Charge balance and acidity regulation during growth of sugar-cane cell suspensions

Abstract. An analysis of nutrient uptake by batch cultures of sugar-cane cells was performed to gain information about the ionic balance during uptake of charged metabolites. Whereas younger cultures (up to 1 week old) have to compensate excess cation influx with proton efflux, older cultures show balanced cation–anion uptake.
Younger cells produce a small amount of carboxylic acids to furnish protons for charge compensation at the cytoplasmic membrane. Older cells synthesize organic acids more abundantly to generate protons necessary for the proton demand of nitrate and sulphate assimilation. Despite these assimilation reactions only a small percentage of carbon, which is taken up mainly as hexose, needs to be oxidized to carboxylic acids for that purpose. In contrast, younger cultures preferentially use the amino acids of the medium instead of assimilating nitrate. The use of amino acids as a nitrogen source does not require a significant part of metabolism for biochemical pH-stability, whereas an efficient proton circulation on the cytoplasmic membrane seems to be of major importance.
A balance study of the main metabolized elements, carbon, nitrogen and sulphur was performed to get a quantitative impression of the fate of these nutrients during growth of cell cultures.     

The membrane-induced proton motive force influences the metal binding ability of Bacillus subtilis cell walls.

Bacillus subtilis 168 is a gram-positive bacterium whose cell wall contains the highly electronegative polymers peptidoglycan (chemotype A1 gamma) and glycerol-based teichoic acid to produce a surface with a net negative charge with high metal binding capacity. During metabolism, a membrane-induced proton motive force continuously pumps protons into the wall fabric. As a result, a competition between protons and metal ions for anionic wall sites occurs, and less metal is bound in living cells than in nonliving cells or those in which the plasma membrane has been uncoupled. This was shown by using two metallic ions, UO2(2+) and Sc3+, on control cells, cells uncoupled with either carbonyl cyanide m-chlorophenylhydrazone or NaN3, or cells killed by gamma radiation. Transmission electron microscopy, energy-dispersive X-ray spectroscopy, and inductively coupled plasma atomic-emission spectroscopy showed that more metal was retained in the walls of nonliving cells and those with deenergized membranes than in their living counterparts.     

The membrane-induced proton motive force influences the metal binding ability of Bacillus subtilis cell walls.

Bacillus subtilis 168 is a gram-positive bacterium whose cell wall contains the highly electronegative polymers peptidoglycan (chemotype A1 gamma) and glycerol-based teichoic acid to produce a surface with a net negative charge with high metal binding capacity. During metabolism, a membrane-induced proton motive force continuously pumps protons into the wall fabric. As a result, a competition between protons and metal ions for anionic wall sites occurs, and less metal is bound in living cells than in nonliving cells or those in which the plasma membrane has been uncoupled. This was shown by using two metallic ions, UO2(2+) and Sc3+, on control cells, cells uncoupled with either carbonyl cyanide m-chlorophenylhydrazone or NaN3, or cells killed by gamma radiation. Transmission electron microscopy, energy-dispersive X-ray spectroscopy, and inductively coupled plasma atomic-emission spectroscopy showed that more metal was retained in the walls of nonliving cells and those with deenergized membranes than in their living counterparts.     

Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis

We present redirection of electron flow to more efficient proton pumping branches within respiratory chains as a generally applicable metabolic engineering strategy, which tailors microbial metabolism to the specific requirements of high cell density processes by improving product and biomass yields. For the example of riboflavin production by Bacillus subtilis, we reduced the rate of maintenance metabolism by about 40% in a cytochrome bd oxidase knockout mutant. Since the putative Yth and the caa(3) oxidases were of minor importance, the most likely explanation for this improvement is translocation of two protons per transported electron via the remaining cytochrome aa(3) oxidase, instead of only one proton via the bd oxidase. The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B. subtilis biomass in fed-batch cultures.     

Oscillatory transepithelial H(+) flux regulates a rhythmic behavior in C. elegans

In C. elegans, rhythmic defecation is timed by oscillatory Ca(2+) signaling in the intestine [1-5]. Here, by using fluorescent biosensors in live, unrestrained worms, we show that intestinal pH also oscillates during defecation and that transepithelial proton movement is essential for defecation signaling. The intestinal cytoplasm is acidified by proton influx from the lumen during defecation. Acidification is predicted to trigger Na(+)/H(+) exchange activity and subsequent proton efflux. The Na(+)/H(+) exchanger NHX-7 (PBO-4) extrudes protons across the basolateral membrane and is necessary for both acute acidification of the pseudocoelom and for strong contractions of the posterior body wall muscles during defecation. This suggests that secreted protons transmit a signal between the intestine and muscle. NHX-2 is a second Na(+)/H(+) exchanger whose distribution is limited to the apical membranes facing the intestinal lumen. RNA interference of nhx-2 reduces the basal pH of the intestinal cells, reduces the rate of proton movement between the lumen and the cytoplasm during defecation, and extends the defecation period. Thus, the cell may integrate both pH and calcium signals to regulate defecation timing. Overall, these results establish the defecation cycle as a model system for studying transepithelial proton flux in tissues that maintain systemic acid-base balance.     

