Regulation of ompC , F and X by OmpR. a) Real-time RT-PCR. The mRNA levels of each indicated gene were compared between ΔompR and WT. This figure shows the increased (positive number) or decreased (minus one) mean fold for each gene in ΔompR relative to WT. b) LacZ fusion reporter. A promoter-proximal region of each indicated gene was cloned into pRW50 containing a promoterless lacZ reporter gene, and transformed into WT or ΔompR to determine the promoter activity (β-galactosidase activity in cellular extracts). The empty plasmid was also introduced into each strain as negative control, which gave extremely low promoter activity (data not shown). Positive and minus numbers indicate the increased and decreased mean folds, respectively, for the detecting promoter activity in ΔompR relative to WT. c) Primer extension. Primer extension assays were performed for each indicated gene using total RNAs isolated from the exponential-phase of WT or ΔompR. An oligonucleotide primer complementary to the RNA transcript of each gene was designed from a suitable position. The primer extension products were analyzed with 8 M urea-6% acrylamide sequencing gel. Lanes C, T, A, and G represent the Sanger sequencing reactions; on the right side, DNA sequences are shown from the bottom (5') to the top (3'), and the transcription start sites are underlined. d) DNase I footprinting. The labeled DNA probe was incubated with various amounts of purified His-OmpR (lanes 1, 2, 3, 4, and 5 contained 0, 5, 10, 15 and 20 pmol, respectively) with the addition of acetyl phosphate, and subjected to DNase I footprinting assay. Lanes G, A, T, and C represent the Sanger sequencing reactions, and theprotected regions (bold lines) are indicated on the right-hand side. The numbers indicate the nucleotide positions upstream of the transcriptional start sites.