ATP-driven sodium pump in Streptococcus faecalis.

Abstract

Sodium extrusion by bacteria is generally attributed to secondary antiport of Na' for H4 energized by the proton circulation. Streptococcs faecalis is an exception, in that sodium expulsion from intact cells requires the generation ofATP but does not depend on the protonmotive force. Unfortunately, studies with everted membrane vesicles failed to reveal the expected sodium pump; instead, the vesicles contained a conventional secondary Na4/H4 antiporter. We report here that everted membrane vesicles prepared in the presence of protease inhibitors retain an ATP-driven sodium transport system. The evidence includes the findings that (i) accumulation of2-Na4 by these vesicles is resistant to reagents that dissipate the protonmotive force but requires ATP and (ii) the vesicles contain a sodium-stimulated ATPase that is distinct from FIFO ATPase, and whose presence is correlated with sodium transport activity. Sodium movements appear to be electroneutral and are accompanied by movement of H4 in the opposite direction. When membranes are incubated in the absence of protease inhibitors, a secondary Na4/H4 antiport activity emerges, possibly by degradation ofthe sodium pump. We suggest that S. faecalis expels Na4 by means of an ATP-driven primary transport system that mediates exchange ofNa4 for H+. The Na+/ H+ antiporter seen in earlier membrane preparation is an artefact of proteolytic degradation.Virtually all cells expel sodium ions from the cytoplasm by active transport ofone kind or another. In animal cells, this is the task of the Na+-K+ ATPase. In bacteria, and probably also-in lower eukaryotes and in plants, sodium is extruded by exchange for protons. Since all these organisms maintain a substantial electrochemical proton gradient (interior alkaline and negative), antiport of Na+ for H4 can expel Na4 against a substantial gradient and no direct coupling to ATP, or other energy donor, should be required. This hypothesis, first proposed by Mitchell (1) in the context of his chemosmotic theory, has received substantial support from bacterial physiologists (for review, see ref.2), and it is generally accepted that Escherichia coli, Azotobacter, alkalophilic bacilli, and other bacteria extrude sodium by secondary Na4/H+ antiport (2-7). The discovery by MacDonald and his colleagues (8, 9) of a light-driven sodium pump in halobacteria was the first indication ofdiversity in bac-. terial sodium transport mechanisms. However, even in this case, the major pathway of sodium extrusion may be secondary Na+/H4 antiport; the light-energized pump appears to make but a minor contribution to the total Na+ flux (10).From the beginning, Na+ extrusion by Streptococcusfaecalis did not quite conform to the hypothesis of secondary Na+/H+ antiport, in that both net Na+ movement and 'Na4/Na+ exchange were observed only in cells capable of generating ATP (11). In a detailed study with intact cells, we (12) demonstrated that glycolyzing cells could extrude Na4 against a 100-fold concentration gradient in the presence of reagents t...

Electrogenic Na+ transport by Enterococcus hirae Na+‐ATPase

“…Na+/H * antiporter functions to extrude Na + from the cytoplasm at low pH where the proton potential is generated by FoFj, H+-ATPase, and Na+-ATPase functions to extrude Na ÷ at high pH where generation of the proton potential is minimal [4]. In the early work on Na+-ATPase, it was speculated that Na+-ATPase functions electroneutrally and may exchange Na + for H + [3]. Later Kakinuma and Harold reported the Na ÷-ATPase-dependent K ÷ uptake (KtrII) activity in whole cells; an apparently equimolar exchange of internal Na + for external K + took place in the absence of proton potential [5].…”

“…Mutant 7683 lacks Na ÷ extrusion activity; that is, both the Na+-ATPase and Na÷/H + antiporter are defective [17]. R-I is a revertant of 7683, which had recovered only the activity of the Na÷/H + antiporter, while R-II is another revertant that had recovered both Na + extrusion activities [13]. Glucose-dependent A N generation of-41 mV, which was resistant to DCCD, was generated in the parent strain 9790, but it was not observed in 7683.…”

“…The Gram-positive bacterium Enterococcus hirae, which lacks the respiratory chain, has two sodium extrusion systems: Na+/H ÷ antiporter [1,2] and Na+-translocating ATPase [3]. Na+/H * antiporter functions to extrude Na + from the cytoplasm at low pH where the proton potential is generated by FoFj, H+-ATPase, and Na+-ATPase functions to extrude Na ÷ at high pH where generation of the proton potential is minimal [4].…”

“…In some experiments, sodium chloride was added to the culture in KTY medium to induce the Na ÷-ATPase activity [4]. In order to induce the arginine deiminase pathway [13], cells were grown on NaTY medium containing 1% arginine and 0.1% galactose instead of 1% glucose.…”

“…hirae ATCC 9790 or mutants derived from this strain were used: these were strain 7683, a mutant defective in sodium extrusion, and its revertants R-1 and R-II [3], and mutant AS25, which is defective in FoFI-ATPase and in proton extrusion [12]. Cells were grown on complex media [5], NaTY (1% tryptone, 0.5% yeast extract, 1% glucose and 0.85% Na2HPO4) or KTY (tryptone, yeast extract and glucose as above, and 1% K2HPO 4 in place of NazHPO4).…”