Changes in host cell energetics in response to bacteriophage PRD1 DNA entry.

Double-stranded DNA bacteriophage PRD1 infects a variety of gram-negative bacteria harboring an IncP-type conjugative plasmid. The plasmid codes for the DNA transfer phage receptor complex in the cell envelope. Our goal was, by using a collection of mutant phage particles for which the variables are the DNA content and/or the presence of the receptor-binding protein, to obtain information on the energy requirements for DNA entry as well as on alterations in the cellular energetics taking place during the first stages of infection. We studied the fluxes of tetraphenylphosphonium (TPP+), phenyldicarbaundecaborane (PCB-), and K+ ions as well as ATP through the envelope of Salmonella typhimurium cells. The final level of the membrane voltage (delta psi) indicator TPP+ accumulated by the infected cells exceeds the initial level before the infection. Besides the effects on TPP+ accumulation, PRD1 induces the leakage of ATP and K+ from the cytosol. All these events were induced only by DNA-containing infectious particles and were cellular ATP and delta psi dependent. PRD1-caused changes in delta psi and in PCB- binding differ considerably from those observed in other bacteriophage infections studied. These results are in accordance with the presence of a specific channel engaged in phage PRD1 DNA transport.     

The membrane potential ofMethanobacterium thermoautotrophicum under different external conditions

The membrane potential (delta psi) of whole cells of Methanobacterium thermoautotrophicum strain delta H was estimated under different external conditions using a TPP(+)-sensitive electrode. The results show that the delta psi values of M. thermoautotrophicum at alkaline pHout (8.5) are comparable with delta psi values under slightly acidic conditions (pH 6.8; 230 and 205 mV, respectively). On the other hand, the size of colonies on Petri dishes was remarkably smaller at pH 8.5 than at 6.8. The delta psi was insensitive to relevant ATPase inhibitors. At pH 6.8, the protonophore 3,3',4',5-tetrachlorosalicylanilide (TCS) strongly inhibited delta psi formation and ATP synthesis driven by methanogenic electron transport. On the other hand, at pH 8.5 the CH4 formation and ATP synthesis were insensitive to TCS and a protonophore-resistant delta psi of approximately 150 mV was determined. The finding of a protonophore-resistant delta psi at pH 8.5 indicates that at alkaline pHout these cells can switch from H(+)-energetics to Na(+)-energetics, when the delta [symbol: see text] H+ becomes limited. The results strongly support the hypothesis that at alkaline pHout Na+ ions might fully substitute for H+ in these cells as the coupling ions.     

Effects of K+ on the proton motive force of Bradyrhizobium sp. strain 32H1.

In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase.     

Membrane potentials in reconstituted cytochrome c oxidase proteoliposomes determined by butyltriphenyl phosphonium cation distribution

Equilibration of the butyltriphenyl phosphonium (BTPP+) cation into cytochrome c oxidase reconstituted proteoliposomes was measured potentiometrically. The maximum membrane potential (delta psi) generated by oxidase activity was estimated to lie between -65 and -90 mV, vesicle interior negative, when internal BTPP+ binding is taken into account. Formation of delta psi was completely prevented by valinomycin and carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone but only 10% inhibited by levels of N',N'-dicyclohexylcarbodiimide that abolish proton pumping by the oxidase. delta psi is thus maintained by at least one charge transfer process that does not involve proton movement. A nonlinear relationship was obtained between oxidase activity and steady-state delta psi. The value of delta psi estimated by BTPP+ distribution was lower than that calculated using the optical probes safranine and a carbocyanine dye. Possible reasons for this discrepancy are discussed.     

