B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli

Eguchi et al. 10.1073/pnas.0705768104.

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Fig. 7. DNaseI footprinting analysis against EvgA. The 602-bp fragment carrying the promoter region of b1500 was incubated with the indicated amount (pmole) of His-tagged EvgA. A+G represents the Maxam-Gilbert sequencing ladder. The protected site is indicated with open bars. The ³²P-end-labeled probe was prepared by PCR as described in the S1 nuclease assay, using primers b1500F and b1500R in SI Table 2. The probes were incubated at 37°C for 10 min with His-tagged EvgA in 25 ml of binding buffer (50 mM Tris×HCl (pH 7.8), 50 mM NaCl, 3 mM magnesium acetate, 5 mM CaCl₂, 0.1 mM EDTA, 0.1 mM DTT, and 25 mg BSA ml^-1), followed by digestion with 5 ng of DNaseI (Takara) at 25°C for 30 sec. The reaction was terminated by addition of 45 ml of DNaseI stop solution [20 mM EDTA, 200 mM NaCl, 1% (vol/vol) SDS, and 250 mg yeast tRNA ml^-1]. The digested products were precipitated by ethanol, extracted by phenol and analyzed by electrophoresis on a 6% (vol/wt) polyacrylamide gel containing 8 M urea. The radioactivity was measured as described for S1 nuclease assay.

Fig. 8. Transcriptional activation of the PhoP regulon by b1500. S1 nuclease assays of slyB, yrb and hemL were performed against cells grown in the presence of 30 mM MgCl₂, with and without the addition of 1 mM IPTG at OD₆₀₀ = 0.4. Lanes 1 and 2 are MG1601 / pQE80L and Lanes 3 and 4 are MG1601 / pQE-b1500.

Table 2. Primers used for constructing plasmids for bacterial two-hybrid assay

Restriction sites are underlined.

Table 3. Promoter-specific primers used in S1 nuclease assay and DNaseI footprinting analysis

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