Relation between the protonmotive force and solute transport in bacteria.
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Biological Transport, Active↗
Biomedical subjects
K J Hellingwerf
Publications and source records attributed to K J Hellingwerf.
Role of the electrochemical proton gradient in genetic transformation of Haemophilus influenzae.
The uptake of homologous DNA by Haemophilus influenzae was studied as a function of the proton motive force in completely competent cultures in the pH range of 6 to 8. The composition and magnitude of the proton motive force were varied by using the ionophores valinomycin and nigericin (in the presence of various potassium ion concentrations) and by using protonophores. No interaction of the ionophores with the DNA transformation system itself was observed. Either component of the proton motive force, the electrical potential or the pH gradient, can drive the uptake of DNA, and the extent of the uptake of DNA is ultimately determined by the total proton motive force. The transformation frequency increases with the proton motive force, which reaches a maximum value at around -130 mV. These results are consistent with an electrogenic proton-DNA symport mechanism, but direct evidence for such a system is not available. The proton motive force was followed during competence development of H. influenzae at pH 8. In the initial phase (up to 50 min), the proton motive force remained constant at about -90 mV, whereas the transformation frequency rose steeply. In the second phase, the proton motive force increased. The transformation frequency in this phase increased with the proton motive force, as in completely competent cultures. These observations and the observed inhibition by NAD of both the proton motive force and the transformation frequency indicate that structural components of the competent state are formed in the initial phase of competence development, whereas the second phase is characterized by an increase of the proton motive force.
Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
The lactose carrier of Escherichia coli functionally incorporated in Rhodopseudomonas sphaeroides obeys the regulatory conditions of the phototrophic bacterium.
Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton-motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.
Alanine↗
The transmembrane electrical potential in Rhodopseudomonas sphaeroides determined from the distribution of tetraphenylphosphonium after correction for its binding to cell components.
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Binding Sites↗
The role of the proton-motive force and electron flow in light-driven solute transport in Rhodopseudomonas sphaeroides.
The initial rate of uptake of alanine was studied in whole cells of Rhodopseudomonas sphaeroides under conditions such that the proton-motive force generated by light-induced cyclic electron transfer, was composed of the membrane potential only. The proton-motive force was varied by varying the light intensity or by pretreatment of the cells with the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone. At constant light intensity a threshold delta psi is required before alanine uptake occurs. Above this threshold value the rate of alanine uptake increases with the delta psi. The threshold value of the delta psi is higher the lower the light intensity. Under conditions of constant delta psi the rate of alanine uptake increases linearly with the light intensity. At low delta psi values a threshold light intensity is required which is higher the lower the delta psi. These results demonstrate that both a proton-motive force and electron transfer are required for alanine uptake.
Alanine↗
Thermodynamic efficiency of microbial growth is low but optimal for maximal growth rate.
Thermodynamic efficiency of microbial growth on substrates that are more oxidized than biomass approaches 24%. This is the theoretical value for a linear energy converter optimized for maximal output flow at optimal efficiency. For growth on substrates more reduced than biomass, thermodynamic efficiencies correspond to those predicted for optimization to maximal growth rate (or yield) only.
Bacteria↗
Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis.
Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.
Adenosine Triphosphatases↗
In vivo 31P and 13C nuclear magnetic resonance studies of acetate metabolism in Chromatium vinosum.
31P and 13C nuclear magnetic resonance (NMR) experiments were performed on suspensions of the phototrophic bacterium Chromatium vinosum incubated anaerobically in the dark. 31P NMR spectra revealed that during prolonged dark incubation high ATP levels are maintained. This phenomenon was independent of the presence of the energy reserves polyglucose and polyphosphate. 13C NMR experiments revealed that the amino acids glutamate, aspartate, and alanine are the major products of acetate incorporation in the dark. Apart from these amino acids, poly-beta-hydroxybutyrate was also formed. Acetate metabolism was markedly stimulated by the presence of polyglucose. The specific 13C activity of glutamate C-2 was approximately 50% that of glutamate C-4. The idea is discussed that this difference is the consequence of the maintenance of redox balance during entry of acetate into cell metabolism.
