 1x binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5), 0.5 mM MgCl₂, 0.5 mM MnSO₄ and 2.5% (v/v) glycerol. To this binding reaction, 50 ηg of the DNA probes were added and the mixture was incubated for 20 minutes on ice. In addition, EMSA were carried out under divalent cation-free conditions by adding EDTA to a final concentration of 2 mM in the above reaction mixture.

Samples were loaded onto a 2% (w/v) agarose gel and submitted to electrophoresis for 30 minutes at 100 Volts in 1x Bis-Tris borate (pH 7.5) buffer containing 0.1 mM MnSO₄. The agarose gels were stained after electrophoresis by soaking then on an ethidium bromide solution (0.5 ug/mL) for 15 minutes with gentle agitation. The DNA bands were visualized under UV light and recorded with a digital photodocumentation system (Gel Doc^TM XR, Biorad). Capture and analysis of the imagens were done with the Image Lab^TM Software version 5.0 (Biorad).