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.     

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.     

The Intracellular pH of Clostridium paradoxum, an Anaerobic, Alkaliphilic, and Thermophilic Bacterium

When the extracellular pH was increased from 7.6 to 9.8, Clostridium paradoxum, a novel alkalithermophile, increased its pH gradient across the cell membrane ((Delta)pH, pH(infin) - pH(infout)) by as much as 1.3 U. At higher pH values (>10.0), the (Delta)pH and membrane potential ((Delta)(psi)) eventually declined, and the intracellular pH increased significantly. Growth ceased when the extracellular pH was greater than 10.2 and the intracellular pH increased to above 9.8. The membrane potential increased to 110 (plusmn) 8.6 mV at pH 9.1, but the total proton motive force ((Delta)p) declined from about 65 mV at pH 7.6 to 25 mV at pH 9.8. Between the extracellular pH of 8.0 and 10.3, the intracellular ATP concentration was around 1 mM and decreased at lower and higher pH values concomitantly with a decrease in growth rate.     

Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force.

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.     

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.     

Measurement of intracellular sodium concentration and sodium transport in Escherichia coli by 23Na nuclear magnetic resonance

Escherichia coli is known to actively extrude sodium ions, but little is known concerning the concentration gradient it can develop. We report here simultaneous measurements, by 23Na NMR, of intracellular and extracellular Na+ concentrations of E. coli cells before and after energization. 23Na spectra in the presence of a paramagnetic shift reagent (dysprosium tripolyphosphate) consisted of two resonances, an unshifted one corresponding to intracellular Na+ and a shifted one corresponding to Na+ in the extracellular medium, including the periplasm. Extracellular Na+ was found to be completely visible despite the presence of a broad component in its resonance; intracellular Na+ was only 45% visible. Measurements of Na+ were made under aerobic and glycolytic conditions. Na+ extrusion and maintenance of a stable low intracellular Na+ concentration were found to correlate with the development and maintenance of proton motive force, a result that is consistent with proton-driven Na+/H+ exchange as a means of Na+ transport. In both respiring and glycolyzing cells, at an extracellular Na+ concentration of 100 mM, the intracellular Na+ concentration observed (4 mM) corresponded to an inwardly directed Na+ gradient with a concentration ratio of about 25. The kinetics of Na+ transport suggest that rapid extrusion of Na+ against its electrochemical gradient may be regulated by proton motive force or intracellular pH.     

Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles

The application of neutral or acidic amino acids to oat coleptiles induced transient depolarizations of the membrane potentials. The depolarizations are considered to reflect H⁺ -amino acid co-transport, and the spontaneous repolarizations are believed to be caused by subsequent electrogenic H⁺ extrusion. The basic amino acids depolarized the cell membrane strongly, but the repolarizations were weak or absent. The depolarizations induced by the basic amino acids were weakly sensitive to manipulations of the extracellular and intracellular pH. The depolarizations induced by the other amino acids, in contrast, were more strongly affected by the pH changes. Several amino acids induced distinct but diminished depolarizations in the presence of 2,4-dinitrophenol or cyanide, but the repolarizations were generally eliminated. These experiments support the co-transport theory but suggest somewhat different mechanisms for the transport of the neutral, acidic, and basic amino acids. We suggest that the neutral amino acids are co-transported with a single H⁺ and that accumulation depends upon both the ΔpH and the membrane potential components of the proton motive force. The acidic amino acids appear to be accumulated by a similar mechanism except that the transport of each molecule may be associated with a cation in addition to a single proton. The permanently protonated basic amino acids appear not to be co-transported with an additional proton. Accumulation would depend only on the membrane potential component of the proton motive force.     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Collapse of the proton motive force in Listeria monocytogenes caused by a bacteriocin produced by Pediococcus acidilactici.

The effect of pediocin JD, a bacteriocin produced by Pediococcus acidilactici JD1-23, on the proton motive force and proton permeability of resting whole cells of Listeria monocytogenes Scott A was determined. Control cells, treated with trypsin-inactivated bacteriocin at a pH of 5.3 to 6.1, maintained a pH gradient and a membrane potential of approximately 0.65 pH unit and 75 mV, respectively. However, these gradients were rapidly dissipated in cells after exposure to pediocin JD, even though no cell lysis had occurred. The pH gradient and membrane potential of the producer cells were also unaffected by the bacteriocin. Whole cells treated with bacteriocin were twice as permeable to protons as control cells were. The results suggest that the inhibitory action of pediocin JD against L. monocytogenes is directed at the cytoplasmic membrane and that inhibition of L. monocytogenes may be caused by the collapse of one or both of the individual components of the proton motive force.     

Dextransucrase secretion in Leuconostoc mesenteroides depends on the presence of a transmembrane proton gradient.

The relationship between proton motive force and the secretion of dextransucrase in Leuconostoc mesenteroides was investigated. L. mesenteroides was able to maintain a constant proton motive force of -130 mV when grown in batch fermentors at pH values 5.8 to 7.0. The contribution of the membrane potential and the transmembrane pH gradient varied depending on the pH of the growth medium. The differential rate of dextransucrase secretion was relatively constant at 1,040 delta mU/delta mg (dry weight) when cells were grown at pH 6.0 to 6.7. Over this pH range, the internal pH was alkaline with respect to the external pH. When cells were grown at alkaline pH values, dextransucrase secretion was severely inhibited. This inhibition was accompanied by an inversion of the pH gradient as the internal pH became more acidic than the external pH. Addition of nigericin to cells at alkaline pH partially dissipated the inverted pH gradient and produced a fourfold stimulation of dextransucrase secretion. Treatment of cells with the lipophilic cation methyltriphenylphosphonium had no effect on the rate of dextransucrase secretion at pH 5.5 but inhibited secretion by 95% at pH 7.0. The reduced rate of secretion correlated with the dissipation of the proton motive force by this compound. Values of proton motive force greater than -90 mV were required for maximal rates of dextransucrase secretion. The results of this study indicate that dextransucrase secretion in L. mesenteroides is dependent on the presence of a proton gradient across the cytoplasmic membrane that is directed into the cell.     