Acetates↗
Thermodynamics of growth. Non-equilibrium thermodynamics of bacterial growth. The phenomenological and the mosaic approach.
Microbial growth is analyzed in terms of mosaic and phenomenological non-equilibrium thermodynamics. It turns out that already existing parameters devised to measure bacterial growth, such as YATP, mu, and Q substrate, have as thermodynamic equivalents flow ratio, output flow and input flow. With this characterisation it becomes possible to apply much of the already existing knowledge of phenomenological non-equilibrium thermodynamics to bacterial growth. One of the conclusions is that the frequent observation that YATP is only 50% of its theoretical maximum does not mean that the microbe corresponds to a thermodynamic system that has been optimized for maximal output power, as has been suggested. Rather, at least in some cases, it corresponds to a system that has been optimized towards maximum growth rate. When the degree of reduction of the (single) carbon source is significantly smaller than that of the biomass produced, the efficiency of biomass synthesis has been kept as high (i.e., about 24%) as is consistent with maximization of the growth rate at optimal efficiency. Mosaic thermodynamics allows an analysis of processes which in microbial metabolism may be responsible for any particular growth behaviour. Equations are derived that predict the effect of uncoupling through leaks, futile cycling, or 'slip' on microbial growth. It turns out that uncoupling is expected to affect both the growth rate-independent and the growth rate-dependent 'maintenance coefficient'. The effect on the latter is different when catabolic substrate limits growth than when anabolic substrate limits growth. In the latter case, the growth rate-dependent maintenance coefficient is negative. It is concluded that mosaic non-equilibrium thermodynamics will be a powerful theoretical tool especially in future experimental analyses of the metabolic basis for microbial growth characteristics and growth regulation.
Adenosine Triphosphate↗
Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of rhodopseudomonas sphaeroides.
13C-nuclear magnetic resonance was used to study the metabolism of [2-(13)C]acetate in suspensions of Rhodopseudomonas sphaeroides. In the dark, in logarithmic-phase cells the 13C label appeared first in butyrate C-2 and C-4 and subsequently in glutamate C-4 and succinate C-2 and C-3. In the light, synthesis of poly(beta-hydroxybutyrate) (PHB) takes place. Butyrate synthesis seems to be independent of PHB synthesis or degradation activity. Starved, logarithmic-phase cells also show massive synthesis of PHB in the dark. Stationary-phase cells incorporate 13C predominantly into glutamate and succinate. No significant butyrate biosynthesis can be detected in the dark or during illumination. The incorporation of label in PHB is very slow in these cells and most probably originates from exchange of 12C for 13C into PHB. This might indicate slow turnover without net synthesis of the polymer occurring under these conditions. The results are discussed in relation to the redox state and the availability of metabolic energy for biosynthetic reactions in the dark and during illumination of cell suspensions of Rps. sphaeroides.
Acetates↗
Energy coupling of facilitated transport of inorganic ions in Rhodopseudomonas sphaeroides.
Within the scope of a study on the effects of changes in medium composition on the proton motive force in Rhodopseudomonas sphaeroides, the energy coupling of sodium, phosphate, and potassium (rubidium) transport was investigated. Sodium was transported via an electroneutral exchange system against protons. The system functioned optimally at pH 8 and was inactive below pH 7. The driving force for the phosphate transport varied with the external pH. At pH 8, Pi transport was dependent exclusively on delta psi (transmembrane electrical potential), whereas at pH 6 only the delta pH (transmembrane pH gradient) component of the proton motive force was a driving force. Potassium (rubidium) transport was facilitated by a transport system which catalyzed the electrogenic transfer of potassium (rubidium) ions. However, in several aspects the properties of this transport system were different from those of a simple electrogenic potassium ionophore such as valinomycin: (i) accumulated potassium leaked very slowly out of cells in the dark; and (ii) the transport system displayed a threshold in the delta psi, below which potassium (rubidium) transport did not occur.
