A protonmotive force as the source of energy for galactoside transport in energy depleted Escherichia coli.

An artificially produced electrochemical potential difference for protons (portonmotive force) provided the energy for the transport of galactosides in Escherichia coli cells which were depleted of their endogenous energy reserves. The driving force for the entry of protons was provided by either a transmembrane pH gradient or a membrane potential. The pH gradient across the membrane was created by acidifying the external medium. The membrane potential (inside negative) was established by the outward diffusion of potassium (in the presence of valinomycin) or by the inward diffusion of the permeant thiocyanate ion. The magnitude of the electrochemical potential difference for protons agreed well with magnitude of the chemical potential difference of the lactose analog, thiomethylgalactoside. The observations are consistent with the view that the carrier-mediated entry of each galactoside molecule is accompanied by the entry of one proton.     

The protonmotive force and beta-galactoside transport in Bacillus acidocaldarius.

The acidophilic and thermophilic bacterium, Bacillus acidocaldarius maintains a cytoplasmic pH between 5.85 and 6.31 over a range of external pH from 2.0 to 4.5. Consistently, the pH optimum of beta-galactosidase, as assayed in cell extracts, is between pH 6.0 and 6.5. An electrical potential (delta-psi), interior positive, is also maintained across the membrane. A delta-psi of approximately 34 mV was calculated from determinations of thiocyanate uptake by cells at pH 3.5. Addition of the proton conductor carbonyl cyanide m-chlorophenylhydrazone increased the delta-psi. Treatment of cells with valinomycin (in the absence of external potassium ions) or high concentrations of thiocyanate, to abolish the delta psi, resulted in collapse of the transmembrane proton gradient (delta pH). Active transport of methylthio-beta, D-galactoside occurred optimally at pH 3.5. Transport of the galactoside was inhibited by various compounds which could dissipate the transmembrane delta pH and by respiratory inhibitors. A decrease in the delta pH and an increase in the delta psi occurred upon addition of methylthio-beta, D-galactoside to cells of B. acidocaldarius. Thus the transport of this solute appears to involve an electrogenic symport with protons. The transport system is most active at 50 degrees C and shows little activity at 25 degrees C, although the delta pH is the same at the two temperatures. Gramicidin inhibits methylthio-beta, D-galactoside transport equally effectively at 50 degrees C and 25 degrees C, while nigericin inhibits only after a lag at 25 degrees C.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.     

Regulation of galactoside transport by the PTS

Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed. It is now well known that non-phosphorylated IIA(Glc) inhibits the transport via direct binding to the transporter. Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIA(Glc). It is concluded that there are multiple interaction sites for IIA(Glc) in these transporters.     

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).     

Evidence for a protonmotive force related regulatory system in Escherichia coli and its effects on lactose transport

Strains of Escherichia coli with mutations in the eup (energy-uncoupled phenotype) locus do not grow on nonfermentable carbon sources, have reduced growth yields on limiting glucose, are insensitive to colicins A and K, exhibit resistance to aminoglycoside antibiotics, and are defective in protonmotive force coupled active transport. eup mutations do not result in lowered protonmotive force. Here we show that deenergization of a eup+ strain results in the appearance of a new low KT, low Vmax form of the lactose carrier; in a strain deleted of the eup locus, deenergization does not evoke the low KT, low Vmax form of the lactose carrier. Cells bearing a eup point mutation and exhibiting the Eup- phenotype possess the low KT, low Vmax form of the lactose carrier even when energized. In addition to affecting the kinetic parameters of the lactose carrier, the eup point mutation also reduces the KT and Vmax of the proline carrier. On the basis of these findings, we suggest that the normal eup gene product mediates a novel regulation of lactose carrier function following deenergization. The defect in proline and lactose transport caused by eup point mutations may stem from an altered eup product aberrantly mediating the regulation under energized conditions. Finally, the pleiotropy associated with eup point mutations may be indicative of those protonmotive force driven functions that are subject to eup regulation.     

The requirement for energy during export of beta-lactamase in Escherichia coli is fulfilled by the total protonmotive force.

The energy requirement for the maturation and export of the plasmid-encoded TEM beta-lactamase in Escherichia coli K12 was shown to be fulfilled by the total protonmotive force. This was demonstrated by assessing the inhibition of proteolytic processing of the precursor form of beta-lactamase caused by perturbation of the energized state of the membrane in cells treated with valinomycin. The magnitude of the membrane potential was manipulated by varying the concentration of KCl in the medium and the pH gradient was manipulated by varying the external pH. Both components were simultaneously affected by addition of the protonophore carbonylcyanide-p- trifluoromethoxy phenylhydrazone (FCCP). Inhibition of processing was demonstrated in a mutant strain having a defective ATP synthase where protonmotive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition was not the result of decreased ATP concentration. Half-maximal accumulation of precursor of beta-lactamase was observed in all cases when the level of protonmotive force was decreased to approximately 150 mV. Under those conditions the membrane potential varied from 65 to 140 mV (internally negative) and the pH gradient from 95 to 25 mV (internally alkaline). Thus, the energy requirement is satisfied by the total protonmotive force, with no specificity for either the membrane potential or the pH gradient.     

