The CorC proteins MgpA (YoaE) and CorC protect from excess-cation stress and are required for egg white tolerance and virulence in Salmonella

Abstract

Cation homeostasis is a vital function. In Salmonella, growth in very low Mg²⁺ induces expression of high-affinity Mg²⁺ transporters and synthesis of polyamines, organic cations that substitute for Mg²⁺. Once Mg²⁺ levels are re-established, the polyamines must be excreted by PaeA. Otherwise, cells lose viability due to a condition we term excess-cation stress. We sought additional tolerance mechanisms for this stress. We show that CorC and MgpA (YoaE) are essential for survival in stationary phase after Mg²⁺ starvation. Deletion of corC causes a loss of viability additive with the paeA phenotype. Deletion of mgpA causes a synthetic defect in the corC background. This lethality is suppressed by loss of the inducible Mg²⁺ transporters, suggesting that the corC mgpA mutant is sensitive to changes in intracellular Mg²⁺. CorC and MgpA function independently of PaeA. A paeA mutant is sensitive to externally added polyamine in stationary phase; loss of CorC and MgpA suppressed this sensitivity. Conversely, the corC mgpA mutant, but not the paeA mutant, exhibited sensitivity to high Mg²⁺ and egg white. The corC mgpA mutant is also attenuated in a mouse model. The corC and mgpA genes are induced in response to increased Mg²⁺ concentrations. Thus, CorC and MgpA play some interrelated role in cation homeostasis. It is unlikely that these phenotypes are due to absolute levels of cations. Rather, the cell maintains relative concentrations of various cations that likely compete for binding to anionic components. Imbalance of these cations affects some essential function(s), leading to a loss of viability.

Keywords: Cations; Egg White; Magnesium; Polyamines; Salmonella; Stationary Phase.

Fig. 1.. Domain architecture of CorC domain-containing proteins in Salmonella.

Conserved domains were predicted using InterPro (https://www.ebi.ac.uk/interpro/). Each domain is represented by a different color, while regions with no domain assignment are shown in white.

Fig. 2.. The ΔcorC strain loses viability in stationary phase after Mg²⁺ starvation, the deletion of mgpA exacerbates the effect.

The indicated strains were grown overnight in N-minimal medium pH 7.4 with 10 mM MgCl₂, washed, diluted into N-minimal medium pH 7.4 with or without 10 mM MgCl₂ (t=0 h), and incubated at 37°C. CFUs were determined at the indicated time points in wild type (A) and ΔpaeA (B) background. Values are mean ± SD, n = 6. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) vs mgpA+ corC+ parent strain at the same time point and at the same Mg²⁺ concentration. Strains used: 14028, JS2692, JS2693, JS2694, JS2695, JS2696, JS2697, and JS2698.

Fig. 3.. Loss of the high-affinity Mg²⁺ transporters MgtA and MgtB, but not CorA, suppresses the corC phenotype in stationary phase after Mg²⁺ starvation.

The indicated strains were grown overnight in N-minimal medium pH 7.4 with 10 mM MgCl₂, washed, diluted into N-minimal medium pH 7.4 with or without 10 mM MgCl₂ (t=0 h), and incubated at 37°C. CFUs were determined at the indicated time points in WT (A), ΔmgtA ΔmgtB (B), Δsynth (C), and ΔcorA (D) background. Values are mean ± SD, n = 6. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) versus the mgpA+ corC+ at the same time point and at the same Mg²⁺ concentration, and (p < 0.05^#, 0.005^##, 0.0005^###) ΔmgpA corC+ versus ΔmgpA ΔcorC at the same time point and at the same Mg²⁺ concentration. Strains used: 14028, JS2692, JS2693, JS2694, JS2713, JS2714, JS2715, JS2716, JS2560, JS2717, JS2718, JS2717, JS2720, JS2721, JS2722, and JS2723.

Fig. 4.. Loss of CorC, but not MgpA increases tolerance to Co²⁺ and Mn²⁺.

The indicated strains were grown overnight in MOPS-minimal medium (pH 7.4) with 1.32 mM KH₂PO₄ and 10 mM MgCl₂, washed, diluted into MOPS-minimal medium (pH 7.4) with or without the indicated amounts of MgCl₂ or CoCl₂ in the absence of KH₂PO₄ and MgCl₂ (t=0h), and incubated at 37°C. CFUs were determined at 0h and 9h. CFU ratio (9h/0h) values are mean ± SD, n = 3. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) versus WT and (p < 0.05^#, 0.005^##, 0.0005^###) versus ΔcorA. Strains used: 14028, JS2692, JS2693, JS2694, JS2720, JS2721, JS2722, JS2723, JS2430, JS2562, JS2560, and JS2563.

Fig. 5.. Loss of CorC and MgpA suppresses polyamine sensitivity in paeA background.