Mechanism of amino Acid uptake by sugarcane suspension cells

The amino acid carriers in sugarcane suspension cells were characterized for amino acid specificity and the stoichiometry of proton and potassium flux during amino acid transport.

Amino acid transport by sugarcane cells is dependent upon three distinct transport systems. One system is specific for neutral amino acids and transports all neutral amino acids including glutamine, asparagine, and histidine. The uptake of neutral amino acids is coupled to the uptake of one proton per amino acid; one potassium ion leaves the cells for charge compensation. Histidine is only taken up in the neutral form so that deprotonation of the charged imidazole nitrogen has to occur prior to uptake. The basic amino acids are transported by another system as uniport with charge-compensating efflux of protons and potassium. The acidic amino acids are transported by a third system. Acidic amino acids bind to the transport site only if the distal carboxyl group is in the dissociated form (i.e. if the acidic amino acid is anionic). Two protons are withdrawn from the medium and one potassium leaves the cell for charge compensation during the uptake of acid amino acids. Common to all three uptake systems is a monovalent positively charged amino acidproton carrier complex at the transport site.

     

The impact of metabolic state on Cd adsorption onto bacterial cells

This study examines the effect of bacterial metabolism on the adsorption of Cd onto Gram-positive and Gram-negative bacterial cells. Metabolically active Gram-positive cells adsorbed significantly less Cd than non-metabolizing cells. Gram-negative cells, however, showed no systematic difference in Cd adsorption between metabolizing and non-metabolizing cells. The effect of metabolism on Cd adsorption to Gram-positive cells was likely due to an influx of protons in and around the cell wall from the metabolic proton motive force, promoting competition between Cd and protons for adsorption sites on the cell wall. The relative lack of a metabolic effect on Cd adsorption onto Gram-negative compared to Gram-positive cells suggests that Cd binding in Gram-negative cells is focused in a region of the cell wall that is not reached, or is unaffected by this proton flux. Thermodynamic modeling was used to estimate that proton pumping causes the pH in the cell wall of metabolizing Gram-positive bacteria to decrease from the bulk solution value of 7.0 to approximately 5.7.     

Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye.

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.     

Kinetics of proton binding to the sarcoplasmic reticulum Ca-ATPase in the E1 state

A new caged proton, 2-methoxy-5-nitrophenyl sulfate, was synthesized and used in time-resolved pH jump experiments to study proton binding in the sarcoplasmic reticulum Ca-ATPase. The major advantage of this compound is that it does not produce significant artifacts in experiments in which the fluorescent styryl dye 2BITC is used to monitor ion movements in the Ca pump. Two rate-limiting processes were resolved and their dependence on pH, Ca(2+) concentration, and temperature investigated. The faster process showed a relaxation time between 4 and 8 ms independent on pH and Ca(2+) concentration, and the time constant of the slower process varied between 31 ms (0 Ca(2+)) and 100 ms (100 microM Ca(2+)). A consistent mechanism to explain the results was derived in agreement with previous studies and the generally accepted Post-Albers scheme of the pump cycle. This mechanism requires that under physiological conditions the ion-binding sites are always occupied and two protons and a Ca(2+) ion replace each other. In the absence of ATP at low pH a nonphysiological state can be induced in which up to four protons bind to the Ca pump in the E(1) conformation. So far it could not be verified whether these additional protons bind to amino acid side chains or are coordinated as hydronium ions.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.     

Genome-scale model for Clostridium acetobutylicum: Part II. Development of specific proton flux states and numerically determined sub-systems

A regulated genome-scale model for Clostridium acetobutylicum ATCC 824 was developed based on its metabolic network reconstruction. To aid model convergence and limit the number of flux-vector possible solutions (the size of the phenotypic solution space), modeling strategies were developed to impose a new type of constraint at the endo-exo-metabolome interface. This constraint is termed the specific proton flux state, and its use enabled accurate prediction of the extracellular medium pH during vegetative growth of batch cultures. The specific proton flux refers to the influx or efflux of free protons (per unit biomass) across the cell membrane. A specific proton flux state encompasses a defined range of specific proton fluxes and includes all metabolic flux distributions resulting in a specific proton flux within this range. Effective simulation of time-course batch fermentation required the use of independent flux balance solutions from an optimum set of specific proton flux states. Using a real-coded genetic algorithm to optimize temporal bounds of specific proton flux states, we show that six separate specific proton flux states are required to model vegetative-growth metabolism and accurately predict the extracellular medium pH. Further, we define the apparent proton flux stoichiometry per weak acids efflux and show that this value decreases from approximately 3.5 mol of protons secreted per mole of weak acids at the start of the culture to approximately 0 at the end of vegetative growth. Calculations revealed that when specific weak acids production is maximized in vegetative growth, the net proton exchange between the cell and environment occurs primarily through weak acids efflux (apparent proton flux stoichiometry is 1). However, proton efflux through cation channels during the early stages of acidogenesis was found to be significant. We have also developed the concept of numerically determined sub-systems of genome-scale metabolic networks here as a sub-network with a one-dimensional null space basis set. A numerically determined sub-system was constructed in the genome-scale metabolic network to study the flux magnitudes and directions of acetylornithine transaminase, alanine racemase, and D-alanine transaminase. These results were then used to establish additional constraints for the genome-scale model.     

