Escherichia coli intracellular pH, membrane potential, and cell growth.

We studied the changes in various cell functions during the shift to alkaline extracellular pH in wild-type Escherichia coli and in strain DZ3, a mutant defective in pH homeostasis. A rapid increase in membrane potential (delta psi) was detected in both the wild type and the mutant immediately upon the shift, when both cell types failed to control intracellular pH. Upon reestablishment of intracellular pH - extracellular pH and growth in the wild type, delta psi decreased to a new steady-state value. The electrochemical proton gradient (delta muH+) was similar in magnitude to that observed before the pH shift. In the mutant DZ3, delta psi remained elevated, and even though delta muH+ was higher than in the wild type, growth was impaired. Cessation of growth in the mutant is not a result of cell death. Hence, the mutant affords an interesting system to explore the intracellular-pH-sensitive steps that arrest growth without affecting viability. In addition to delta muH+, we measured respiration rates, protein synthesis, cell viability, induction of beta-galactosidase, DNA synthesis, and cell elongation upon failure of pH homeostasis. Cell division was the only function arrested after the shift in extracellular pH. The cells formed long chains with no increase in colony-forming capacity.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride

Endocytic vesicles possess an electrogenic proton pump, and measurements of ATPase activity suggest that Cl- may stimulate proton pump activity. This study was undertaken to measure the steady-state pH, potential (delta psi), and total proton electrochemical gradients established by the rat liver multivesicular body (MVB) proton pump and to examine the effects of Cl- (0.5-140 mM) on these gradients. Radiolabeled [( 14C] methylamine and 36Cl-) and fluorescent (fluorescein isothiocyanate-conjugated low density lipoproteins) probes were used to assess internal pH (pHi) and delta psi. In the absence of ATP, pHi averaged 7.37 +/- 0.05 (extracellular pH 7.31 +/- 0.02), and delta psi ranged from -32 to -71 mV; but neither pHi nor delta psi varied consistently with [Cl-]. In the presence of ATP, pHi decreased progressively with increasing [Cl-] to a plateau value of about 5.89 at greater than or equal to 25 mM Cl-, and MVB exhibited an interior positive delta psi that was maximal at the lowest Cl- concentration (+65.5 mV) and decreased as medium Cl- increased. The total ATP-dependent proton electrochemical gradient (proton-motive force (delta p] averaged 118.0 +/- 4.3 mV and did not change in any consistent manner as [Cl-] varied almost 300-fold. However, initial rates of MVB acidification increased with increasing [Cl-]. These studies indicate that: (a) in the absence of ATP, isolated MVB exhibited a negative delta psi, probably a Donnan potential; (b) in the presence of ATP and at a [Cl-] similar to that in hepatocyte cytoplasm (25 mM), MVB pHi was 5.89, and delta psi was +9.6 mV; and (c) over the range of [Cl-] tested, the magnitudes of delta pH and delta psi were inversely related, apparently related to Cl- availability, but the ATP-dependent delta p did not vary. Therefore, it is concluded that Cl- increases the initial rate of vesicle acidification in MVB and also affects the relative chemical and electrical contributions of the steady-state proton pump-determined delta p. Cl-, however, does not alter steady-state delta p.     

Effects of potassium ions on proton motive force in Rhodobacter sphaeroides.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (delta psi) only. At pH 7.9 the PMF was composed of a high delta psi of -98 mV and was partially compensated by a reversed pH gradient (delta pH) of +37 mV. ATPase inhibitors did not affect the delta psi, which was most likely the result of a K+ diffusion potential. Under energized conditions in the presence of K+ the delta psi depolarized due to electrogenic K+ uptake. This led to the generation of a delta pH (inside alkaline) in the external pH range of 6 to 8. This delta pH was dependent on the K+ concentration and was maximal at external K+ concentrations larger than 1.2 mM. In energized cells in 50 mM KPi buffer containing 5 mM MgSO4, a delta pH (inside alkaline) was present at external pHs from pH 6 to 8. As a result the overall magnitude of the PMF at various external pHs remained constant at -130 mV, which was significantly higher than the PMF under anaerobic-dark conditions. In the absence of K+, in 50 mM NaPi buffer containing 5 mM MgSO4, no depolarization of the delta psi was found and the PMF was composed of a large delta psi and a small delta pH. The delta pH became even reversed (inside acidic) at alkaline pHs (pH>7.3), resulting in a lowering of the PMF. These results demonstrate that in R. sphaeroides K+ uptake is essential for the generation of a delta pH and plays a central role in the regulation of the internal pH.     

Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force

The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse-induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse-induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse-induced ATP synthesis required a minimum total proton motive force of -200 to -250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans.     

Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli.

An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios. We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment. The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi. (b) An increase in [K+] from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity. (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM [K+], causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV. From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM. (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution. (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors. (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.     

The electrochemical proton gradient in Mycoplasma cells

The electrochemical proton gradient, delta mu H+ generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, delta pH, and the membrane potential, delta psi, were measured using several methods. The determination of the delta pH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extra-cellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of - 85 mV, interior negative. The delta mu H+ in M. mycoides var. Capri cells is thus about - 115 mV. When the external pH was altered from 7.7 to 5.7 delta psi decreased from - 90 mV to - 60 mV. On other hand although the internal pH decreased, delta pH was found to increase from 0.2 to 1.0 pH unit. Since the changes in delta psi were largely compensated by the changes in delta pH, delta mu H+ remained practically constant at about - 115 mV throughout the pH range tested. Finally, inhibition of delta pH by N,N'-dicyclohexylcarbodiimide, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.     

Oxygen taxis and proton motive force in Salmonella typhimurium

The aerotactic response of Salmonella typhimurium SL3730 has been quantitatively correlated with a change in the proton motive force (delta p) as measured by a flow-dialysis technique. At pH 7.5, the membrane potential (delta psi) in S. typhimurium changed from -162 +/- 13 to -111 +/- 15 mV when cells grown aerobically were made anaerobic, and it returned to the original value when the cells were returned to aerobiosis. The delta pH across the membrane was zero. At pH 5.5, delta psi was -70 mV in aerobiosis and -20 mV in anaerobiosis, and delta pH was -118 and -56 mV for aerobic and anaerobic cells, respectively. A decrease in delta p resulted in increased tumbling, and an increase in delta p resulted in a smooth swimming response at either pH. Inhibition of aerotaxis at pH 7.5 by various concentrations of KCN correlated with a decreased delta p, due to a decreased delta psi in aerobiosis and little change in delta psi in anaerobiosis. At concentrations up to 100 mM, 2,4-dinitrophenol decreased delta psi, but did not inhibit aerotaxis because the difference between delta psi in aerobic and anaerobic cells remained constant. Considered as a whole, the results indicate that aerotaxis in S. typhimurium is mediated by delta p.     

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force.

The magnitude of the proton motive force generated during in vitro substrate oxidation by Coxiella burnetii was examined. The intracellular pH of C. burnetii varied from about 5.1 to 6.95 in resting cells over an extracellular pH range of 2 to 7. Similarly, delta psi varied from about 15 mV to -58 mV over approximately the same range of extracellular pH. Both components of the proton motive force increased during substrate oxidation, resulting in an increase in proton motive force from about -92 mV in resting cells to -153 mV in cells metabolizing glutamate at pH 4.2. The respiration-dependent increase in proton motive force was blocked by respiratory inhibitors, but the delta pH was not abolished even by the addition of proton ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone or 2,4-dinitrophenol. Because of this apparently passive component of delta pH maintenance, the largest proton motive force was obtained at an extracellular pH too low to permit respiration. C. burnetii appears, therefore, to behave in many respects like other acidophilic bacteria. Such responses are proposed to contribute to the extreme resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome of eucaryotic cells in which this organism multiplies.     

Proton motive force and the physiological basis of delta pH maintenance in thiobacillus acidophilus.

