Increasing intracellular magnesium levels with the 31-amino acid MgtS protein

Abstract

Synthesis of the 31-amino acid, inner membrane protein MgtS (formerly denoted YneM) is induced by very low Mg²⁺ in a PhoPQ-dependent manner in Escherichia coli Here we report that MgtS acts to increase intracellular Mg²⁺ levels and maintain cell integrity upon Mg²⁺ depletion. Upon development of a functional tagged derivative of MgtS, we found that MgtS interacts with MgtA to increase the levels of this P-type ATPase Mg²⁺ transporter under Mg²⁺-limiting conditions. Correspondingly, the effects of MgtS upon Mg²⁺ limitation are lost in a ∆mgtA mutant, and MgtA overexpression can suppress the ∆mgtS phenotype. MgtS stabilization of MgtA provides an additional layer of regulation of this tightly controlled Mg²⁺ transporter and adds to the list of small proteins that regulate inner membrane transporters.

Keywords: FtsH; MgtA; PhoP; small protein; transporters.

Fig. 1.

MgtS contributes to cell growth and intracellular Mg²⁺ upon Mg²⁺ limitation. (A) The mgtS gene is located between ydeE and mgrR, encoding a PhoPQ-regulated sRNA. mgtS and mgrR do not overlap. (B) Expression of MgtS-SPA is induced by Mg²⁺ starvation in a PhoP-dependent manner. Northern and immunoblot analyses were carried out for mgtS-SPA phoP⁺ (GSO767) and mgtS-SPA ∆phoP::kan (GSO768) strains grown in N medium with 500 µM Mg²⁺ or without added Mg²⁺ as described in SI Appendix. (C) ΔmgtS strain shows survival defect upon Mg²⁺ limitation. The OD₆₀₀ of the cultures of wild-type (MG1655), ΔmgtS::kan (GSO229), ∆mgrR::kan (GSO769), and ∆phoP::kan (GSO766) cells grown in N medium containing 7.5 μM Mg²⁺ was measured at the indicated times. The average of three cultures is shown. (D) Cells lacking MgtS exhibit elevated Mg²⁺-reporter activity upon Mg²⁺ limitation. β-Galactosidase activity was assayed for cultures of wild-type (GSO770), ΔmgtS::kan (GSO772), ∆mgrR::kan (GSO773), and ∆phoP::kan (GSO771) cells carrying the chromosomal P_lac-5′-UTR_mgtA-lacZ fusion grown in N medium with 500 µM Mg²⁺ or without added Mg²⁺ as described in SI Appendix. The average of three independent assays is shown (error bars represent one SD).

Fig. 2.

MgtA Mg²⁺ transporter copurifies with MgtS. (A) MgtS is present in multiple enterobacteriaceae species. The sequences shown were aligned using ClustalW (www.ebi.ac.uk/Tools/msa/clustalw2/). MgtS contains one predicted TM domain with the C terminus in the cytoplasm (16). The arrows indicate the alanine-substitution mutants generated. The sequences of the DD-FLAG and GG-FLAG derivatives are shown below the alignment, with the modified FLAG tag indicated in bold. (B) D30A and D31A derivatives of MgtS are defective. (C) DD-FLAG and GG-FLAG derivatives of MgtS are active. For both B and C, β-galactosidase activity was assayed for cultures of P_lac-5′-UTR_mgtA-lacZ ∆mgtS (GSO774) cells carrying pBAD24 expressing indicated derivatives of MgtS as described in SI Appendix. The average of two independent trials is shown (error bars represent one SD). (D) MgtA copurifies with tagged MgtS. Cultures of ∆mgtS (GSO775) cells carrying pBAD24 expressing indicated derivatives of MgtS were grown in N medium with 15 μM Mg²⁺. Lysates were prepared and mixed with M2 anti-FLAG antibodies conjugated to Sepharose beads as described in SI Appendix. Bound proteins were analyzed by SDS/PAGE, and prominent bands were identified as MgtA, HflK, HflC, and PspA by mass spectrometry.

