Characterization of PhoP-Regulated Promoters

The promoters of phoP and pbgP1 were amplified by PCR from the genomic DNA of TT01 with the primers (Supplementary Table 5), and purified with the High Pure PCR Product Purification kit (ROCHE). The 5′ ends of the DNA were labeled with [γ-³²P] ATP and T4 polynucleotide kinase (Promega). The PhoP-His protein was purified and phosphorylated in vitro by incubation with acetyl phosphate56 . Radioactive DNA probe (2000 cpm.ml⁻¹), 200 ng of poly(dI-dC)-poly(dI-dC) (SIGMA) and various amounts of PhoP-His were mixed in binding buffer (50 mM Tris-HCl pH 8, 50 mM KCl, 50 μg.ml⁻¹ BSA), in a total volume of 20 μl, and incubated for 20 minutes at room temperature. The mixture was then loaded onto a native 6% (w/v) polyacrylamide TBE precast gel (Invitrogen) and subjected to electrophoresis in 1% TBE (Tris-borate-EDTA) buffer for 1 h at 100 V. Radioactive species were detected by autoradiography. PhoP-His was activated by in vitro phosphorylation with acetyl phosphate57 (link).