Activated by different signals, the PhoP/PhoQ two-component system differentially regulates metal uptake

Abstract

The PhoP/PhoQ two-component system controls several physiological and virulence functions in Salmonella enterica. This system is activated by low Mg(2+), acidic pH, and antimicrobial peptides, but the biological consequences resulting from sensing multiple signals are presently unclear. Here, we report that the PhoP/PhoQ system regulates different Salmonella genes depending on whether the inducing signal is acidic pH or low Mg(2+). When Salmonella experiences acidic pH, the PhoP/PhoQ system promotes Fe(2+) uptake in a process that requires the response regulator RstA, activating transcription of the Fe(2+) transporter gene feoB. In contrast, the PhoP-induced RstA protein did not promote feoB expression at neutral pH with low Mg(2+). The PhoP/PhoQ system promotes the expression of the Mg(2+) transporter mgtA gene only when activated in bacteria starved for Mg(2+). This is because mgtA transcription promoted at high Mg(2+) concentrations by the acidic-pH-activated PhoP protein failed to reach the mgtA coding region due to the mgtA leader region functioning as a Mg(2+) sensor. Our results show that a single two-component regulatory system can regulate distinct sets of genes in response to different input signals.

FIG. 1.

Model illustrating Fe²⁺ and Mg²⁺ uptake regulated by the PhoP/PhoQ system and RstA protein in Salmonella. (A) When activated at millimolar Mg²⁺ concentrations by the acidic-pH signal, the phosphorylated PhoP protein promotes transcription of the rstA and mgtA genes. The induced RstA protein is activated, possibly by phosphorylation. The increase in feoB expression resulting from RstA binding to the feoA promoter enhances the FeoB-mediated Fe²⁺ uptake. In contrast, mgtA transcription fails to proceed due to Mg²⁺ binding to the mgtA leader. (B) The low-Mg²⁺ signal activates the PhoP/PhoQ system at neutral pH. Activation of mgtA transcription by phosphorylated PhoP increases production of the full length of the mgtA mRNA, leading to expression of the MgtA protein, which promotes Mg²⁺ uptake. In contrast, the RstA protein induced at neutral pH lacks the activity to promote transcription of the feoB gene.

FIG. 2.

The PhoP/PhoQ system activated by acidic pH or low Mg²⁺ promotes expression of the rstA gene. Salmonella strains were grown to an OD₆₀₀ of 0.5 to 0.6 in N-minimal medium containing 2 mM (H, high) or 50 μM (L, low) Mg²⁺ and adjusted to pH 7.7 (H, high) or 5.7 (L, low). (A) The rstA mRNA levels in wild-type (14028s) and phoPQ deletion (DS267) strains were determined using qRT-PCR. Shown are the mean values and standard deviations from three independent experiments. (B) Western blot analysis of crude extracts prepared from wild-type (DS604) and phoPQ mutant (EN267) strains and probed with anti-FLAG and anti-DnaK antibodies.

FIG. 3.

Activation of feoB transcription at acidic pH is dependent on the PhoP/PhoQ and RstA proteins but independent of the RstB protein. Wild-type (WT; 14028s), rstA deletion (JH101), phoPQ deletion (DS267), and rstB deletion (DS603) strains were grown in N-minimal medium at pH 7.7 (H, high) or 5.7 (L, low) with 2 mM (H, high) or 50 μM (L, low) Mg²⁺. The feoB mRNA levels were determined using qRT-PCR. Shown are the mean values and standard deviations from three independent experiments.

FIG. 4.

RstA-promoted feoB transcription requires the acidic-pH signal and its putative phosphorylation site even when the rstA gene is transcribed from a heterologous promoter. Plasmids pEN105 [pRstA(WT)] and pEN106 [pRstA(D52A)] express the FLAG-tagged wild-type RstA and D52A RstA protein, respectively, from the lac promoter. Strains were grown in N-minimal medium containing 2 mM Mg²⁺ and buffered at pH 7.7 (H, high) or 5.7 (L, low) in the presence (+) or absence (−) of 0.1 mM IPTG. (A) Western blot analysis of crude extract prepared from the ΔrstA (JH101) strain harboring pEN105 or pEN106 and probed with anti-FLAG and anti-DnaK antibodies. (B) feoB mRNA levels produced by the ΔrstA (JH101) strain harboring pEN105 or pEN106. Shown are the mean values and standard deviations from three independent experiments.

FIG. 5.

The PhoP/PhoQ system is not required for RstA-promoted feoB transcription at acidic pH. (A) feoB mRNA levels produced by the ΔrstA (JH101) and ΔrstA ΔphoPQ (EN293) strains harboring pJH4, which expresses RstA from the lac promoter. The strains were grown in N-minimal medium containing 2 mM Mg²⁺ and buffered at pH 7.7 (H, high) or 5.7 (L, low) in the presence of 0.1 mM IPTG. (B) rstA and feoB mRNA levels produced by the ΔphoPQ strain (DS267) harboring pDS303, which expresses the PhoP* protein from the lac promoter. The strain was grown in N-minimal medium containing 2 mM Mg²⁺ and buffered at pH 7.7 (H, high) or 5.7 (L, low) in the presence (+) or absence (−) of 0.5 mM IPTG. Shown are the mean values and standard deviations from three independent experiments.

FIG. 6.

FeoB induction at acidic pH by the PhoP/PhoQ and RstA proteins enhances Salmonella growth upon iron starvation. (A) Growth of Salmonella strain (JH380) lacking both the sitABCD and mntH genes in N-minimal medium at pH 7.7 or 5.7 with 2 mM or 50 μM Mg²⁺. (B) Growth of the JH380 strain and its isogenic feoB (EN258), rstA (EN256), and phoPQ (EN290) deletion mutants in N-minimal medium at pH 5.7 with 2 mM Mg²⁺. All media contained 2 mM sodium ascorbate and 10 μM diethylenetriaminepentaacetic acid. The results shown are representative of three experiments.

FIG. 7.

The leader sequence allows the mgtA gene to be expressed only when Salmonella is starved for Mg²⁺. (A) Schematic representation of the leader and coding regions of the mgtA gene. The double-headed arrows marked as “a” and “b” indicate the regions amplified by qRT-PCR using two pairs of primers. A part of the leader replaced with the “scar” sequence (10) in strain EN323 is also shown. (B) The wild-type Salmonella strain (14028s) and its phoPQ deletion mutant (DS267), mgtA leader mutant (EN323), and EN323 with the phoPQ deletion (EN331) were grown to an OD₆₀₀ of 0.4 in N-minimal medium at pH 7.7 with 2 mM Mg²⁺ and transferred to medium at pH 7.7 or 5.7 in the presence or absence of Mg²⁺. After 30 min, RNA was isolated from the strains, and the levels of mgtA mRNA corresponding to region “a” were determined by qRT-PCR. (C) By conducting qRT-PCR on the same RNA samples used for the experiment described for panel B, the mgtA transcripts corresponding to region “b” were quantified. Shown are the mean values and standard deviations from three independent experiments. UTR, untranslated region; WT, wild type; +, present; −, absent.