Fig. 3.

MgtS copurifies with MgtA. (A) MgtS-GG-FLAG copurifies with MgtA-His₆ in cell extracts. ∆mgtS mutant cells (GSO776) carrying pBAD24 derivatives expressing MgtS-GG-FLAG or MgtS-D30A-GG-FLAG were mixed with ∆mgtS mutant cells (GSO775) carrying pBAD24 derivatives expressing MgtA-His₆ or AcrB-His₆ at a ratio of 3:1. The cells were then homogenized and applied to Ni-NTA columns as described in SI Appendix. Control unmixed cells were treated similarly except that buffer was added to give the same volume. Bound proteins were eluted and analyzed on immunoblots using anti-His or M2 anti-FLAG antibodies. (B) Chromosomally encoded MgtS-GG-FLAG and MgtA-HA copurify. Cells expressing MgtS-GG-FLAG or MgtS-D30A-GG-FLAG with or without MgtA-HA (GSO777, GSO779, GSO778, and GSO780), all from the chromosome, grown for 2.5 h in N medium without added Mg²⁺, were homogenized and applied to Pierce anti-HA agarose as described in SI Appendix. Aliquots of cells (c) taken before lysis and bound proteins eluted from the beads (b) were analyzed on immunoblots using anti-His or M2 anti-FLAG antibodies.

Fig. 4.

MgtS acts via MgtA. (A) The ability of MgtS overexpression to complement the ∆mgtS growth curve defect requires mgtA. The OD₆₀₀ of cultures of ΔmgtS (GSO775) and ∆mgtS ∆mgtA (GSO781) carrying pBAD24 or pBAD-MgtS were measured at the indicated times as in Fig. 1C. The average of three cultures is shown. (B) Repression of Mg²⁺-reporter activity by MgtS is dependent on mgtA. β-Galactosidase activity was assayed for cultures of P_lac-mgtA 5′-UTR-lacZ ΔmgtS (GSO774) or P_lac-mgtA 5′-UTR-lacZ ΔmgtS ΔmgtA (GSO782) cells carrying pBAD24 or pBAD24-MgtS cells as described in SI Appendix. The average of three independent trials is shown, and the error bars represent one SD.

Fig. 5.

MgtS modulates MgtA stability. (A) Chromosomally encoded MgtA levels are lower in a ∆mgtS strain upon Mg²⁺ depletion. Cultures of mgtS⁺ mgtA-HA (GSO784) and ∆mgtS mgtA-HA (GSO786) were grown in N medium with 1 mM Mg²⁺ to OD₆₀₀ ∼0.4–0.6, whereupon one aliquot was taken (0 min). The remaining cells were washed and incubated with N medium without added Mg²⁺ as described in SI Appendix. (B) Chromosomally encoded MgtA levels are elevated upon MgtS overexpression. Cultures of ∆mgtS mgtA-HA (GSO786) cells carrying pBAD24 or pBAD24-MgtS were treated as in A, except that after cells were washed, arabinose was added. (C) MgtA accumulation requires continued MgtS production. Cultures of ∆mgtS mgtA-HA (GSO786) cells carrying pBAD24-MgtS were grown as in B, except that after 30 min in N medium with arabinose but lacking Mg²⁺, one aliquot was taken (0 min), and the remaining culture was split and one fraction was washed and grown in N medium lacking Mg²⁺ and arabinose, whereas the second fraction was washed and grown in N medium lacking Mg²⁺ with arabinose. (D) MgtA is stabilized by overexpression of the FtsH substrate cII. Overnight cultures of ∆mgtS mgtA-HA (GSO786) cells carrying pBAD24 or pBAD24-cII were treated as in B. In A–D, aliquots taken at each time point indicated were pelleted, resuspended to OD₆₀₀ ∼50 in SDS loading buffer, and separated by SDS/PAGE for immunoblot analysis using anti-HA antibodies.