FIG 2.

The PorX response regulator binds to the sigP promoter region. (A) The mRNA levels of the sigP gene in the 33277 wild-type strain and the ΔporX mutant (YS19181) carrying pT-COW, p-porX, or p-sigP. Results are representative of three independent experiments. (B) EMSA analysis for binding of PorX to the sigP promoter. ³²P-labeled sigP DNA fragment (40 fmol) was incubated with PorX-_C-His₆ protein at the indicated amount. Lane 5 is the same as lane 4 but supplemented with nonlabeled (cold) sigP DNA fragment (1 pmol). The PorX/DNA mixtures were subjected to 5% PAGE. The location of DNA migration was detected by autoradiography. Arrows indicate the shifted bands after DNA fragments were associated with the PorX-_C-His₆ protein. The experiment was repeated twice. (C) DNase footprinting analysis of the sigP promoter fragment amplified with primers ³²P-3043 and 3044 for the coding strand and increasing amounts of PorX-c-His₆ protein. Products were separated in polyacrylamide DNA sequencing electrophoresis and the bands were detected by autoradiography. The bracket indicates the region protected by the PorX-_C-His₆ protein. Underlined DNA sequence (right of gel) indicates the PorX-protected nucleotides in the sigP promoter. The ladder M corresponds to the same ³²P-labeled sigP promoter fragment and degraded by the Maxam and Gilbert reaction. Results were repeated multiple times. (D) The DNA sequence of the sigP promoter region. Underlining corresponds to the PorX-protected region characterized in (C). Capital letters represent the sigP start codon. Numbering begins from the adenine nucleotide of the start codon. Highlighted sequences are shared by the PorX-binding site in the porT promoter (also shown in panel E). (E) The homologous sequences of the PorX-binding sites in the sigP and porT promoters. Vertical lines represent the identical nucleotides in the two sequences. Arrows represent the complementary nucleotides exhibited in the two sequences. Highlighted sequences are shared in these two promoters.