Evidence for Na(+) influx via the NtpJ protein of the KtrII K(+) uptake system in Enterococcus hirae

Abstract

The ntpJ gene, a cistron located at the tail end of the vacuolar-type Na(+)-ATPase (ntp) operon of Enterococcus hirae, encodes a transporter of the KtrII K(+) uptake system. We found that K(+) accumulation in the ntpJ-disrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (DeltaPsi; inside negative) by 3, 3'-dihexyloxacarbocyanine iodide fluorescence revealed that the DeltaPsi was hyperpolarized at pH 10 in JEM2; the DeltaPsi values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K(+) or Rb(+) in the presence of valinomycin, were -118 and -160 mV, respectively. DeltaPsi generation at pH 10 was accomplished by an electrogenic Na(+) efflux via the Na(+)-ATPase, whose levels in the two strains were quite similar. Na(+) uptake driven by an artificially imposed DeltaPsi (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na(+) movement in addition to K(+) movement. Finally, the growth of JEM2 arrested in K(+)-limited high-Na(+) medium at pH 10 was restored by addition of valinomycin. These results suggest that NtpJ mediates electrogenic transport of K(+) as well as Na(+), that it likely mediates K(+) and Na(+) cotransport, and that Na(+) movement via NtpJ is the major Na(+) reentry pathway at high pH values.

FIG. 1

Effects of valinomycin on K⁺ accumulation at pH 10. Strains ATCC 9790 (A) and JEM2 (B) were grown in NaTY medium (pH 10), loaded with Na⁺, and suspended in 0.1 M Na⁺-CHES buffer (pH 10) at a cell density equivalent to 1 mg of protein/ml. The suspension was (○) or was not (▵) supplemented with 10 mM glucose at 0 min. Valinomycin (30 μM) was added together with glucose at 0 min (●); K⁺ uptake was initiated by addition of 2 mM KCl at 10 min. The cellular K⁺ contents were determined by flame photometry.

FIG. 2

Time course of changes in DiOC₆(3) fluorescence in E. hirae cells. Sodium-loaded cells of ATCC 9790 (A), JEM2 (B), and Nak1 (C) were incubated at a density of 0.5 mg/ml in 0.1 M Na⁺-CHES (pH 10) with 1 μM DiOC₆(3). Fluorescence quenching was initiated by addition of 10 mM glucose (glc) followed by addition of valinomycin (val; 30 μM) and KCl (10 mM). In the experiment shown in panel C, valinomycin and KCl were added simultaneously.

FIG. 3

K⁺ accumulation in the presence of valinomycin at various concentrations. Sodium-loaded cells of strains ATCC 9790 (A) and JEM2 (B) cultured in NaTY medium (pH 10) were suspended in 0.1 M Na⁺-CHES buffer (pH 10) at a cell density equivalent to 1 mg of protein/ml. The suspension was supplemented with 10 mM glucose and 30 μM valinomycin at −10 min; K⁺ accumulation was initiated by addition of KCl at various concentrations at 0 min. The established K⁺ concentration gradients are shown in parentheses. ○, 20 mM KCl; ●, 10 mM KCl; ▵, 5 mM KCl; ▴, 2 mM KCl; □, 1 mM KCl; ■, 0.5 mM KCl; ×, 0.2 mM KCl.

FIG. 4

Western blotting of cell lysates after denaturing polyacrylamide gel electrophoresis. The cell lysates were prepared from strains ATCC 9790 (lanes 1 and 2) and JEM2 (lanes 3 and 4) grown in NaTY medium (pH 10) as described elsewhere (24). Lysates (5 μg, lanes 1 and 3; 10 μg, lanes 2 and 4) were electrophoresed, immunoblotted with antiserum against purified V₁-ATPase (dilution, 1:3,000), and visualized by the alkaline phosphatase system.

FIG. 5

Movements of Na⁺ and K⁺ at pH 10. (A and B) Passive Na⁺ uptake. Sodium uptake was initiated by suspending potassium-loaded cells of strain ATCC 9790 (A) or JEM2 (B) into 200 mM Na⁺-CHES (pH 10) (●), 180 mM N-methylglucamine-CHES containing 20 mM Na⁺-CHES (pH 10) (▴), or 200 mM N-methylglucamine-CHES containing 2 mM Na⁺-CHES (pH 10) (■), respectively. (C and D) ΔΨ-driven Na⁺ uptake. Potassium-loaded cells of strain ATCC 9790 (C) or JEM2 (D) were suspended in 50 mM N-methylglucamine-CHES (pH 10) buffer containing 2 mM NaCl at −10 min, and the cellular Na⁺ (triangles) and K⁺ (circles) contents were monitored; 30 μM valinomycin was added at 0 min (closed symbols).

FIG. 6

Effects of valinomycin on the growth of E. hirae at pH 10. Strains ATCC 9790 (A) and JEM2 (B) were cultured in mNaTY (K⁺-limited NaTY) complex medium at pH 8 (○). At an optical density at 540 nm of 0.08, the medium pH was shifted to 10 by addition of 80 mM Na₂CO₃ (●). Valinomycin (30 μM) (▵) or KCl (20 mM) (▴) was added 2 min after the addition of the Na₂CO₃. The cell growth was monitored by cell density measurement.

FIG. 7

Sodium circulation and potassium accumulation in E. hirae at a high pH: an interpretation. (A) Wild type. Elements shown are the Na⁺-translocating vacuolar ATPase; the KtrII (NtpJ) (J) system for electrogenic accumulation of K⁺ as well as Na⁺ (cotransport), which presumably interacts with a KtrA-like component modulated by NAD(H); and a leaky pathway for Na⁺. (B) NtpJ mutant. Valinomycin (V)-mediated K⁺ accumulation was monitored. The low-affinity K⁺ transport system described in the text was omitted.