Model illustrating the regulatory interactions between the PhoP/PhoQ and PmrA/PmrB two-component systems and the shunt protein PmrD. Transcription of PmrA-activated genes is promoted during growth in low Mg²⁺ via the PhoP/PhoQ system, the PmrD protein, and the PmrA/PmrB system (green arrow), and in the presence of iron via the PmrA/PmrB system, independently of the PhoP/PhoQ system and the PmrD protein. The PmrA protein represses transcription of the pmrD gene under conditions that activate PmrA independently of the PhoP/PhoQ system (red line).

Mutation of the PmrA-binding site in the pmrD promoter abolishes PmrA-mediated repression of pmrD transcription. (A) β-Galactosidase activity (Miller units) from a pmrD⁺-lacZY transcriptional fusion expressed by bacteria grown in N-minimal medium, pH 5.8, with 10 μM Mg²⁺ was determined in wild-type (EG13659), pmrA (EG13660), and phoP (EG13663) strains harboring a wild-type upstream PmrA-binding site in the pmrD promoter and in wild-type (EG13723), pmrA (EG13724), and phoP (EG13725) strains harboring a mutant upstream PmrA-binding site in the pmrD promoter. (B) β-Galactosidase activity (Miller units) from a pmrD⁺-lacZY transcriptional fusion expressed by bacteria grown in N-minimal medium, pH 5.8, with 10 μM Mg²⁺ was determined in wild type (EG13659), a strain with a mutant upstream PmrA-binding site (EG13723), and a strain with a mutant downstream PmrA-binding site (EG13728) in the pmrD promoter.

The PmrA/PmrB system represses PmrD expression. (A) Western blot analysis with anti-FLAG antibodies of cell extracts prepared from a strain harboring a chromosomally encoded PmrD-FLAG protein (EG13654) grown in N-minimal medium, pH 7.7, with 10 μM (L) or 10 mM (H) Mg²⁺. (B) PmrD-FLAG protein expressed by wild-type (EG13654), pmrA (EG13655), and pmrB (EG13656) strains grown in N-minimal medium, pH 5.8, with 10 μM Mg²⁺ detected by Western blot with anti-FLAG antibodies. (C) β-Galactosidase activity (Miller units) from a pmrD⁺-lacZY transcriptional fusion expressed by bacteria grown in N-minimal medium, pH 5.8, with 10 μM Mg²⁺ were determined in wild-type (EG13659), pmrA (EG13660), pmrB (EG13661), and phoP (EG13663) strains. (D) β-Galactosidase activity (Miller units) from a pmrD⁺-lacZY transcriptional fusion expressed by strain EG13659 grown in N-minimal medium, pH 5.8, with 10 mM Mg²⁺ (H) or 10 μM Mg²⁺(L) in the presence (+) or absence of (−) of 100 μM FeSO₄. (E) S1 nuclease-protection assay of RNAs extracted from bacteria grown in N-minimal medium, pH 5.8, with 10 μM Mg²⁺. Lanes G, A, T, and C correspond to dideoxy chain-termination sequence reactions corresponding to this region. The sequence spanning the transcription start site is shown, and the start site is marked with an arrow.

The PmrA and PhoP proteins footprint the pmrD promoter. (A) DNA sequence of the promoter region of the pmrD gene. +1 corresponds to the transcription start site, the underlined sequence is the predicted −10 region for the pmrD promoter, the sequence in bold matches the PhoP-binding consensus sequence (PhoP box) (26, 27), and the boxed sequences are the PmrA-binding consensus sequence (PmrA box) (23, 25). Mutated nucleotides at the PmrA-binding sites (upstream and downstream) are indicated above the sequence. A putative UP element may be represented by the AT-rich PmrA boxes and the sequences immediately surrounding them. (B) DNase I footprinting analysis of the pmrD promoter performed by using probes A (coding strand) and B (noncoding strand) (see Methods). The amount of PmrA and phospho-PmrA (PmrA-P) protein added to DNA probes A and B was 0, 10, 25, 50, and 100 pmol. A solid line represents the PmrA-binding region. Lanes G, A, T, and C are dideoxy chain-termination sequences corresponding to probes A and B. (C) DNase I footprinting analysis of the pmrD promoter performed by using probes C (coding strand) and D (noncoding strand) (see Methods). The amount of PhoP and PmrA proteins was 25 and 5 pmol, respectively. A wide bar and a thin line represent the PhoP-binding and PmrA-binding regions, respectively. Lanes G, A, T, and C are dideoxy chain-termination sequences corresponding to probes C and D.