Biological Transport, Active↗
Wavelength dependence of energy transduction in Rhodopseudomonas sphaeroides: action spectrum of growth.
We determined the wavelength dependence of the specific growth rate of Rhodopseudomonas sphaeroides (the action spectrum of growth). A half-maximal (light-limited) growth rate was obtained when the culture vessel was illuminated with photon intensities between 0.8 x 10(14) and 3.5 x 10(14) photons cm-2 s-1 in the wavelength region between 400 and 950 nm. In the action spectrum, measured at 1.25 x 10(14) photons cm-2 s-1, distinct peaks could be observed at 480, 580, 800, and 870 nm, and minima could be found at 420, 540, 640 to 730, 830, and 940 nm. Both the pigment content and pigment composition of R. sphaeroides varied, depending on the wavelengths of the actinic light used for growth. This demonstrates that chromatic adaptation occurs in this bacterium.
Kinetics↗
Role of proton motive force in genetic transformation of Bacillus subtilis.
This study explored the role of the proton motive force in the processes of DNA binding and DNA transport of genetic transformation of Bacillus subtilis 168 strain 8G-5 (trpC2). Transformation was severely inhibited by the ionophores valinomycin, nigericin, and 3,5-di-tert-4-hydroxybenzylidenemalononitrite (SF-6847) and by tetraphenylphosphonium. The ionophores valinomycin and nigericin also severely inhibited binding of transforming DNA to the cell envelope, whereas SF-6847 and carbonylcyanide-p-trifluoromethoxyphenylhydrazone hardly affected binding. The proton motive force, therefore, does not contribute to the process of DNA binding, and valinomycin and nigericin interact directly with the DNA binding sites at the cell envelope. The effects of ionophores, weak acids, and tetraphenylphosphonium on the components of the proton motive force and on the entry of transforming DNA after binding to the cell envelope was investigated. DNA entry, as measured by the amount of DNase I-resistant cell-associated [3H]DNA and by the formation of DNA breakdown products, was severely inhibited under conditions of a small proton motive force and also under conditions of a small delta pH and a high electrical potential. These results suggest that the proton motive force and especially the delta pH component functions as a driving force for DNA uptake in transformation.
Bacillus subtilis↗
Linear relations between proton current and pH gradient in bacteriorhodopsin liposomes.
The dependence of proton movement across the membrane of bacteriorhodopsin liposomes on the pH gradient was investigated. Under the appropriate experimental conditions, endogenous proton (or hydroxyl) leakage, proton movement catalyzed by protonophore or nigericin, and light-driven proton translocation depend linearly on the pH gradient. This justifies the use of linear proton flux vs. protonmotive force relations in a recent mosaic thermodynamic description of ion translocation in bacteriorhodopsin liposomes [Westerhoff, H. V., Scholte, B. J., & Hellingwerf, K. J. (1979) Biochim. Biophys. Acta 547, 544-560]. Since bacteriorhodopsin liposomes are a model system for all biological energy transducing systems in which proton pumps are involved, these findings also explain linear relations between proton flux and protonmotive force observed in and postulated for those systems. In cases where the membrane potential is not clamped at a low value, an initial phase of rapid proton movement occurs, followed by a phase of slower proton movement. The rate of proton movement during the slow phase is again linear with the pH gradient. Such a linear relation is not observed for the fast phase. Since the rapid proton movement phase is also observed in liposomes without bacteriorhodopsin, it is not due (only) to dissociation of scalar protons from bacteriorhodopsin. We suggest that during the initial phase of proton movement, the proton flux is not yet electrically compensated by the fluxes of other ions.
Bacteriorhodopsins↗
Quantitative agreement between the values for the light-induced delta pH in Rhodopseudomonas sphaeroides measured with automated follow-dialysis and 31P NMR.
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Cell Membrane↗
Mosaic nonequilibrium thermodynamics describes biological energy transduction.