The indicated strains were grown in N-minimal medium (pH 7.4) with (A) 10 mM or (B) 50 μM MgCl₂ for 24 hours at 37°C, washed, diluted into pH 8.5-buffered saline containing indicated polyamines (t=0h), and incubated at 37°C. CFUs were determined at 0h and 24h. CFU ratio (24h/0h) values are mean ± SD, n = 3. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) versus WT, (p < 0.05^#, 0.005^##, 0.0005^###) ΔmgpA corC+ versus ΔmgpA ΔcorC, and (p < 0.05^♮, 0.005 ^{♮ ♮}, 0.0005 ^{♮ ♮ ♮}) mgpA+ ΔcorC versus ΔmgpA ΔcorC. Strains used: 14028, JS2695, JS2724, JS2725, and JS2726.

Fig. 6.. The ΔmgpA ΔcorC strain is sensitive to egg white.

The indicated strains were pre-grown to mid-exponential phase in N-minimal medium (pH 7.4) with 10 mM MgCl₂ at 37°C, washed, diluted into 80% egg white solution or PBS (t=0 h), and incubated at 37°C. CFUs were determined at 0h and 24h. CFU ratio (24h/0h) values are mean ± SD, n = 6. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) versus WT same treatment. Strains used: 14028, JS2728, JS2729, JS2730, JS2699, JS2731, JS2732, JS2733, JS2430, JS2560, JS2562, JS2563, and JS2694.

Fig. 7.. The loss of viability in ΔcorC and ΔmgpA ΔcorC strains during stationary phase after Mg²⁺ starvation is reduced in acidic medium and exacerbated in alkaline medium.

The indicated strains were grown overnight in N-minimal medium pH 7.4 with 10 mM MgCl₂, washed, diluted into N-minimal medium (A) pH 5.5 and (B) pH 8.5 with or without 10 mM MgCl₂ (t=0 h), and incubated at 37°C. CFUs were determined at the indicated timepoints. Values are mean ± SD, n = 6. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) versus corresponding WT and (p < 0.05^#, 0.005^##, 0.0005^###) ΔmgpA corC+ versus ΔmgpA ΔcorC at the same time point and at the same Mg²⁺ concentration. Strains used: 14028, JS2692, JS2693, and JS2694.

Fig. 8.. pH affects growth under high Mg²⁺ conditions.

The indicated strains were pre-grown to mid-exponential phase in N-minimal medium pH 7.4 with 1 mM MgCl₂, washed, and diluted into N-minimal medium pH 7.4 or pH 8.5 with (A) 1 mM, (B) 10 mM, and (C) 100 mM MgCl₂ (t=0h), and incubated at 37°C. OD₆₀₀ was determined at the indicated timepoints. Values are mean ± SD, n = 3. Unpaired t test (p < 0.05*, 0.005**, 0.0005***) versus corresponding WT at the same timepoint. Strains used: 14028, JS2692, JS2693, and JS2694.

Fig 9.. Expression of mgpA and corC are upregulated under high Mg²⁺ conditions.

(A) The corC and mgpA loci. Dark bars indicate the sequences cloned upstream and in-frame with lacZ. The (B) corC’-’lacZ and (C) mgpA’-’lacZ fusion strains were pre-grown to mid-exponential phase in N-minimal medium pH 7.4 with 1 mM MgCl₂ at 37°C, washed, diluted in N-minimal medium pH 5.5, pH 7.4, and pH 8.5 with or without the indicated amount of MgCl₂, and incubated at 37°C for 24h. β-galactosidase activity was measured as described in Materials and Methods section. Values are mean ± SD, n = 6. Paired t test (p < 0.05*, 0.005**, 0.0005***) versus 0 mM Mg²⁺ at the same pH. Strains used: JS2734 and JS2735.

Fig 10.. The ΔcorC strain shows a lower free-Mg level at stationary phase when grown with 100 mM MgCl₂, and the deletion of mgpA exacerbates the effect.

The indicated strains harboring pS2513-MagFRET, pre-grown to mid-exponential phase in N-minimal medium (pH 7.4) with 1 mM MgCl₂, were washed and diluted into N-minimal medium pH 7.4 with the indicated amounts of MgCl₂, and incubated at 37 °C. Aliquots (1.5 mL) were collected after (A) 3, (B) 7.5, and (C) 24 hours, and the fluorescence ratio (Citrine/Cerulean) was determined at OD₆₀₀ = 0.1. A higher ratio indicates a higher free-Mg level. Values are mean ± SD, n = 3. Unpaired t test (P < 0.05*; P < 0.005**; P < 0.0005***) versus the wild-type, (P < 0.05^#; P < 0.005^##; P < 0.0005^###) of mgpA + ΔcorC versus ΔmgpA ΔcorC, or (P < 0.05^♮; P < 0.005 ^{♮ ♮}; P < 0.0005 ^{♮ ♮ ♮}) of ΔmgpA corC + versus ΔmgpA ΔcorC at the same condition. Paired t test (P < 0.05 ♭; P < 0.005 ♭♭; P < 0.0005 ♭♭♭) versus 1 mM Mg²⁺ in the wild-type. Strains used: JS2736, JS2737, JS2738, and JS2739.