Failure of an alkalophilic bacterium to synthesize ATP in response to a valinomycin-induced potassium diffusion potential at high pH

Starved whole cells of the obligately alkalophilic Bacillus firmus RAB synthesize ATP upon addition of L-malate at pH 9.0 as expected of an aerobic organism that grows oxidatively on nonfermentable carbon sources at pH values as high as 11.0. The current study was a detailed examination of the perplexing inability of such cells to exhibit ATP synthesis in response to a valinomycin-mediated potassium diffusion potential at pH 9.0. While there were minor differences in the patterns of generation of the potential and the proton influx that accompanies its generation in the three different buffering systems employed, the magnitude of the transmembrane electro-chemical potential of protons was at least as high as pH 9.0 as at pH 7.0. Nevertheless, a diffusion potential consistently energized ATP synthesis at pH 7.0 but not at 9.0; these findings were independent of the presence or absence of Tris or of Na+. By contrast, the artificial electron donor ascorbate, in the presence of phenazine methosulfate, energized ATP synthesis by the starved whole cells at both pH values. The same phenomenon, i.e., efficacy of a respiration-derived potential but not of a diffusion potential at pH 9.0, was demonstrated in ADP + Pi-loaded membrane vesicles. On the other hand, electrogenic Na+-coupled solute transport could be energized by both ascorbate/phenazine and methosulfate and a diffusion potential in the vesicles at pH 9.0. The results are discussed in connection with models of a localized path of proton flow between proton pumps and the ATP synthase.     

The fluorescence intensity of the lipophilic probe N-phenyl-1-naphthylamine responds to the oxidation-reduction state of purified Escherichia coli cytochrome o incorporated into phospholipid vesicles

N-Phenyl-1-naphthylamine (NPN), a reagent which has been used previously to probe the fluidity or microviscosity of the membrane lipids of intact cells of Escherichia coli, was found to respond to the redox state of purified cytochrome o incorporated into lipid vesicles formed from purified or E. coli phospholipids. NPN was bound to the proteoliposomes to produce a steady-state level of fluorescence intensity. Addition of the substrate ascorbate, in the presence of phenazine methosulfate as an electron donor, did not alter the fluorescence. However, following complete removal of oxygen from the medium by oxidation of the substrate by molecular oxygen catalyzed by cytochrome o, there was an increase in the fluorescence of NPN. This coincided with the reduction of cytochrome o. Reoxidation of the cytochrome by addition of oxygen decreased the fluorescence to steady-state levels until the oxidant had been completely reduced. The fluorescence changes were dependent on the incorporation of cytochrome o into phospholipid vesicles but were insensitive to the state of energization of the vesicle membrane.     

Reconstitution of the histidine periplasmic transport system in membrane vesicles. Energy coupling and interaction between the binding protein and the membrane complex

The periplasmic histidine transport system of Salmonella typhimurium has been reconstituted in isolated right-side-out membrane vesicles. The reconstituted system is entirely dependent on both the periplasmic protein, HisJ, and the membrane-bound complex, composed of proteins HisQ, HisM, and HisP. Transport is also dependent on the presence of ascorbate and phenazine methosulfate, which provide the energy for transport. Ascorbate oxidation generates a proton-motive-force, which allows ATP synthesis. ATP (or a cogenerated molecule) appears to be the immediate energy donor. Dissipation of the proton-motive-force or reduction of the level of ATP by a variety of treatments results in inhibition of transport. Vanadate inhibits transport, indicating that ATP utilization is necessary to energize transport. The interaction between liganded HisJ and the membrane complex has been measured directly: it displays Michaelis-Menten type kinetics, with a K1/2 of approximately 65 microM. The significance of this finding in terms of transport properties of whole cells is discussed.     

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.     

