The mgtCBR mRNA Leader Secures Growth of Salmonella in Both Host and Non-host Environments

Abstract

Upon intracellular cues, bacterial mRNA leaders often form secondary structures that determine expression of a downstream protein-coding region(s), thereby providing bacteria with a mechanism to control the amounts of necessary proteins in the right locales. Here we describe a polycistronic mRNA leader that secures bacterial growth by preventing dysregulated expression of the protein-coding regions. In Salmonella, the mgtCBR mRNA encodes the virulence protein MgtC and the Mg²⁺ transporter MgtB. A mutant designed to produce leaderless mgtCBR mRNA induced MgtC and MgtB in conditions that promote mgtC transcription. The dysregulated expression of MgtC and MgtB impaired bacterial growth under all such non-host environments. While MgtC, but not MgtB, normally reduces ATP levels in a process requiring the F₁F₀ ATP synthase, dysregulated MgtC and MgtB reduced ATP levels independently of the F₁F₀ ATP synthase, which correlated with the mutant's growth defect. The mutant showed dysregulated MgtC expression and attenuated survival inside macrophages. While MgtB normally does not affect the phenotype, MgtB impaired intramacrophage survival of the mutant in the presence of MgtC. We provide an example showing that a polycistronic mRNA leader prevents the dysregulated function of protein-coding regions to allow bacteria to proliferate across complex niches.

Keywords: ATP; MgtB; MgtC; Salmonella; mRNA leader.

FIGURE 1

Dysregulated induction of MgtC and MgtB impairs growth of Salmonella in low Mg²⁺. (A) Schematic of the mgtCBR operon in the wild-type Salmonella (WT, 14028s) and ΔLD mutant (DN557). (B) Immunoblot analysis using anti-MgtC antibodies of crude extracts prepared from ΔLD and ΔLD phoP^– (DN558) strains. Bacteria were grown in N-minimal medium with 10 mM or 10 μM Mg²⁺ at pH 7.5 for 4 h. (C,E) Growth curves of wild-type (WT), ΔLD, ΔLD phoP^–, phoP^– (MS7953s), ΔLD ΔmgtC (HK111), and ΔLD ΔmgtB (DN581) strains. Bacteria were grown in N-minimal medium with 10 μM Mg²⁺ at pH 7.5, and OD₆₀₀ values were determined at the indicated time points. Means and standard deviations from three independent experiments are shown. (D) Immunoblot analysis using anti-MgtC and anti-MgtB antibodies of crude extracts prepared from wild-type (WT) and ΔLD strains grown in N-minimal medium with 10 μM Mg²⁺ at pH 7.5 and harvested at the indicated time points. The band indicated with an asterisk (^∗) corresponds to a protein displaying cross-reactivity against the anti-MgtC antibody and serves as an internal loading control. Numbers below the blots correspond to relative levels of MgtC and MgtB at a given time point.

FIGURE 2

Dysregulated expression of MgtC and MgtB reduces ATP to abnormal levels in a process that does not require F₁F₀ ATP synthase. ATP levels were determined in wild-type (WT, 14028s), ΔmgtC (EN397), ΔmgtB (EN481), ΔLD (DN557), ΔLD ΔmgtC (HK111), ΔLD ΔmgtB (DN581), and ΔLD ΔmgtCB (DN608) strains (A) as well as in ΔatpB (HK468), ΔLD ΔatpB (DN575), ΔLD ΔmgtC ΔatpB (MS575), ΔLD ΔmgtB ΔatpB (MS576), and ΔLD ΔmgtCB ΔatpB (DN686) strains (B). Bacteria were grown in N-minimal medium with 10 μM Mg²⁺ at pH 7.5 for 4 h. Data depicted in arbitrary units (AU) are means and standard deviations from three independent experiments. ^∗∗P < 0.01, ^∗∗∗P < 0.001, two-tailed t-test with each sample vs. WT (A) and with each sample vs. ΔatpB (B), ns, not significant. (C) Growth curves of Salmonella strains, ΔatpB (HK468), ΔLD ΔatpB (DN575), ΔLD ΔmgtC ΔatpB (MS575), and ΔLD ΔmgtB ΔatpB (MS576). Bacteria were grown in N-minimal medium with 10 μM Mg²⁺ at pH 7.5, and OD₆₀₀ values were determined at the indicated time points. Data are representative of three independent experiments, and means and standard deviations from three independent experiments.

