A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus

Abstract

We cloned several genes encoding an Na+/H+ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+ conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.

FIG. 1

Effect of Na⁺ and Li⁺ on the growth of cells. E. coli TG1 (■), KNabc (○), and KNabc/pNAS20 (•) were grown in L(K) medium containing various concentrations of NaCl (A) or LiCl (B) under aerobic conditions at 37°C.

FIG. 2

Na⁺/H⁺ antiport and Li⁺/H⁺ antiport activities in membrane vesicles. Everted membrane vesicles were prepared from E. coli KNabc and KNabc/pNAS20 cells by the French press method. Antiport activities were measured by the quinacrine fluorescence quenching method. At the time point indicated by the first arrow on the left, potassium lactate (final concentration, 5 mM) was added to the assay mixture to initiate respiration. At time point indicated by the second arrow from the left, NaCl (final concentration, 5 mM) or LiCl (final concentration, 5 mM) was added.

FIG. 3

Plasmids and restriction map. Physical maps of the DNA insert derived from the S. aureus chromosome in each plasmid are shown. The DNA inserts are aligned. Restriction sites are present at the same horizontal position in each insert. Locations and directions of each ORF (mnhA to mnhG) which were revealed by sequencing are shown at the bottom. The probe used for the Southern analysis is also shown. The growth capability of E. coli KNabc harboring each plasmid in L(K) medium supplemented with 0.2 M NaCl is shown on the right. Symbols: +, cell growth occurred; −, no cell growth occurred.

FIG. 4

Hydropathy patterns of deduced Mnh proteins. Hydropathy values were calculated by the method of Kyte and Doolittle (28) for the deduced amino acid sequences of MnhA to MnhG. The values were plotted from the NH₂ terminus to the COOH terminus. The portions above and below the midpoint line indicate hydrophobic and hydrophilic regions, respectively. The hydrophobic regions are shaded.

FIG. 5

Na⁺ extrusion from cells elicited by respiration. Extrusion of Na⁺ from cells of E. coli TG1 (A and B), KNabc (C and D), and KNabc/pNAS20 (E and F) was measured with an Na⁺ electrode. At the time point indicated by the arrow labeled H₂O₂, a small amount (5 μl of a 0.5% solution) of H₂O₂ was added to the cell suspension (2.5 ml) to supply O₂ and to initiate respiration. In three cases (B, D, and F), CCCP was present at 100 μM in the assay medium. A downward deflection represents Na⁺ extrusion from the cells.

FIG. 6

Effects of pH on cell growth of E. coli HITΔAB⁻ and HITΔAB⁻/pNAS20. E. coli HITΔAB⁻ (○) and HITΔAB⁻/pNAS20 (•) were grown in minimal medium supplemented with glycerol at the indicated pHs at 37°C under aerobic conditions.