Ascorbate-phenazine methosulfate-dependent membrane energization in respiratory chain mutants of Escherichia coli

Ascorbate with phenazine methosulfate was able to energize the membrane of inside-out membrane vesicles from cytochrome-containing but not cytochrome-deficient cells of the E., coli, hem A^? mutant SASX76 as measured by the quenching of the fluorescence of acridine dyes. This substrate could also energize vesicle membranes from the ubiquinone-deficient mutant E., coli AN59 in the absence of exogenous ubiquinone. These results suggest that there is site of membrane energization coupled to substrate oxidation in the respiratory chain of E., coli in the cytochrome region between ubiquinone and oxygen.     

Energization of amino acid transport in energy-depleted Ehrlich cells and plasma membrane vesicles

We redirect attention to contributions to the energization of the active transport of amino acids in the Ehrlich cell, beyond the known energization by down-gradient comigration of Na⁺, beyond possible direct energization by coupling to ATP breakdown, and beyond known energization by exchange with prior accumulations of amino acids. We re-emphasize the uphill operation of System L, and by prior depletion of cellular amino acids show that this system must receive energy beyond that made available by their coupled exodus. After this depletion the Na⁺-independent accumulation of the norbornane amino acid, 2-aminobicycloheptane-2-carboxylic acid becomes strongly subject to stimulation by incubation with glucose. Energy transfer between Systems A and L through the mutual substrate action of ordinary amino acids was minimized although not entirely avoided by the use of amino acid analogs specific to each system.When 2,4-dinitrophenol was included in the depleting treatment, and pyruvate, phenazine methosulfate, or glucose used for restoration, recovery of uptake of the norbornane amino acid was independent of external Na⁺ or K⁺ levels. Restoration of the uptake of 2-(methylamino)isobutyric acid was, however, decreased by omission of external K⁺. Contrary to an earlier finding, restoration of uptake of each of these amino acids was associated with distinct and usually correlated rises in cellular ATP levels. ATP addition failed to stimulate exodus of the norbornane amino acid from plasma membrane vesicles, although either NADH or phenazine methosulfate did stimulate exodus. ATP production and use is thus associated with transport energization, although evidence for a direct role failed to appear.     

Amino acid transport systems in animal cells: Interrelations and energization

After summarizing the discrimination of the several transport systems of neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends to run forward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali-ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate-aspartate shuttle. After depletion of the energy reseves in the Ehrilich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restoration by pyruvate but in poor correlation with the restoration of alkali-ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly senstitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali-ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na⁺-independent system, as well as to one entering by the principal Na⁺-dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali-ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali-ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.     

Energy coupling sites in the electron transport chain of Zymomonas mobilis

Abstract Energy-coupling sites in the electron transport chain of the obligately fermentative aerotolerant bacterium Zymomonas mobilis were examined. The H⁺ /O stoichiometry of the electron transport chain in intact bacteria oxidizing ethanol was close to 3.3. Cytoplasmic membrane vesicles coupled NADH oxidation to ATP synthesis. With ascorbate/phenazine methosulfate they showed oxygen uptake which was sensitive to antimycin A, but no significant ATP synthesis could be detected. Cells with a defective coupling site I, prepared by cultivation on a sulfate-deficient medium, showed a decreased rotenone sensitivity of respiration, and they lacked almost all the respiration-driven proton translocation and ATP synthesis. We conclude that, despite the reported composition of the electron transport chain, only energy coupling site 1 was functional in Z. mobilis .     

Interrelation between salvage of purine nucleotides and protein synthesis in rat heart cells

The effect of transition from a respiring to a respiration-inhibited state on the rate of protein synthesis was investigated in glycolyzing, cultured rat heart cells. The rate was found to be significantly lower after blocking respiration, and it was further decreased by L-lactate. In contrast, pyruvate or phenazine methosulfate prevented the drop in the rate caused by lack of respiration. The changes in the respiratory state also affected the steady-state concentration of ATP, which varied in the same sense as the rate of protein synthesis. Pyruvate or phenazine methosulfate induced an increment in the concentration of ATP of respiration-inhibited cells. This increment could not be accounted for by more extensive phosphorylation of the available purine nucleotides, but required repletion of the pool by synthesis of purine nucleotides through the salvage pathway. Pyruvate and phenazine methosulfate were found to stimulate incorporation of labeled hypoxanthine into the purine nucleotide fraction in general, and into the nucleotide triphosphates in particular. Under similar incubation conditions an increase in the ATP/ADP ratio was also noted. The stimulatory effect of pyruvate on protein synthesis and on the cellular level of ATP was also observed in respiration-inhibited 3T6 cells and in human fibroblasts, but not in human fibroblasts deficient in the salvage enzyme, hypoxanthine-guanine-phosphoribosyltransferase. Based on the demonstrated influence of L-lactate, pyruvate, and phenazine methosulfate on the salvage synthesis of purine nucleotides [K. Ravid, P. Diamant, and Y. Avi-Dor, (1984) Arch. Biochem. Biophys. 229, 632-639] and on the present findings, the connection between protein synthesis and the salvage activity is discussed.     