The constitutive K+ pump in Serratia marcescens

Transport of K+ and H+ in the anaeronically and aerobically grown bacterium Serratia marcescens has been studied. The volumes of one cell of the anaerobically and aerobically grown bacterium were 3.7 X 10(-13) cm3 and 2.4 X 10(-13) cm3, respectively. Irrespective of the growth conditions the bacteria manifested the same respiration rate. However, the values of membrane potential for the anaerobically and aerobically grown bacterium were different and equal to -130 mV and -175 mV (interior negative), respectively, in the absence of an exogenic energy source. KCN + DCCD decreases delta psi down to almost zero in both species. DCCD alone decreases delta psi partially in anaerobes and increases delta psi in aerobes, whereas KCN alone reduces delta psi partially in both species. The introduction of glucose into the medium containing K+ reduces the absolute value of delta psi to [-160] mV in aerobes and to [-20] mV in anaerobes. The effect is not observed without external K+. In the presence of arsenate a delta psi is not reduced after the addition of glucose. At pH 7.5-7.8 the ATP level in aerobes grows notably faster than in anaerobes. The H+ extrusion becomes intensified when K+ uptake is activated by the increase in external osmotic pressure. Apparent Km and Vmax for K+ accumulation are 1.2 mM and 0.4 mM.min-1.g-1. The decrease of delta psi by glucose or KCN + DCCD have no effect on the K+ uptake whereas CCCP inhibits potassium accumulation. At the same time, arsenate stabilizes the delta psi value, but blocks K+ uptake. The accumulation of K+ correlates with the potassium equilibrium potential of -200 mV calculated according to the Nernst equation, whereas the delta psi measured was not more than [-25] mV. The calculated H+/ATP stoichiometry was 3.3 for aerobes. It was assumed that a constitutive K+ pump having a K+/ATP ratio equal to 2 or 3 operates in S. marcescens membranes.     

The effect of lowering the pH on the composition and metabolism of a community of nine oral bacteria grown in a chemostat

Nine oral bacteria, associated with both healthy and diseased sites in the mouth, were grown at D = 0.05 h-1 (mean generation time 13.9 h) in a glucose-limited chemostat. After an initial period of steady-state growth at pH 7.0, pH control was discontinued. The pH then decreased until it stabilized at pH 4.1 after 9 d (16 generations), while the Eh rose from -165 mV to +160 mV. The lowering in pH resulted in the composition and metabolism of the flora being altered and in increased bacterial aggregation. At pH 7.0, 'Streptococcus mitior', Veillonella alcalescens and S. sanguis were most numerous while at pH 4.1 the counts of all bacteria fell except for Lactobacillus casei, which became predominant. The proportions of S. mutans within the community also increased while S. sanguis was recovered only occasionally and Bacteroides intermedius was not detected below pH 4.6. The survival at pH 4.1 of several other species would not have been predicted from earlier pure culture studies. Relative to pH 7.0, the community growing at pH 4.1 produced more lactic acid, washed cells had a greater glycolytic activity over a wider pH range but amino acid metabolism decreased. In general, when pH control was restored, so were the original patterns of metabolism and bacterial counts, except for B. intermedius, which was still not detected. The inverse relationship between S. sanguis and S. mutans, and the increase in proportions of L. casei and S. mutans during growth in a low pH environment parallel observations made in vivo and suggest that the chemostat can be used as a model for microbial behaviour in dental plaque.     

L-malate transport and proton symport in vesicles prepared from Pseudomonas putida

In vesicles from glucose-grown Pseudomonas putida, L-malate is transported by nonspecific physical diffusion. L-Malate also acts as an electron donor and generates a proton motive force (delta p) of 129 mV which is composed of a membrane potential (delta psi) of 60 mV and a delta pH of 69 mV. In contrast, vesicles from succinate-grown cells transport L-malate by a carrier-mediated system with a Km value of 14.3 mM and a Vmax of 313 nmol X mg protein-1 X min-1, generate no delta psi, delta pH, or delta p when L-malate is the electron donor, and produce an extravesicular alkaline pH during the transport of L-malate. A kinetic analysis of this L-malate-induced proton transport gives a Km value of 16 mM and a Vmax of 667 nmol H+ X mg protein-1 X min-1. This corresponds to a H+/L-malate ratio of 2.1. The failure to generate a delta p in these vesicles is considered, therefore, to be consistent with the induction in succinate-grown cells of an electrogenic proton symport L-malate transport system.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

Tetraphenylphosphonium ion is a true indicator of negative plasma-membrane potential in the yeast Rhodotorula glutinis. Experiments under osmotic stress and at low external pH values.

In the yeast Rhodotorula glutinis, accumulation of the tetraphenylphosphonium ion (TPP+) was increased under conditions of osmotic stress, indicating a hyperpolarization of the negative membrane potential (delta psi). The following observations were consistent with the occurrence of hyperpolarized delta psi: enhanced accumulation of glucosamine, the uptake of which is also driven by delta psi; increased respiratory rate. The accumulation of TPP+ was gradually decreased by lowering the pH of cell suspensions. At pH values below 4.5, no TPP+ was taken up, but instead thiocyanate (SCN-) was accumulated, indicating a positive delta psi. The pH-dependent influx of glucosamine followed the pattern of TPP+ accumulation both in the wild-type and in the nystatin-resistant mutant, M67, which displayed a negative delta psi down to pH 3. Thus TPP+ accumulation in Rh. glutinis reflected actual electrical potential difference across the plasma membrane.     

