Adenosine 5''-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli.

Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Protonmotive force as the source of energy for adenosine 5''-triphosphate synthesis in Escherichia coli.

Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).     

The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles

The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated. Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential). Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.     

Calcium transport driven by a proton gradient and inverted membrane vesicles of Escherichia coli.

Calcium transport into inverted vesicles of Escherichia coli was observed to occur without an exogenous energy source when an artificial proton gradient was used. The orientation of the proton gradient was acid inside and alkaline outside. Either phosphate or oxalate was necessary for transport, as was found for respiratory-driven or ATP-driven uptake (Tsuchiya, T., and Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692). Phosphate accumulation was found to occur in conjunction with calcium accumulation. Calcium transport driven by an artificial proton gradient was stimulated by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ATPase (EC 3.6.1.3). Valinomycin, which catalyzes electrogenic potassium movement, stimulated calcium accumulation, while nigericin, which catalyzes electroneutral exchange of potassium and protons, inhibited both artificial proton gradient-driven transport and respiratory-driven transport. Other properties of the proton gradient-driven system and the previously reported energy-linked calcium transport system are similar, indicating that calcium is transported by the same carrier whether energy is supplied through an artificial proton gradient or an energized membrane state. These results suggest the existence of a calcium/proton antiport.     

Evidence for an electrogenic 3-deoxy-2-oxo-D-gluconate--proton co-transport driven by the protonmotive force in Escherichia coli K12.

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.     

Efficient membrane assembly of the KcsA potassium channel in Escherichia coli requires the protonmotive force

Very little is known about the biogenesis and assembly of oligomeric membrane proteins. In this study, the biogenesis of KcsA, a prokaryotic homotetrameric potassium channel, is investigated. Using in vivo pulse–chase experiments, both the monomeric and tetrameric form could be identified. The conversion of monomers into a tetramer is found to be a highly efficient process that occurs in the Escherichia coli inner membrane. KcsA does not require ATP hydrolysis by SecA for insertion or tetramerization. The presence of the protonmotive force (pmf) is not necessary for transmembrane insertion of KcsA; however, the pmf proved to be essential for the efficiency of oligomerization. From in vivo and in vitro experiments it is concluded that the electrical component, Δψ, is the main determinant for this effect. These results demonstrate a new role of the pmf in membrane protein biogenesis.     

Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force.

The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (delta p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a delta p, inside acid and positive. delta p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. delta psi and delta pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed delta p. These results support a model [Schiebel, E., Driessen, A.J.M., Hartl, F.-U. and Wickner, W. (1991) Cell, 64, 927-939] in which the delta p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the delta p challenges models involving delta p-dependent membrane destabilization and provides further evidence for a role of the delta p as driving force in precursor protein translocation.     

ATP synthesis by an artificial proton gradient in right-side-out membrane vesicles of Escherichia coli.

Membrane vesicles formed from spheroplasts of E. coli lysed in the presence of ADP and P_i produced ATP when an artificial proton gradient (acid outside) was formed across the membrane. ATP synthesis required Mg²⁺ and ADP, was inhibited by dicyclohyxylcarbodiimide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and stimulated by valinomycin in the presence of KCl. Synthesis was absent in a mutant lacking the Mg²⁺-ATPase. The optimum external pH was 2.5 when the internal pH was 8.2. Oxidative phosphorylation driven by D-lactate or succinate was also observed.     

Magnitude of the protonmotive force in respiring Staphylococcus aureus and Escherichia coli.

The membrane potential and pH gradient developed across the plasma membranes of whole cells of Staphylococcus aureus and spheroplasts of Escherichia coli were estimated. The distributions of potassium ions in the presence of valinomycin and the pH gradient across the membrane were determined from the changes in pK and pH observed in the external medium during transition from the energized respiring state to the de-engerized resting condition. The protonmotive force in respiring cells was estimated at 211 mV for S. aureus and 230 mV for E. coli at external pH values of approximately 6.5. The adequacy of these protonmotive forces as a driving force for substrate accumulation or adenosine 5'-triphosphate synthesis is discussed.     

Glutamate transport driven by an electrochemical gradient of sodium ion in membrane vesicles of Escherichia coli B.

Membrane vesicles prepared from $\text{E. coli}$ B strain 29–78 require Na⁺ for the accumulation of glutamate. Respiratory-driven transport of glutamate but not lysine is sensitive to the ionophore monensin. An artificially-imposed sodium gradient and/or membrane potential drives glutamate uptake. These results suggest that glutamate is accumulated via a Na⁺/glutamate symport.     

Adenosine 5'-triphosphate synthesis in Escherichia coli submitted to a microsecond electric pulse

The total cytoplasmic ATP content (bound and free) increased in Escherichia coli when the bacteria were submitted to electric pulses with field strengths of 1-6 kV/cm and a decay time of 7-20 microseconds. The electron-transport chain was blocked by cyanide, and ATP synthesis was detected by a luminescence assay. The amount of newly formed ATP depends on the field strength. A total of 150 pmol of ATP was formed per milligram of bacteria submitted to a 3 kV/cm pulse. Synthesis was blocked by uncouplers and ionophores (valinomycin). The F1F0-ATP synthase inhibitor dicyclohexylcarbodiimide blocked a large part of this synthesis. Synthesis was not induced in unc mutants (unc B, unc D). The synthesis of ATP is related to the induced transmembrane potential, not to the Joule heating. A minimum 35-50-mV increase in membrane potential must be maintained for at least 12 microseconds to trigger this synthesis. This very fast energy transduction in bacteria is in good agreement with our previous results concerning submitochondrial particles. Because of the localized character of the induced membrane potential, these results are in agreement with the recent hypothesis of "mosaic proton coupling".     

A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2 coded lysis protein dissipates the protonmotive force in Escherichia coli membrane vesicles by generating hydrophilic pores.

The RNA phage MS2 encodes a protein, 75 amino acids long, that is necessary and sufficient for lysis of the host cell. DNA deletion analysis has shown that the lytic activity is confined to the C-terminal half of the protein. We have examined the effects of a synthetic peptide, covering the C-terminal 25 amino acids of the lysis protein, on the electrochemical potential, generated in Escherichia coli membrane vesicles and in liposomes reconstituted with cytochrome c oxidase. In all cases the peptide dissipates the electrochemical potential. The peptide also induces the release of carboxyfluorescein (376 daltons), but not of inuline (5500 daltons), from protein-free liposomes. The phenomena are observed at a lipid to peptide molar ratio of approximately 100:1. The possible connection between the dissipation of the proton-motive force and bacteriolysis is discussed.     

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.