Sodium and proton transport in Mycoplasma gallisepticum

Abstract

When washed cells of Mycoplasma gallisepticum were incubated at 37°C in 250 mM 22NaCl, the intracellular Na+ increased, and the K+ decreased. The addition of glucose to these Na+-loaded cells caused Na+ efflux and K+ uptake (both ions moving against concentration gradients). This effect of glucose was blocked by the ATPase inhibitor dicyclohexylcarbodiimide, which prevents the generation of a proton motive force in these cells. In additional experiments, Na+ extrusion was studied by diluting the 22Na+-loaded cells into Na+-free media and following the loss of 2Na' from the cells. Glucose stimulated 22Na' extrusion in such cells by a dicyclohexylcarbodiimide-sensitive mechanism. Proton movement was studied by measuring the pH gradient across the cell membrane with the 9-atninoacridine fluorescence technique. Glucose addition to cells preincubated with cations other than Na+ resulted in cell alkalinization (which was prevented by dicyclohexylcarbodiimide). This observation is consistent with the operation of a proton-extruding ATPase. When glucose was added to Na+-loaded cells and diluted into Na+-free media, intracellular acidification was observed, followed several minutes later by a dicyclohexylcarbodiimide-sensitive alkalinization process. The initial acidification was probably due to the operation of an Na+-H+ antiport, since Na+ exit was occurring simultaneously with H+ entry. When Na+-loaded cells were diluted into Na+-containing media, the subsequent addition of glucose resulted in a weak acidification, presumably due to H+ entry in exchange for Na+ (driven by the ATPase) plus a continuous passive influx of Na+. All of the data presented are consistent with the combined operation of an ATP-driven proton pump and an Na+-H+ exchange reaction.

Characterization and solubilization of the membrane-bound ATPase of Mycoplasma gallisepticum

“…The evidence presented here and in other investigations from this laboratory (25,26,34) is consistent with the view that the M. gallisepticum ATPase functions as an electrogenic H+ pump, a physiological process found in virtually all microorganisms (11). The evidence for this conclusion can be summarized as follows.…”

Analysis of energy sources forMycoplasma penetransgliding motility

“…Arsenate enters prokaryotic and eukaryotic cells via phosphate transporters (Rosen, ) and inhibits many reactions involving phosphate. These reactions include substrate‐level phosphorylation events leading to ATP synthesis via the glycolysis (Warburg & Christian, ) and arginine dihydrolase (Knivett, ) pathways, the only two means of ATP synthesis available to M. penetrans (Lo et al ., ; Sasaki et al ., ), as mycoplasma membrane ATP synthase actually hydrolyses ATP to create a proton gradient (Linker & Wilson, ). To confirm toxicity of arsenate to M. penetrans , cells were cultured in the presence of 10 mM sodium arsenate or sodium phosphate, pH 7.2.…”

“…Furthermore, M. penetrans cells were still able to glide well after 8 h in the presence of arsenate and at concentrations fivefold greater than those tested for M. mobile , both of which are conditions under which ATP is nearly completely depleted through inhibition of the reactions catalyzed by glyceraldehyde 3‐phosphate dehydrogenase (Warburg & Christian, ) and ornithine carbamoyltransferase (Knivett, ). As mycoplasma membrane ATP synthase actually operates in reverse to maintain a proton gradient functioning in sodium extrusion and cell volume maintenance (Linker & Wilson, ) and is therefore not involved in ATP synthesis, it is overwhelmingly likely that ATP is depleted under our experimental conditions, which include incubation in 25 times the concentration of arsenate that prevents growth. These data suggest that ATP hydrolysis is at best an indirect source of energy for motility in M. penetrans , perhaps only providing the energy necessary to replenish less stable molecular components of the motor and/or to maintain these components, such as by phosphorylation, which is essential for normal function of motility‐associated proteins in M. pneumoniae (Schmidl et al ., ).…”

Sodium ion transport decarboxylases and other aspects of sodium ion cycling in bacteria.

“…In contrast, the activity of the ATPase of M. gallisepticumn was only slightly stimulated by Na+ and Kions (37%c at maximum) and the effect was not specific since each monovalent cation slightly increased the catalytic rate (126). The experimental evidence points to Nai movements in this organism by the combined action of a proton-translocating ATIPase and an Nat/Ht antiporter (127).…”