GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter

Abstract

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.

FIG. 1

Effects of different concentrations of NaCl or KCl on the growth of E. coli strain KNabc or TK2420 transformed with control plasmid or pGerN. (A) Growth of E. coli KNabc transformants in the presence of increasing NaCl concentrations was measured after 15 h at 37°C. (B) Growth of E. coli TK2420 transformants in the presence of increasing KCl concentrations was measured after 15 h at 37°C. Open circles, control vector; solid circles, pGerN.

FIG. 2

Na⁺ or Li⁺/H⁺ antiport activity in everted membrane vesicles prepared from E. coli KNabc transformed with pGEM3Zf(+) or pGerN. Formation of a ΔpH was monitored via AO quenching at pH 8.0 after the addition of 2 mM Tris-d-lactate to a mixture containing 70 μg of vesicle protein. The figure shows the effects of adding (at the arrows) various amounts of KCl, LiCl, or NaCl after the steady-state level of ΔpH (100% quenching) had been established.

FIG. 3

Double-reciprocal plot of NaCl concentration versus percent dequenching in the AO fluorescence assay. The assay was performed as for Fig. 2 at pH 7.0 (●), 7.5(▴), or 8.0 (○) by measuring dequenching after addition of NaCl at various concentrations.

FIG. 4

Effect of KCl on ACMA fluorescence in right-side-out membrane vesicles prepared from E. coli KNabc or TK2420 transformed with pGEM3Zf(+) or pGerN. Right-side-out membrane vesicles were prepared with 10 mM NaCl inside and assayed for changes in ACMA fluorescence as described in Materials and Methods. Where indicated, 10 mM KCl was included in the assay buffer. (Top) Fluorescence in vesicles prepared from E. coli KNabc transformants. (Bottom) Fluorescence in vesicles prepared from E. coli TK2420 transformants.

FIG. 5

Uptake of ²²Na⁺ by everted membrane vesicles from E. coli KNabc transformed with pGerN upon energization and/or loading with K⁺. Membrane vesicles were either loaded (right) or not loaded (left) with K⁺ as indicated in Materials and Methods. Uptake of ²²Na⁺ was measured either with no further additions (○) or in the presence of either 10 μM CCCP (●), 2.5 mM Tris-d-lactate (▵), or lactate plus CCCP (▴).

FIG. 6

Effect of loading with K⁺ on a double-reciprocal plot of ²²Na⁺ uptake by everted membrane vesicles from E. coli KNabc transformed with pGerN. Initial uptake (10 s) of ²²Na⁺ was measured in Tris-d-lactate-energized membrane vesicles loaded (●) or not loaded (○) with K⁺.

FIG. 7

Effect of thiocyanate on ²²Na⁺ uptake by K⁺-loaded everted membrane vesicles prepared from E. coli KNabc transformed with pGerN. Vesicles were loaded with 2 mM KP_i (circles) or 2 mM KSCN (triangles) and diluted 1:10 into buffer containing 2 mM ²²NaP_i (circles) or 2 mM ²²NaSCN (triangles) in the absence (open symbols) or presence (closed symbols) of 2.5 mM Tris-d-lactate.

FIG. 8

Effects of combinations of energization, Na⁺ in trans, and CCCP on GerN-dependent efflux of ⁸⁶Rb⁺ from everted membrane vesicles prepared from E. coli TK2420 transformants. Vesicles were loaded with K⁺-⁸⁶Rb⁺ as indicated in Materials and Methods. Efflux was initiated by 10-fold dilution into buffer containing no Na⁺ or 10 mM Na⁺ as indicated. Efflux was performed either with no further additions (○) or in the presence of either 2.5 mM Tris-d-lactate (●), 10 μM CCCP (▵), or both lactate and CCCP (▴).

FIG. 9

Proposed antiport activities of GerN. Two modes of GerN-mediated antiport are shown. (Left) GerN-mediated Na⁺/H⁺ antiport. The assignment of a stoichiometry of 2H⁺ entering in exchange for 1 Na⁺ effluxing is completely hypothetical and represents the apparent electrogenicity of the antiport, i.e., the number of total coupling ions translocated per turnover is greater than the number of effluxing Na⁺ ions, so that a net positive charge moves inward. The geometric figures surrounding the two coupling ions suggest that these ions have distinct binding sites. The pH profile of Na⁺/H⁺ antiport suggests that protons compete with Na⁺ on the cytoplasmic side of the membrane. (Right) Na⁺/H⁺-K⁺ antiport by GerN is supported by the the finding of GerN-mediated and Na⁺-dependent Rb⁺ (K⁺) translocation with K⁺ replacing some, but not all, of the H⁺ ions that are transported in antiport with Na⁺. In the diagram, K⁺ is shown, hypothetically, as able to compete with H⁺ at only one of the H⁺ binding sites, and the antiport is shown as still requiring the full complement of coupling ions in this mode. GerN-mediated Na⁺/H⁺-K⁺ antiport is much more rapid than Na⁺/H⁺ antiport; K⁺ increases the velocity of the antiport without affecting the K_m for Na⁺.