Proteins were incubated with DNA targets during 30 min at 25°C in the final reaction mixture volume of 15 μl. 900 ng of each GadE and RcsBD56E protein are used for yhiM and aslB. B. Gel mobility shift assays with HdfR or AdiY proteins. Quantities JIB04 concentration of purified HdfR or AdiY proteins are indicated above each lane (in ng). Gel mobility shift assays (A and B) were performed with 0.1 ng [γ32P]-labelled
DNA fragment and loaded on a 6% polyacrylamide native gel. An arrow points out the position of the DNA-regulatory protein complex. An asterisk marks the position of the unbound probe. Identification of the targets directly controlled by HdfR or AdiY Real-time quantitative RT-PCR analysis showed that HdfR regulates aslB and gltBD, while AdiY regulates several genes involved in acid stress resistance (adiA, adiC, aslB, gadA, gadBC,
gltBD, hdeAB, hdeD and slp-dctR) (Table 4). To establish whether these regulators control the expression of these genes by direct binding to their promoter regions, gel mobility shift assays were performed with purified HdfR and AdiY proteins. It was found that HdfR binds to the promoter region of gltBD and that AdiY binds to the promoter find more DMXAA datasheet regions of gltBD, adiA and gadABC (Figure 1B). However, no band shift was observed even with higher concentration of regulator with HdfR on the promoter region of aslB and with AdiY on the promoter regions of adiC, aslB, hdeABD and slp-dctR (Figure 1B), suggesting an indirect regulation for these genes. Identification of the targets directly controlled by H-NS H-NS modulates the
expression of several regulators controlling acid stress resistance including HdfR, RcsD, EvgA, YdeO, YdeP, GadE, GadW, GadX, AdiY and CadC. However, the direct control by H-NS has not yet been established for the majority of these regulators, except for GadX  and HdfR . Furthermore, slp-dctR and yhiM could also be directly repressed by H-NS, as deletion of their regulators, RcsB-P/GadE complex and/or AdiY, in hns-deficient strain was not sufficient to restore their wild-type mRNA level (Table PJ34 HCl 4) . Competitive gel mobility shift assays were performed with purified H-NS protein on PCR fragments, corresponding to assayed promoters, and restriction fragments derived from the pBR322 plasmid, used as negative competitors for binding to H-NS protein except for one 217-bp DNA fragment corresponding to the bla promoter used as positive internal control . A preferential binding of H-NS was observed to the promoter regions of adiY, cadBA, cadC, evgA, gadE, gadW, hdfR, rcsD, slp-dctR, ydeO, ydeP, yhiM, confirming the direct control by H-NS of these genes (Figure 2). Figure 2 Competitive gel mobility shift assay with H-NS, target promoter fragments and restriction fragments derived from plasmid pBR322. The cleaved plasmid and promoter fragments were incubated with the indicated concentrations of purified H-NS protein (in μM).