Tetraphenylphosphonium is an indicator of negative membrane potential in Candida albicans

The characteristics of the uptake of lipophilic cations tetraphenylphosphonium (TPP+) into Candida albicans have been investigated to establish whether TPP+ can be used as a membrane potential probe for this yeast. A membrane potential (delta psi, negative inside) across the plasma membrane of C. albicans was indicated by the intracellular accumulation of TPP+. The steady-state distribution of TPP+ was reached within 60 min and varied according to the expected changes of delta psi. Agents known to depolarize membrane potential caused a rapid and complete efflux of accumulated TPP+. The initial influx of TPP+ was linear over a wide range of TPP+ concentrations (2.5-600 microM), indicating a non mediated uptake. Thus, TPP+ is a suitable delta psi probe for this yeast.     

Factors determining the plasma-membrane potential of lymphocytes.

1. Lymphocytes from pig mesenteric lymph node have low permeability to K+ (Rb+), Na+ and Cl-. None of these ions is in Nernst equilibrium with the plasma-membrane potential (delta psi p). 2. delta psi p can be calculated from the transmembrane distribution of the permeant cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to abolish uptake into intracellular mitochondria. In normal culture medium delta psi p is 56 mV. 3. A similar potential is found in T-enriched pig cells and in mouse thymocytes. 4. The contribution of electrogenic (Na+ + K+)ATPase to delta psi p is about 7 mV. 5. The remainder of the lymphocyte delta psi p is a polyionic potential set up by K+ and Cl- with a permeability coefficient for Cl- of similar magnitude to that for K+.     

Sites of action of glucagon and other Ca2+ mobilizing hormones on the malate aspartate cycle

Data from a number of laboratories suggest that the exchange of glutamate for aspartate across the mitochondrial inner membrane is stimulated by glucagon and by Ca2+-mobilizing hormones. The purpose of this study was to determine the site of action of these hormones. Two possibilities were considered and tested. The first hypothesis is that the mitochondrial membrane electrical potential gradient (delta psi m) in the cells is increased by the hormones; and that the putative increase in delta psi m stimulates aspartate efflux. The second possibility is that Ca2+ mediates decreases in cellular levels of alpha-ketoglutarate, secondary to stimulation of alpha-ketoglutarate dehydrogenase, and that the decrease in alpha-ketoglutarate stimulates aspartate production by mitochondria. The effect of glucagon on delta psi m was estimated in intact hepatocytes using the lipophilic cation tetraphenyl phosphonium. No increase in delta psi m was observed due to hormone treatment. On the other hand, alpha-ketoglutarate was found to be an effective competitive inhibitor of aspartate formation via glutamate transamination by isolated liver mitochondria (Ki = 0.55 mM).     

Proton motive force in growing Streptococcus lactis and Staphylococcus aureus cells under aerobic and anaerobic conditions.

Measurements of the electrochemical gradient of hydrogen ions, which gives rise to the proton motive force (PMF), were carried out with growing Streptococcus lactis and Staphylococcus aureus cells. The facultative anaerobe was chosen in order to compare the PMF of cells growing aerobically and anaerobically. It was expected that during aerobic growth the cells would have a higher PMF than during anaerobic growth, because the H+-translocating ATPase (BF0F1) operates in the direction of H+ influx and ATP synthesis during respiration, whereas under anaerobic conditions the BF0F1 hydrolyzes glycolytically generated ATP and establishes the proton gradient by extruding H+. The electrical component of the PMF, delta psi, and the chemical gradient of H+, delta pH, were measured with radiolabeled tetraphenylphosphonium and benzoate ions. In both S. lactis and S. aureus cells, the PMF was constant during the exponential phase of batch growth and decreased in the stationary phase. In both species of bacteria, the exponential-phase PMF was not affected by varying the growth rate by adding different sugars to the medium. The relative contributions of delta psi and delta pH to the PMF, however, depended on the pH of the medium. The internal pH of S. aureus was constant at pH 7.4 to 7.6 under all conditions of growth tested. Under aerobic conditions, the delta psi of exponential phase S. aureus remained fairly constant at 160 to 170 mV. Thus, the PMF was 250 to 270 mV in cells growing aerobically in media at pH 6 and progressively lower in media of higher pH, reaching 195 to 205 mV at pH 7. Under anaerobic conditions, the delta psi ranged from 100 to 120 mV in cells at pH 6.3 to 7, resulting in a PMF of 150 to 140 mV. Thus, the mode of energy metabolism (i.e., respiration versus fermentation) and the pH of the medium are the two important factors influencing the PMF of these gram-positive cells during growth.     

Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.     

Plasma membrane potential of murine erythroleukemia cells: approach to measurement and evidence for cell-density dependence

The plasmamembrane potential (delta psi p) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, AR exp) was measured by adding 2 microM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to AR exp, evaluated by adding carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or 2,4-dinitrophenol (DNP), was apparently negligible in standard cultures, AR exp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong AR exp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit AR exp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to AR exp values, delta psi p of these cells was then measured; this potential varying from -65 mV to -16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of delta psi p on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (PK).     

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.     

Ammonium/urea-dependent generation of a proton electrochemical potential and synthesis of ATP in Bacillus pasteurii.

The influence of ammonium and urea on the components of the proton electrochemical potential (delta p) and de novo synthesis of ATP was studied with Bacillus pasteurii ATCC 11859. In washed cells grown at high urea concentrations, a delta p of -56 +/- 29 mV, consisting of a membrane potential (delta psi) of -228 +/- 19 mV and of a transmembrane pH gradient (delta pH) equivalent to 172 +/- 38 mV, was measured. These cells contained only low amounts of potassium, and the addition of ammonium caused an immediate net decrease of both delta psi and delta pH, resulting in a net increase of delta p of about 49 mV and de novo synthesis of ATP. Addition of urea and its subsequent hydrolysis to ammonium by the cytosolic urease also caused an increase of delta p and ATP synthesis; a net initial increase of delta psi, accompanied by a slower decrease of delta pH in this case, was observed. Cells grown at low concentrations of urea contained high amounts of potassium and maintained a delta p of -113 +/- 26 mV, with a delta psi of -228 +/- 22 mV and a delta pH equivalent to 115 +/- 20 mV. Addition of ammonium to such cells resulted in the net decrease of delta psi and delta pH without a net increase in delta p or synthesis of ATP, whereas urea caused an increase of delta p and de novo synthesis of ATP, mainly because of a net increase of delta psi. The data reported in this work suggest that the ATP-generating system is coupled to urea hydrolysis via both an alkalinization of the cytoplasm by the ammonium generated in the urease reaction and a net increase of delta psi that is probably due to an efflux of ammonium ions. Furthermore, the findings of this study show that potassium ions are involved in the regulation of the intracellular pH and that ammonium ions may functionally replace potassium to a certain extent in reducing the membrane potential and alkalinizing the cytoplasm.     

Measurement of 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.     

Serotonin transport in isolated platelet granules. Coupling to the electrochemical proton gradient

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in intact 5-hydroxytryptamine (serotonin) containing dense granules. The granules were isolated and purified from other subcellular organelles under isotonic conditions utilizing a newly developed continuous density gradient of Percoll. As measured by [14C]methylamine distribution, isolated granules suspended in a highly buffered medium at pH 7.0 had an intragranular pH of 5.40, independent of the pH of the external medium. This pH gradient could be collapsed by the addition of 60 mM ammonia. In the presence of Mg-ATP, a transmembrane potential (delta psi) of 30-40 mV, inside positive, was generated and sustained for over 30 min, as measured by [14C]thiocyanate distribution. The addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a proton translocator, resulted in the reversal of the potential to negative values. The Mg-ATP-dependent generation of the delta psi was prevented by addition of dicyclohexylcarbodiimide and trimethyltin, inhibitors of proton-translocating ATPases in this and other subcellular organelles. Ammonia (1-50 mM) addition to highly buffered suspensions of serotonin granules caused a dose-dependent decrease in the delta pH, while thiocyanate added at varying concentrations produced a dose-related collapse of the delta psi and had no effect upon the delta pH. Both the delta pH and delta psi were found to independently drive accumulation of [14C]serotonin into the granules; stepwise collapse of each gradient resulted in a corresponding diminution of [14C]serotonin accumulation. The maximum rate and extent of [14C]serotonin uptake, however, were observed in the presence of both the delta pH and delta psi. The conclusions provide support for the existence of a proton-translocating ATPase in the serotonin granule membrane responsible for the generation of the delta pH and delta psi. Moreover, the results demonstrate a primary role for the electrochemical proton gradient (delta mu H+) in the carrier-mediated active transport of 5-hydroxytryptamine into the platelet granule.     

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.