Active transport of manganese in isolated membrane vesicles of Bacillus subtilis.

Membrane vesicles isolated from cells of bacillus subtilis W23 accumulate manganese in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrozone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 muM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCL2 or Mgcl2.     

C-terminal propeptide of the Caldariomyces fumago chloroperoxidase: an intramolecular chaperone?

The Na(+)/H(+) antiport activity encoded by the seven-gene mrp operons of Bacillus subtilis and alkaliphilic Bacillus pseudofirmus OF4 were cloned into a low copy plasmid, were expressed in several Escherichia coli mutant strains and compared side-by-side with similarly cloned nhaA, a major secondary antiporter from E. coli. All three antiporter systems exhibited electron donor-dependent antiport in a fluorescence-based vesicle assay, with NhaA being the most active. In whole cells of the same antiporter-deficient strain from which the vesicles were made, E. coli KNabc, Mrp-mediated Na(+) exclusion was significantly more protonophore-resistant than that conferred by NhaA. The Mrp systems were also more efficacious than NhaA: in supporting anaerobic Na(+) resistance in wild type and a terminal oxidase mutant strain of E. coli (SBS2115); and in increasing non-fermentative growth of an NADH dehydrogenase-minus E. coli mutant (ANN0222). The results suggest the possibility that the Mrp systems may have both secondary and primary energization capacities.     

Bacterial Calcium Transport: Energy-Dependent Calcium Uptake by Membrane Vesicles from Bacillus megaterium

Membrane vesicles capable of energy-dependent calcium uptake have been prepared from Bacillus megaterium cells in log-phase growth or when undergoing sporulation. The uptake is dependent on the calcium concentration and appears saturable in vesicles from cells in log-phase growth. Both ascorbate and phenazine methosulfate are needed as a source of electrons for the energy-dependent increase in calcium uptake. Addition of 8 mM sodium cyanide inhibited the energy-dependent uptake. If this calcium uptake mechanism is a component of the sporulation-specific calcium accumulation process, the latter's functional expression would appear to be inhibited during log-phase growth.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

Patterns of electrochemical proton gradient formation by membrane vesicles from an obligately acidophilic bacterium

Isolated membrane vesicles from the obligately acidophilic bacterium Bacillus acidocaldarius generated an electrochemical gradient of protons (delta mu- H+) upon energization with ascorbate-phenazine methosulfate at pH 6.0 or 3.0. At pH 6.0, there was little or no transmembrane pH gradient (delta pH), but a transmembrane electrical potential (delta psi) of ca. -77 mV, positive out, was observed. At pH 3.0, a delta pH equivalent to - 100 mV, acid out, and a delta psi of -73 mV, positive out, were observed upon energization. The total magnitude of the delta mu- H+ was higher than that of whole cells at acid pH, but the very large delta pHs and the reversed delta psi s, i.e., inside positive, that are typical of acidophile cells were not observed in the vesicles. The vesicles exhibited energy-dependent accumulation of alpha-aminoisobutyric acid that was inhibited by both nigericin and valinomycin (plus K+) at pH 3.0 but was inhibited little by nigericin at pH 6.0.     

Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.     

Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c

Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c-oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme.     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50 degrees C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H(+) into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H(+). 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH(4) (+)) prevented net H(+) translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD(+) supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H(+) by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.