A procedure, called "mosaic nonequilibrium thermodynamics," for describing ion movement and energy transduction in biological membranes is tested in a model system: bacteriorhodopsin liposomes. The important steps in the theoretical derivations are summarized; one of the experimental tests of the postulated fundamental flow-force relationships is shown. Furthermore, how the quantitative method, even if used only qualitatively, facilitates analysis and understanding of experimental results (in this case, the effect of medium composition on the development of pH gradient and membrane potential in the bacteriorhodopsin liposomes) is shown. The main advantage of this method lies in its quantitative description of the effect of variation of system parameters on the performance of, in this case, the reconstituted proton pump bacteriorhodopsin. As an example, the method is shown to explain quantitatively the dependence of the steady-state pH gradient on the light intensity. Even in more refined analyses of experiments, the quantitative theoretical description is in full accordance with the experimental results; this is illustrated by considering the effect of valinomycin on the dependence of the initial rate of proton uptake into bacteriorhodopsin liposomes on light intensity. It is concluded that mosaic nonequilibrium thermodynamics describes ion movement and energy transduction in the model system of bacteriorhodopsin liposomes and, therefore, may be applied to any other biological system performing such processes.
Bacteriorhodopsins↗
The electrochemical proton gradient generated by the fumarate-reductase system in Escherichia coli and its bioenergetic implications.
Proton translocation, coupled to electron transfer in the fumarate reductase system, generates and electrochemical potential gradient for protons (delta approximately mu H+). The magnitude of this free energy gradient has been determined in the Escherichia coli strains ML 208-225 and AN 283. The measurements were performed in (inverted) membrane particles, right-side out membrane vesicles and EDTA-treated intact cells in external media of various ionic compositions and pH. The maximal values of delta approximately mu H+ in these three systems were +103, -101 and -105 mV, respectively. This implicates that in E. coli, upon transition from oxygen to fumarate as electron acceptor, the magnitude of the delta approximately mu H+ decreases considerably. This change of delta approximately mu H+ has substantial consequences for the cellular metabolism.
Cell Membrane↗
31P nuclear magnetic resonance studies of energy transduction in Rhodopseudomonas sphaeroides.
31P nuclear magnetic resonance spectra of th phototrophic bacterium Rhodopseudomonas sphaeroides reveal the presence of inorganic phosphate, sugar phosphates and two non-identified P,P1-diesterified pyrophosphate compounds. Due to the presence of paramagnetic cations the resonances of these compounds can only be detected after repeated washing of the bacterial cells with a buffer, containing EDTA plus excess Mg2+. Washing with Mg2+-free EDTA buffer deteriorates the structural integrity of the membranes of Rps. sphaeroides. This is indicated by the appearance of an extra resonance peak in the spectra of these cells in a region where the phospholipids absorb and by a fivefold increase in proton permeability of the cytoplasmic membrane of Rps. sphaeroides under these conditions. Upon illumination of the cell suspension in the NMR tube the generation of a transmembrane pH gradient can be inferred from the shift in the resonances of extracellular and intracellular inorganic phosphate. Intracellular inorganic phosphate shows one homogeneous resonance peak upon illumination. This demonstrates that the mixing system, which has been developed for this application, functions efficiently. The magnitude of the light-dependent pH difference is 0.8 at the external pH 6. The width at half height of the internal inorganic phosphate peak is essentially independent of internal pH from pH 5--8, remains unchanged upon addition of uncoupler and is inversely proportional to the number of EDTA washings applied. These observations indicate that the inorganic phosphate NMR peak width is predominantly determined by the presence of a residual amount of paramagnetic cations, rather than by a broad distribution of internal pH values over the cells. Ionophores have an effect on the light-dependent pH-gradient in accordance with the chemiosmotic theory: valinomycin increases, and carbonylcyanide p-trifluoromethoxyphenylhydrazone decreases, the magnitude of this gradient.
Adenosine Triphosphate↗