At optimal growth pH (3.0) Thiobacillus acidophilus maintained an internal pH of 5.6 (delta pH of 2.6 units) and a membrane potential (delta psi) of some +73 mV, corresponding to a proton motive force (delta p) of -83 mV. The internal pH remained poised at this value through external pH values of 1 to 5, so that the delta pH increased with decreasing external pH. The positive delta psi increased linearly with delta pH: above a delta pH of 0.6 units, some 60% of the increase in delta pH was compensated for by an opposing increase in delta psi. The highest magnitude of delta pH occurred at an external pH of 1.0, where the cells could not respire. Inhibiting respiration by CN- or azide in cells at optimal pH decreased delta pH by only 0.4 to 0.5 units and caused a corresponding opposite increase in delta psi. Thus, a sizable delta pH could be maintained in the complete absence of respiration. Treatment of cells with thiocyanate to abolish the delta psi resulted in a time-dependent collapse of delta pH, which was augmented by protonophores. We postulate that T. acidophilus possesses unusual resistance to ionic movements. In the presence of a large delta pH (greater than 0.6 pH units), limited diffusion of H+ into the cell is permitted, which generates a positive delta psi because of resistance to compensatory ionic movements. This delta psi, by undergoing fluctuations, regulates the further entry of H+ into the cell in accordance with the metabolic state of the organism. The effect of protonophores was anomalous: the delta p was only partially collapsed, and respiration was strongly inhibited. Possible reasons for this are discussed.     

Electrochemical proton gradient and lactate concentration gradient in Streptococcus cremoris cells grown in batch culture.

The lactate concentration gradient and the components of the electrochemical proton gradient (delta micro H+) were determined in cells of Streptococcus cremoris growing in batch culture. The membrane potential (delta psi) and the pH gradient (delta pH) were determined from the accumulation of the lipophilic cation tetraphenylphosphonium and the weak acid benzoate, respectively. During growth the external pH decreased from 6.8 to 5.3 due to the production of lactate. Delta pH increased from 0 to -35 mV, inside alkaline (at an external pH of 5.7), and fell to zero directly after growth stopped. Delta psi was nearly constant at -90 mV during growth and also dissipated within 40 min after termination of growth. The internal lactate concentration decreased from 200 mM at the beginning of growth (at pH 6.8) to 30 mM at the end of growth (at pH 5.3); the external lactate concentration increased from 8 to 30 mM due to the fermentation of lactose. Thus, the lactate gradient decreased from 80 mV to zero as growth proceeded and the external pH decreased. From the data obtained on delta psi, delta pH, and the lactate concentration gradient, the H+/lactate stoichiometry (n) was calculated. The value of n varied with the external pH from 1.9 (at pH 6.8) to 0.9 (at pH values below 6). This implies that especially at high pH values the carrier-mediated efflux of lactate supplies a significant quantity of metabolic energy to S. cremoris cells. At pH 6.8 this energy gain was almost two ATP equivalents per molecule of lactose consumed if the H+/ATP stoichiometry equals 2. These results supply strong experimental evidence for the energy recycling model postulated by Michels et al.     

The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.     

Effects of aerobiosis and nitrogen source on the proton motive force in growing Escherichia coli and Klebsiella pneumoniae cells.

The electrochemical gradient of hydrogen ions, or proton motive force (PMF), was measured in growing Escherichia coli and Klebsiella pneumoniae in batch culture. The electrical component of the PMF (delta psi) and the chemical component (delta pH) were calculated from the cellular accumulation of radiolabeled tetraphenylphosphonium, thiocyanate, and benzoate ions. In both species, the PMF was constant during exponential phase and decreased as the cells entered stationary phase. Altering the growth rate with different energy substrates had no effect on the PMF. The delta pH (alkaline inside) varied with the pH of the culture medium, resulting in a constant internal pH. During aerobic growth in media at pH 6 to 7, the delta psi was constant at 160 mV (negative inside). The PMF, therefore, was 255 mV in cells growing at pH 6.3, and decreased progressively to 210 mV in pH 7.1 cultures. K. pneumoniae cells and two E. coli strains (K-12 and ML), including a mutant deficient in the H+-translocating ATPase and a pleiotropically energy-uncoupled mutant with a normal ATPase, had the same PMF during aerobic exponential phase. During anaerobic growth, however, both species had delta psi values equal to 0. Therefore, the PMF in anaerobic cells consisted only of the delta pH component, which was 75 mV or less in cells growing at pH 6.2 or greater. These data thus met the expectation that cells deriving metabolic energy from respiration have a PMF above a threshold value of about 200 mV when the ATPase functions in the direction of H+ influx and ATP synthesis; in fermenting cells, a PMF below a threshold value was expected since the enzyme functions in the direction of H+ extrusion and ATP hydrolysis. K. pneumoniae cells growing anaerobically had no delta psi whether the N source added was N2, NH+4 or one of several amino acids; the delta pH was unaffected. Therefore, any energy cost incurred by the process of nitrogen fixation could not be detected as an alteration of the proton gradient.     

Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulolytic ruminal bacterium.

The utilization of cellulose or cellobiose by Bacteroides succinogenes S85 was severely inhibited at pH values of less than 5.7. Since low pH inhibited the utilization of both cellobiose and cellulose, changes in cellulase activity could not explain the effect. At an extracellular pH of 6.9, the pH gradient (delta pH) across the cell membrane was only 0.07 U. As extracellular pH declined from 6.9 to 5.7, intracellular pH decreased to a smaller extent than extracellular pH and delta pH increased. Below pH 5.7, there was a linear and nearly proportional decrease in intracellular pH. B. succinogenes took up the lipophilic cation tetraphenylphosphonium ion (TPP+) in the presence of cellobiose, and uptake was sensitive to the ionophore valinomycin. As pH was decreased with phosphoric acid, the cells lost TPP+ and electrical potential, delta psi, decreased. From extracellular pH 6.9 to 5.7, the decrease in delta psi was compensated for by an increase in delta pH, and the proton motive force ranged from 152 to 158 mV. At a pH of less than 5.7, there was a large decrease in proton motive force, and this decrease corresponded to the inhibition of cellobiose utilization.     

Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulolytic ruminal bacterium

The utilization of cellulose or cellobiose by Bacteroides succinogenes S85 was severely inhibited at pH values of less than 5.7. Since low pH inhibited the utilization of both cellobiose and cellulose, changes in cellulase activity could not explain the effect. At an extracellular pH of 6.9, the pH gradient (delta pH) across the cell membrane was only 0.07 U. As extracellular pH declined from 6.9 to 5.7, intracellular pH decreased to a smaller extent than extracellular pH and delta pH increased. Below pH 5.7, there was a linear and nearly proportional decrease in intracellular pH. B. succinogenes took up the lipophilic cation tetraphenylphosphonium ion (TPP+) in the presence of cellobiose, and uptake was sensitive to the ionophore valinomycin. As pH was decreased with phosphoric acid, the cells lost TPP+ and electrical potential, delta psi, decreased. From extracellular pH 6.9 to 5.7, the decrease in delta psi was compensated for by an increase in delta pH, and the proton motive force ranged from 152 to 158 mV. At a pH of less than 5.7, there was a large decrease in proton motive force, and this decrease corresponded to the inhibition of cellobiose utilization.     

Comparison of polyvinyl chloride membrane electrodes sensitive to alkylphosphonium ions for the determination of the electrical difference (delta psi) of Streptococcus mutans and Lactobacillus casei

Polyvinyl chloride membrane electrodes sensitive to tetraphenyl phosphonium (TPP+), butyltriphenyl phosphonium ( bTPP +), and methyltriphenyl phosphonium ( mTPP +) ions have been compared for the determination of the electrical potential difference (delta psi) of the oral bacteria, Streptococcus mutans DR0001 /6 and Lactobacillus casei RB1014 . All three types of electrode proved suitable for determining delta psi, although the TPP+-sensitive electrode was particularly susceptible to interference by protonmotive force (delta p) dissipators known to inhibit sugar uptake by the bacteria. The mTPP +-sensitive electrode was the least affected. Similarly, both strains had a high nonspecific binding capacity for TPP+ and bTPP + ions, and this increased for all three ions when the bacteria were heated to 80 degrees C for 1 h to abolish glucose uptake and metabolism. This heat-treatment procedure is therefore not a suitable control for determination of nonspecific binding to cells. However, 1% (v/v) toluene, 20 microM gramicidin, or 10 microM valinomycin effectively depolarized the bacteria without interfering with nonspecific binding. The ionophores were therefore used subsequently for the determination of nonspecific binding of the lipid-soluble cations. The mTPP + ion and corresponding electrode proved the most effective system, and delta psi values of -89 and -107 mV were obtained for S. mutans and L. casei, respectively, harvested from glucose-limited continuous cultures and incubated in 100 mM Hepes-KOH buffer (pH 7.0), containing 1 mM dithiothreitol and 10 mM glucose. Although the delta psi of S. mutans decreased significantly in the presence of Mes-KOH and potassium phosphate buffers at pH 7.0, it increased to -119 mV in Tris-HCl buffer (pH 7.0).(ABSTRACT TRUNCATED AT 250 WORDS)