FIGURE 4.

The CpxR protein enhances amiA transcription by binding to the amiA promoter. A, primer extension for mapping the transcription start site of amiA. The cDNA products were synthesized using ³²P-labeled primer 1472 and total RNA templates isolated from wild-type (14028s) carrying pUHE21 (vector) (lane 2) and pnlpE (pYS2132) (lane 3) and cpxR mutant carrying pnlpE (lane 4). Transcription starts at −71 and −62 are shown in boldface capital letters. B, EMSA. A ³²P-labeled DNA fragment of wild-type amiA promoter was incubated with different amounts (0, 25, and 50 pmol) of His₆-CpxR, shown in lanes 1–3. Lane 4, same as lane 3 but supplemented with “cold” amiA promoter fragment. His₆-CpxR-DNA mixtures were subjected to 4% polyacrylamide electrophoresis. The location of DNA migration was detected by autoradiography. C, DNase I footprinting analysis of the wild-type amiA promoter with probes for the coding and noncoding strands and increasing amounts of His₆-CpxR protein (50 and 100 pmol). Solid vertical lines correspond to the CpxR-binding sites, RI and RII. D, DNase I footprinting analysis of the CpxR box (box 1)-substituted amiA promoter with probes for the coding strand and increasing amounts of His₆-CpxR protein (50 and 100 pmol). Solid vertical lines correspond to the substituted CpxR box. E, DNA sequence of the amiA promoter region. The underlines correspond to the CpxR-binding sites, RI and RII. The boxes correspond to sequences (box 1) resembling the consensus CpxR box. The putative −35 and −10 boxes for the transcription started from −62 described in A are labeled with solid braces. The highlighted sequences correspond to an alternative CpxR box (box 2), and the putative −35 and −10 regions for the transcription started from −71 are labeled with dashed braces. Numbering in C–E is from the predicted start codon of amiA.