FIGURE 3

Dysregulated induction of MgtC and MgtB impairs growth of Salmonella in high Mg²⁺ with acidic pH or an antimicrobial peptide. Bacteria were grown in N-minimal medium with 1 mM Mg²⁺ at pH 5.5 (A–C) or with 1 mM Mg²⁺ and 5 μg/ml C18G peptide at pH 7.5 (D–F). (A,D) Immunoblot analysis of MgtC levels in wild-type (WT), ΔLD (DN557), and ΔLD phoP^– (DN558) strains at 1 and 6 h after growth. The band indicated with an asterisk (^∗) corresponds to a protein displaying cross-reactivity against the anti-MgtC antibody and serves as an internal loading control. (B,E) Growth curves of wild-type, ΔLD, ΔLD ΔmgtC (HK111), and ΔLD ΔmgtB (DN581) strains. OD₆₀₀ values were determined at the indicated time points. Means and standard deviations from three independent experiments are shown. (C,F) ATP levels were determined in wild-type (WT), ΔLD, ΔLD ΔmgtC, and ΔLD ΔmgtB strains at 4 h after growth. Data depicted in arbitrary units (AU) are means and standard deviations from three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, two-tailed t-test with each sample vs. WT; ns, not significant.

FIGURE 4

Dysregulated induction of MgtC and MgtB impairs Salmonella inside macrophages. (A) Immunoblot analysis of MgtC and RpoA determined in the wild-type (WT, 14028s), ΔLD (DN557), and mgtM(UAG) (EG19307) strains at 1 and 6 h after engulfment by J774A.1 macrophages. The band indicated with an asterisk (^∗) corresponds to a protein displaying cross-reactivity against the anti-MgtC antibody. (B) Growth behaviors of wild-type, ΔmgtC (EN397), and ΔLD strains inside macrophages. After infection of macrophages, intracellular numbers of bacteria were determined at the indicated time points. (C,D) Intramacrophage survival of wild-type, ΔmgtC, ΔmgtB (EN481), ΔLD, ΔLD ΔmgtC (HK111), ΔLD ΔmgtB (DN581), ΔLD ΔmgtCB (DN608), ΔatpB (HK468), ΔLD ΔatpB (DN575), ΔLD ΔmgtC ΔatpB (MS575), ΔLD ΔmgtB ΔatpB (MS576), and ΔLD ΔmgtCB ΔatpB (DN686) strains. After infection of macrophages, the intracellular numbers of bacteria at 18 h were divided by those at 1 h. The percentage of survival of each mutant relative to the wild-type strain is presented. Means and standard deviations from three independent experiments are shown.

FIGURE 5

A conformation of the mgtCBR mRNA leader that causes dysregulated induction of MgtC and MgtB impairs Salmonella in host and non-host environments. (A,B) Immunoblot analysis of MgtC and MgtB in wild-type (WT, 14028s), ΔLD (DN557), and mgtM(UAG) (EG19307) strains. Bacteria were grown in N-minimal medium with 10 μM Mg²⁺ at pH 7.5 and harvested at the indicated time points (A) or at 4 h (B). The band indicated with an asterisk (^∗) corresponds to a protein displaying cross-reactivity against the anti-MgtC antibody. In panel (B), numbers above the blots correspond to relative levels of MgtC and MgtB in each lane. (C–E) Growth curves of wild-type, mgtM(UAG) (EG19307), mgtM(UAG) ΔmgtC (DN649), and mgtM(UAG) ΔmgtB (DN612) strains. Bacteria were grown in N-minimal medium with 10 μM Mg²⁺ at pH 7.5 (C), with 1 mM Mg²⁺ at pH 5.5 (D), or with 1 mM Mg²⁺ and 5 μg/ml C18G peptide at pH 7.5 (E), and OD₆₀₀ values were determined every 5 min using a plate reader. Data are representative of three independent experiments, which gave similar results. (F) Intramacrophage survival of wild-type, mgtM(UAG), mgtM(UAG) ΔmgtC, mgtM(UAG) ΔmgtB, and mgtM(UAG) ΔmgtCB (DN652) strains was determined as described in the legend of Figure 4. Means and standard deviations from three independent experiments are shown.