Evidence that an ATPase and a protonmotive force function in the transport of acetylcholine into storage vesicles

PC12, a clonal line of rat pheochromocytoma, accumulates newly synthesized acetylcholine in storage granules. The accumulation of acetylcholine in PC12 granules, but not acetylcholine synthesis, was inhibited by treatment of the cells with any of several inhibitors of energy metabolism. These included nigericin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, dicyclohexylcarbodiimide, and iodoacetate. Valinomycin alone and oligomycin were without effect. Except for iodoacetate, these agents did not exert their effects on acetylcholine storage by depleting the cells of ATP.     

Absence of a unique relationship between active transport of lactose and protonmotive force in E. coli

The relationship between active transport of lactose via the lactose permease and the protonmotive force has been determined in E. coli cells using either the respiratory chain inhibitor cyanide or protonophores to decrease the protonmotive force progressively. In contradiction with the prediction of the delocalized chemiosmotic theory, two different relationships were obtained depending on the method used.     

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).     

The pH in the neighborhood of membranes generating a protonmotive force

The chemiosmotic mechanism provides a way whereby energy inherent in a chemical combustion process is extracted and transduced: first into the energy of electron X volts of the electron redox system and second into proton X volts as protons are forced to leave the interior of the cell, creating an electro-chemical protonic potential (the protonmotive force). Here we consider the distribution of potential and pH across the membrane and the phases bathing the membrane in more detail. The distribution of hydrogen ions parallel to the surface is also described. It is shown that the voltage and pH gradients due to the proton extrusion occur near to the membrane (approximately 2 nm). This implies that the pH is much lower immediately outside the membrane than in the cytoplasm or in usual neutral growth or isotonic media. It provides a link between the points of view of Mitchell and Williams. It requires that literature models for the role of the protonmotive force in the maintenance of wall thickness in Gram-positive organisms, the adhesion of microbes to surfaces, and the transport of auxin in plants be modified.     

Fast measurement of galactoside transport by lactose permease

Lactose permease of Escherichia coli was reconstituted into vesicles of dimyristoylphosphatidylcholine, and the rate of galactoside counterflow was measured in the millisecond time range. The turnover number and the half-saturation constant for transport agree with the values known for cells. This result demonstrates that lactose permease is the sole protein necessary for galactoside transport. Furthermore, lactose permease seems not to require a high level of negatively charged lipids or a certain degree of unsaturation of the lipid hydrocarbon chains. However, the lipids must be in the fluid state, because the transport rate drastically decreases below the lipid ordered fluid phase transition.     

A sodium ion gradient as energy source for Peptostreptococcus asaccharolyticus

The determination of enzymatic activities in cell-free extracts of Acidaminococcus fermentans and Peptostreptococcus asaccharolyticus led to a refined scheme for the pathway of glutamate fermentation via (R)-2-hydroxyglutarate to acetate and butyrate. From the ratio of these products the amount of ATP generated by substrate level phosphorylation was calculated. Growth experiments with the organisms including Clostridium symbiosum and Clostridium tetanomorphum indicated that a sodium gradient contributed additional energy for growth. The high growth yields found in organisms containing the biotin dependent sodium pump glutaconyl-CoA decarboxylase could be reduced by the sodium ionophor monensin. In P. asaccharolyticus energy equivalent up to 0.6 mol ATP per mol of glutaconyl-CoA decarboxylated was conserved via the Na⁺ gradient. The data may explain the growth promoting effects of monensin in cattle.     

Formate transport,growth inhibition and the membrane protonmotive force in two methylotrophs (T15 and L3)

Summary Formate transport and the effect of formate on growth and the membrane protonmotive force were studied in two ribulose-monophosphate-type obligate methylotrophs (bacterial strains T15 and L3). Formate was accumulated very fast by the membrane pH according to the general transport mechanism of short-chain organic acids. Formate accumulation was reduced or abolished by a number of factors (protonophores, high extracellular pH, cell-starvation conditions) that reduced or abolished the pH. Formate transport was accompanied by removal of protons from the medium by the cells. Since protons are released by the cells upon substrate oxidation, the stoichiometry of proton uptake upon formate transport could not be directly determined, although data suggest that formate is cotransported with one proton. The net rate of proton removal from the medium by the cells due to formate transport and oxidation increased with increasing formate concentrations or decreasing medium-pH values. The membrane protonmotive force of strain T15 was also studied as a function of the pH. High formate concentrations (of 100 to 400 mM) reduced the membrane pH by ca. 20 to 60% and the growth rate by ca. 20 to 100% for both strains but to a different extent. For example, 400 mM formate inhibited growth by ca. 60% in strain T15 and by 100% in strain L3. The nature of growth inhibition by formate is discussed in some detail.