Genetic screens reveal novel major and minor players in magnesium homeostasis of Staphylococcus aureus

Abstract

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.

Fig 1. Import and export are two independent systems.

Serial dilutions of overnight cultures of each strain were spotted on Mueller Hinton medium (MH) supplemented with increasing amount of MgCl₂. Plates were incubated for 24h at 37°C.

Fig 2. Complementation of ΔmgtEΔcorAΔcorA2 by either pcorA2 or pmgtE mgtE, corA and corA2 were cloned on pCN47 plasmids, a medium to high copy number plasmid (Charpentier et al., 2004).

Since MgtE is in an operon, we cloned the ORF under the control of a constitutive promoter (Hu) while both CorAs were cloned with their natural promoter. Serial ten-fold dilutions of overnight cultures of each strain were spotted on Mueller Hinton medium (MH) supplemented with erythromycin and increasing amount of MgCl₂. Plates were incubated for 24h at 37°C.

Fig 3. Internal free magnesium concentration is affected in magnesium transporter mutants.

Fluorescence of cultures of different strains carrying a plasmid harboring a fusion between GFP and BSmgtE promoter where measured mid-exponential phase. Bacteria where grown in MH medium supplemented with indicated amount of MgCl₂. The GFP fluorescence is given in arbitrary unit. The value was calculated as the average of three independent measurements (N = 3), subtracted of the background noise, i.e. the inherent fluorescence of a GFP-less culture. The results presented here are representative of at least three different experiments. N/A indicates the fluorescence could not be analysed since the strain does not grow in said condition. Unpaired t-test (R program) was used to calculate p-values. Numerical data are visible in S1 Appendix. * p-value<0.05 **p-value<0.01.

Fig 4. mpfA is epistatic to corA2 in ΔcshB suppression.

Three dilutions of overnight cultures of each strain were spotted on Mueller Hinton medium (MH) or RPMI medium supplemented with uracil. Plates were incubated for 24h at 37°.

Fig 5. Overexpression of MgtE affect both ΔcshB and ΔmpfA mutants.

SamgtE was clone under a constitutive promoter (pHU) on a pCN47 derived vector, carrying a tetracycline cassette. All plates were supplemented with tetracycline and incubated for 24h at 37°C. A: Three dilutions of overnight cultures of ΔcshB carrying either an empty plasmid or the fusion were spotted on Mueller Hinton medium (MH) or RPMI medium supplemented with uracil. B: Three dilutions of overnight cultures of WT and ΔcshB were spotted on Mueller Hinton (MH) plates supplemented with the indicated amount of MgCl₂.

Fig 6. MpfB is an active protein.

Serial dilutions of overnight cultures of each strain were spotted on Mueller Hinton medium (MH), supplemented in uracil and eventually supplemented with 40 or 80 mM MgCl₂.Plates were incubated for 24h at 37°C. A: In absence of mpfB, tRNA^CAC mutation does not suppress ΔmpfA magnesium sensitivity. B: Expression of MpfB from a multi-copy plasmid (pCN47) complements ΔmpfA magnesium sensitivity. Plates were supplemented with erythromycin. C. An AUG start codon for mpfB suppresses ΔmpfA magnesium sensitivity.

Fig 7. Current model of magnesium homeostasis in S. aureus.

Data presented here allows us to propose the following model. Magnesium import is carried mainly by CorA2 and MgtE with additional marginal role of CorA. We have isolated mutated alleles of CorA2 which are able to increase magnesium import. Export is ensured by MpfA, although it remains to be determined whether MpfA directly transport Mg²⁺ or regulates another player. MpfB, a paralog of MpfA plays an additional minor role. Arrows indicate the proposed directionality of the transporter, full arrows to show the main transporters and dotted arrows for the secondary actors.