Closing the loop: the PmrA/PmrB two-component system negatively controls expression of its posttranscriptional activator PmrD

Abstract

A fundamental question in biology is how an organism integrates multiple signals to mediate an appropriate cellular response. The PmrAPmrB two-component system of Salmonella enterica can be activated independently by Fe(3+), which is sensed by the PmrB protein, and in low Mg(2+), which is sensed by the PhoQ protein. The low-Mg(2+) activation requires pmrD, a PhoPPhoQ-activated gene that activates the response regulator PmrA at a posttranscriptional level. We now report that pmrD expression is negatively regulated by the PmrAPmrB system. Conditions that activate the PmrA protein independently of pmrD, such as exposure to Fe(3+), resulted in lower levels of pmrD transcription. The PmrA protein footprinted the pmrD promoter upstream of the PhoP-binding site but did not interfere with binding of the PhoP protein. Mutation of the PmrA-binding site in the pmrD promoter abolished PmrA-mediated repression. Negative regulation of the PhoPPhoQ-activated pmrD gene by the PmrAPmrB system closes a regulatory circuit designed to maintain proper cellular levels of activated PmrA protein and constitutes a singular example of a multicomponent feedback loop.

Figure 1

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).

Figure 2

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.

Figure 3

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.

Figure 4

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.