FIG. 5.

NikR binds the HP1511-1512 intergenic region. (A) In silico analysis identified a putative NikR consensus sequence (10) upstream of HP1512 within the 154-bp HP1511-1512 intergenic region (proposed operator regions in bold). (B) The nikR sequence was cloned into pET30a_nikR plasmid and expressed in E. coli BL21(DE3)pLysS. Recombinant protein was purified by Ni-NTA affinity purification under native conditions (tagged NikR) followed by recombinant enterokinase digestion/capture to remove fusion tags. The resulting recombinant H. pylori NikR (rNikR) protein (∼17 kDa) contains a single N-terminal alanine from the expression vector. Target DNA, encompassing the HP1511-1512 intergenic region, was amplified from H. pylori 26695 chromosomal DNA via PCR, labeled with digoxigenin (DIG), and used in gel shift assays with the recombinant H. pylori NikR. Binding reaction mixtures consisted of approximately 15 fmol of DIG-labeled target DNA and fourfold dilutions of recombinant NikR protein ranging from 16 μM to 63 nM. Binding reaction mixtures were electrophoresed on 7% nondenaturing polyacrylamide gels; nickel was added to the gel and running buffer to a final concentration of 100 μM NiCl₂. (C) Proteins and DNA were electroblotted onto a positively charged nylon membrane and visualized via chemiluminescence detection with alkaline phosphate and disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2′-(5′-chloro)tricyclo]3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD).