Magnesium and the role of MgtC in growth of Salmonella typhimurium

Abstract

Salmonella typhimurium has three distinct transport systems for Mg2+: CorA, MgtA, and MgtB. The mgtCB operon encodes two proteins, MgtC, a hydrophobic protein with a predicted molecular mass of 22.5 kDa, and MgtB, a 102-kDa P-type ATPase Mg2+ transport protein. The mgtCB locus has been identified as part of a new Salmonella pathogenicity island, SPI-3. Transcription of mgtCB is regulated by extracellular Mg2+ via the two-component PhoPQ regulatory system important for virulence. To elucidate MgtC's role in a low-Mg2+ environment, we looked at growth and transport in strains lacking the CorA and MgtA Mg2+ transporters but expressing MgtB, MgtC, or both. mgtC mgtB+ and mgtC+ mgtB+ strains exhibited growth in N minimal medium without added Mg2+ with a 1- to 2-h lag phase. An mgtC+ mgtB strain was also able to grow in N minimal medium without added Mg2+ but only after a 24-h lag phase. In N minimal medium containing 10 mM Mg2+, all strains grew after a short lag phase; the mgtC+ mgtB strain grew to a higher optical density at 600 nm than an mgtC+ mgtB+ strain and was comparable to wild type. The lengthy lag phase before growth in an mgtC+ mgtB strain was not due to lack of expression of MgtC. Western blot analysis indicated that substantial MgtC protein is present by 2 h after suspension in N minimal medium. Surprisingly, in an mgtC+ mgtB+ strain, MgtC was undetectable during Mg2+ starvation, although large amounts of MgtB were observed. The lack of expression of MgtC is not dependent on functional MgtB, since a strain carrying a nonfunctional MgtB with a mutation (D379A) also did not make MgtC. Since, during invasion of eukaryotic cells, S. typhimurium appears to be exposed to a low-pH as well as a low-Mg2+ environment, the growth of an mgtC+ mgtB strain was tested at low pH with and without added Mg2+. While significant quantities of MgtC could be detected after suspension at pH 5.2, the mgtC+ mgtB strain was unable to grow at pH 5.2 whether or not Mg2+ was present. Finally, using 63Ni2+ and 57Co2+ as alternative substrates for the unavailable 28Mg2+, cation uptake could not be detected in an mgtC+ mgtB strain after Mg2+ starvation. We conclude that MgtC is not a Mg2+ transporter and that it does not have a primary role in the survival of S. typhimurium at low pH.

FIG. 1

Growth curves of strains possessing or lacking a functional mgtC. Strains MM1442 (wild-type LT2) (▾), MM281 (corA mgtA mgtC mgtB) (•), MM196 (mgtC mgtB) (■), and MM197 (mgtC⁺ mgtB) (▴) were grown overnight in N minimal medium supplemented with 10 mM or 10 μM Mg²⁺. Cultures were washed three times with N minimal medium without added Mg²⁺ and resuspended in a volume to give the same initial optical density (OD₆₀₀). The samples were diluted 1:200 in N medium containing various Mg²⁺ concentrations and incubated at 37°C. Aliquots were removed at the indicated times, and the OD₆₀₀ was measured. Data similar to that at 10 μM Mg²⁺ were obtained at Mg²⁺ concentrations below 0.5 mM.

FIG. 2

Growth analysis of strains grown in the presence of absence of mgtB. Cultures of MM1490 (mgtC⁺ mgtB⁺) (•) and MM1665 (mgtC⁺ mgtB) (■) were grown overnight in N minimal medium supplemented with 10 mM or 70 μM Mg²⁺. Cultures were processed as described in the legend to Fig. 1 and grown at various Mg²⁺ concentrations at 37°C. Aliquots were removed at the indicated times, and the OD₆₀₀ was measured. Growth similar to that seen at 70 μM Mg²⁺ was seen at other Mg²⁺ concentrations below 500 μM.

FIG. 3

Growth phenotype of the strain carrying only mgtC. LB plates were streaked with the following strains and incubated at 37°C for 24 h. Strains: 1, LT2 (wild type); 2, MM281 (corA mgtA mgtC mgtB); 3, MM1490 (mgtC⁺ mgtB⁺); 4, MM1665 (mgtC⁺ mgtB); 5, MM1733 (mgtC mgtB⁺); 6, MM1542 (mgtC⁺ mgtB[D379A]) The plate was scanned and contrast was adjusted by using Adobe Photoshop 4.0.

FIG. 4

Effect of mgtB on the detection of MgtC protein in whole cells. Strains MM1490 (A and C) and MM1665 (B) were grown overnight in 10 mM Mg²⁺, washed three times in N minimal medium without added Mg²⁺, resuspended in the same medium in the absence of Mg²⁺, and incubated at 37°C. Cell aliquots were removed at the indicated time points and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5 or 7.5% polyacrylamide gels followed by transfer to nitrocellulose. Proteins were visualized with anti-MgtC (A and B) or anti-MgtB (C) antibodies as described in Materials and Methods. The internal control was a band that reacted with both the preimmune serum and the anti-MgtC antibody.

FIG. 5

MgtC detection in an D379A MgtB mutant. Strain MM1542 was grown and samples were processed as described in the legend to Fig. 4.

FIG. 6

Effects of pH 5.2 and low Mg²⁺ concentration on growth in the presence of mgtC. MM1665 (•, ■) and MM281 (▴) were grown overnight in N minimal medium supplemented with 10 mM or 70 μM Mg²⁺. Cultures were washed with N minimal medium (pH 7.4 or pH 5.2) three times and resuspended in a volume to give the same initial OD₆₀₀. The samples were diluted 1:200 in N minimal medium at pH 7.2 (•) or pH 5.2 (■) containing various Mg²⁺ concentrations and incubated at 37°C. Aliquots were removed at the various time points, and the OD₆₀₀ was measured. Growth of MM281 at pH 7.4 was used as a negative control. Data similar to that shown for 70 μM Mg²⁺ were obtained at Mg²⁺ concentrations below 500 μM.

FIG. 7

Effect of pH on the detection of MgtC in whole cells. Strains MM1665 (A), MM1490 (B and C), and MM1737 (D) were grown in N minimal medium in the absence of Mg²⁺ at pH 5.2 (A to C) or pH 7.2 (D). Cell aliquots were removed at the indicated time points for electrophoresis and visualization of MgtC and MgtB proteins was performed as described in the legend to Fig. 4.