Thyroid hormone effects on the State IV proton motive force in rat liver mitochondria

In order to further investigate the mechanisms regulating the control of mitochondrial respiration by thyroid hormone, the proton motive force was measured during State IV respiration in liver mitochondria isolated from euthyroid, hyperthyroid, hypothyroid and T₃-treated hypothyroid rats. The proton motive force was significantly higher in the hyperthyroid group due to an increased pH. The proton motive force of hypothyroid mitochondria was lower than controls due to a decreased membrane potential. The proton motive force for the T3-treated hypothyroid group did not differ from the euthyroid group due to negating changes in the pH gradient and the membrane potential. The intramitochondrial volume was decreased in the hyperthyroid group and unchanged in the other groups. The results indicate that the thyroid status alters the proton motive force in State IV through individual changes in the pH and membrane potential components of the force. The component that changes in hyperthyroid mitochondria is different from that changing in hypothyroid mitochondria.     

Apoplastic pH during low-oxygen stress in Barley

BACKGROUND AND AIMS: Anoxia leads to an energy crisis, tolerance of which varies from plant to plant. Although the apoplast represents an important storage and reaction space, and engages in the mediation of membrane transport, this extracellular compartment has not yet been granted a role during oxygen shortage. Here, an attempt is made to highlight the importance of the apoplast during oxygen stress and to test whether information about it is transferred systemically in Hordeum vulgare. METHODS: Non-invasive ion-selective microprobes were used which, after being inserted through open stomata, directly contact the apoplastic fluid and continuously measure the apoplastic pH and changes to it. KEY RESULTS: (a) Barley leaves respond to oxygen stress with apoplastic alkalinization and membrane depolarization. These responses are persistent under anoxia (N2; O2 < 3%) but transient under hypoxia. (b) Being applied to the root, the information 'anoxia' is signalled to the leaf as an increase in pH, whereas 'hypoxia' is not: flooding of the roots within the first 2 h has no effect on the leaf apoplastic pH, whereas anoxia (N2) or chemical anoxia (NaCN/salicylic hydroxamic acid) rapidly increase the leaf apoplastic pH. (c) Under anoxia, the proton motive force suffers a decrease by over 70 %, which impairs H(+) -driven transport. CONCLUSIONS: Although anoxia-induced apoplastic alkalinization is a general response to stress, its impact on the proton motive force (reduction) and thus on transport mediation of energy-rich compounds is evident. It is concluded that anoxia tolerance depends on how the plant is able to hold the proton motive force and H(+) turnover at a level that guarantees sufficient energy is harvested to overcome the crisis.     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.     

植物乳杆菌素L-1对单核细胞增生李斯特氏菌作用机理的研究

以凝胶层析纯化的植物乳杆菌素作用单核细胞增生李斯特氏菌,结果表明该细菌素可以导致能量化的敏感细胞胞内K 、无机磷离子、乳酸脱氢酶、紫外吸收物质和ATP发生不同程度的泄漏,相应地破坏了膜Δψ和部分ΔpH,引起PMF的耗散,结果导致细胞的死亡。综合所测指标,可以推测植物乳杆菌素L-1对单增李斯特氏菌的作用目标主要是细胞膜,通过形成非选择性孔洞使得选择性离子和小分子生命物质外泄,从而打破原有平衡,最终引起细胞的衰亡。     

Control of intracellular pH. Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora

Recessed-tip microelectrodes were used to measure internal pH (pHi) in the fungus Neurospora, and to examine the response of pHi to several kinds of stress: changes of extracellular pH (pHo), inhibition of the principal proton pump in the plasma membrane, and inhibition of respiration. Under control conditions, at pHo = 5.8, pHi in Neurospora is 7.19 +/- 0.04. Changes of pHo between 3.9 and 9.3 affect pHi linearly but with a slope of only approximately 0.1 unit pHi per unit pHo, stable pHi being reached within 3 min of changed pHo. Despite a postulated high passive permeability of the Neurospora membrane to protons (Slayman, 1970), neither active nor passive H+ transport appears critical to pHi because (alpha) specific inhibition of the proton pump by orthovanadate has little effect on pHi, and (b) cytoplasmic acidification produced by respiratory blockade is unaffected by the size or direction of proton gradient. To convert measured changes in pHi into net proton fluxes, intracellular buffering capacity (beta i) was measured by the weak acid/weak base technique. At pHi = 7.2, beta i was (-) 35 mmol H+ (liter cell water)-1 (pH unit)-1, but beta i increased substantially in both the acid and alkaline directions, which suggests that amino acid side chains are the principal source of buffer.     

Proton motive force and Na+/H+ antiport in a moderate halophile.

The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.     

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae

Picrophilus oshimae is an extremely acidophilic, thermophilic archaeon that grows optimally at 60°C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane. Received: July 18, 1997 / Accepted: November 25, 1997