Copyright � 1996, American Society for Microbiology Sodium-Coupled Energy Transduction in the Newly Isolated (original) (raw)
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Sodium-coupled energy transduction in the newly isolated thermoalkaliphilic strain LBS3
Journal of …, 1996
Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50؇C. Since a high concentration of Na ؉ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na ؉ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na ؉ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na ؉ and Li ؉ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed.
2019
Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50؇C. Since a high concentration of Na ؉ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na ؉ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na ؉ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na ؉ and Li ؉ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed.
Solute transport and energy transduction in bacteria
Antonie van Leeuwenhoek, 1994
In bacteria two forms of metabolic energy are usually present, i.e. ATP and transmembrane ion-gradients, that can be used to drive the various endergonic reactions associated with cellular growth. ATP can be formed directly in substrate level phosphorylation reactions whereas primary transport processes can generate the ion-gradients across the cytoplasmic membrane. The two forms of metabolic energy can be interconverted by the action of ion-translocating ATPases. For fermentative organisms it has long been thought that ion-gradients could only be generated at the expense of ATP hydrolysis by the FoF1-ATPase. In the present article, an overview is given of the various secondary transport processes that form ion-gradients at the expense of precursor (substrate) and/or end-product concentration gradients. The metabolic energy formed by these chemiosmotic circuits contributes to the 'energy status' of the bacterial cell which is particularly important for anaerobic/fermentative organisms.
Purification and Reconstitution of Na+-translocating Vacuolar ATPase from Enterococcus hirae
Journal of Biological Chemistry, 1997
Vacuolar ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. An unusual member of this family, a sodium-translocating ATPase, has been found in the eubacterium Enterococcus hirae. We report here the purification of enterococcal Na ؉-ATPase from the plasma membrane of cells, whose ATPase content was highly amplified by expression of the cloned ntp operon that encodes this Na ؉-ATPase (ntpFIKECGABDHJ). The purified enzyme appears to consist of nine Ntp polypeptides, all the above except for the ntpH and ntpJ gene products. ATPase activity was strictly dependent on the presence of Na ؉ or Li ؉ ions and was inhibited by nitrate, N-ethylmaleimide, and the peptide antibiotic destruxin B. When the purified ATPase was reconstituted into liposomes prepared from Enterococcus faecalis phospholipids, ATPdriven Na ؉ uptake was observed; uptake was blocked by nitrate, destruxin B, and monensin, but it accelerated by carbonyl cyanide m-chlorophenylhydrazone and valinomycin. These data demonstrate that E. hirae Na ؉-ATPase is an electrogenic sodium pump of the vacuolar type. This is a promising system for research on the fundamental molecular structure and mechanism of vacuolar ATPase.
K+-dependent Na+ transport driven by respiration in Escherichia coli cells and membrane vesicles
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996
Respiration-driven Na + transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K +. Cells from an E. coli mutant deficient in three major K + transport systems were incapable of accumulating K + or expelling Na + unless valinomycin was added. Membrane vesicles from an E. coli mutant from which the genes encoding the two known electrogenic Na+/nH + antiporters nhaA and nhaB were deleted transported Na + as well as did vesicles from wild-type cells. Quantitative analysis of A~b and ApH showed a high driving force for electrogenic Na+/nH + antiport whether K + was present or not, although Na + transport occurred only in its presence. These results suggest that an Na+/nH + antiporter is not responsible for the Na + transport. Respiration-driven effiux of Na + from vesicles was found to be accompanied by primary uphill effiux of K +. Also, no respiration-dependent effiux of K + was observed in the absence of Na +. Such coupling between Na + and K + fluxes may be explained by the operation of an Na +, K+/H + antiporter previously described in E. coli membrane vesicles (Verkhovskaya, M.L., Verkhovsky, M.I. and WikstrSm, M. (1995) FEBS Lett. 363, 46-48). Active Na + transport is abolished when A~H~ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na + transport. Such an effect may be expected if the Na +, K+/H + antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump.
Journal of Bacteriology, 1994
Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of-76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 ,uM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 ,M. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 ,LM. At Na+ concentrations above 250 ,uM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na+-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.
Na+/solute symport in membrane vesicles from Bacillus alcalophilus
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1982
The characteristics of cx-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited 0l-aminoisobutyric acid uptake upon energization with ascorbate and N, N,N',N'-tetramethyl-p-phenylenediamine. The presence of Na ÷ caused a pronounced reduction in the K m for a-aminoisobutyric acid in wildtype but not KM23 vesicles; the maximum velocity (I0 was unaffected in vesicles from both strains. Passive efflux and exchange of ÷x-aminoisobutyric acid from wild-type vesicles were Na÷-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of a-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na÷-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in L-methionine transport indicated that the porters, i.e., the solnte-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na÷-independent, H÷-coupled porters upon mutation of B. alcalophilus to non-alkalophily.