Metastable proton pools in thylakoids and their importance for the stability of Photosystem II

After isolated chloroplast thylakoids have been transferred to a medium which is more alkaline than their storage medium, they retain considerable amounts of unequilibrated protons for often longer than 10 min. Essentially all of these protons are released upon uncoupler addition when the thylakoids are osmotically swollen, but only a portion of them when they are in a shrunken state. Osmotic swelling also greatly accelerates the inactivation of the water-oxidizing system enzyme of Photosystem II, and its depletion of functional Cl^?, at alkaline pH. Analyses of the mestable proton gradient in terms of stoichiometry, temperature dependence, and effect on fluorescent amine probes, suggest that most of the protons involved are bound and exchange readily with the bulk phases only when the thylakoids are swollen. It is concluded that, in shrunken thylakoids, the water-oxidizing enzymes are buried in special H⁺-sequestering domains which probably are formed by cavities in the inner surface of the thylakoid membrane. An observed cooperative action of alkaline pH and divalent cations during Cl^?-extraction from Photosystem II is interpreted as revealing an involvement of both a negatively charged surface region and positively charged groups in maintaining the functional integrity of the site of water oxidization.     

Formation of Molecular Complexes Between a Structurally Defined M Protein and Acylated or Deacylated Lipoteichoic Acid of Streptococcus pyogenes

The orientation of lipoteichoic acid (LTA) molecules on the surface of bacterial cells undoubtedly is determined by the ability of the LTA, during its transit through the cell wall, to bind via its polyglycerophosphate backbone or its glycolipid moieties to other constituents of the cytoplasmic membrane and the cell wall. We have investigated the possibility that LTA may become anchored to the cell surface by binding through its polyanionic backbone to positively charged regions of cell wall proteins. LTA was found to prevent the precipitation of partially purified HCl extracts of several strains of streptococci as well as a structurally defined streptococcal M protein molecule (pep M24) in 83% solutions of ethanol. The formation of complexes between LTA and M protein was demonstrated further by immunoelectrophoresis of pep M24 protein with increasing concentrations of radiolabeled LTA and by using antiserum against pep M24 to develop precipitin arcs. Pep M24 electrophoresed alone produced a single precipitin arc close to the origin. In contrast, when electrophoresed as a mixture with LTA or deacylated LTA, the M protein produced a second precipitin arc toward the anode coinciding with the area of migration of the radioactive LTA. Increasing concentrations of LTA or deacylated LTA shifted increasing amounts of the pep M24 antigen to the region of the second arc. Maleylation of M protein to block the positively charged free amino groups before mixing it with LTA prevented the formation of complexes. The complexes formed by the M protein with LTA, but not with deacylated LTA, showed the capacity to bind bovine serum albumin; LTA had been shown previously to bind to the fatty acid binding sites on bovine serum albumin. These results indicate that the LTA molecule is able to bind via its polyanionic backbone to positively charged residues of surface proteins of cells of S. pyogenes. The implications of such interaction as to the orientation of LTA molecules on the surface of cells of S. pyogenes are discussed.     

Effect of extracellular calcium,pH and borate on growth oscillations in Lilium formosanum pollen tubes

Calcium ions (Ca(2+)), protons (H(+)), and borate (B(OH)(4)(-)) are essential ions in the control of tip growth of pollen tubes. All three ions may interact with pectins, a major component of the expanding pollen tube cell wall. Ca(2+ )is thought to bind acidic residues, and cross-link adjacent pectin chains, thereby strengthening the cell wall. Protons are loosening agents; in pollen tube walls they may act through the enzyme pectin methylesterase (PME), and either reduce demethylation or stimulate hydrolysis of pectin. Finally, borate cross-links monomers of rhamnogalacturonan II (RG-II), and thus stiffens the cell wall. It is demonstrated here that changing the extracellular concentrations of Ca(2+), H(+) and borate affect not only the average growth rate of lily pollen tubes, but also influence the period of growth rate oscillations. The most dramatic effects are observed with increasing concentrations of Ca(2+) and borate, both of which markedly reduce the rate of growth of oscillating pollen tubes. Protons are less active, except at pH 7.0 where growth is inhibited. It is noteworthy, especially with borate, that the faster growing tubes exhibit the shorter periods of oscillation. The results are consistent with the idea that binding of Ca(2+) and borate to the cell wall may act at a similar level to alter the mechanical properties of the apical cell wall, with optimal concentrations being high enough to impart sufficient rigidity to the wall so as to prevent bursting in the face of cell turgor, but low enough to allow the wall to stretch quickly during periods of accelerating growth.     

Potentiation of TRPM7 inward currents by protons

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from -100 to -40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an approximately 10-fold increase at pH 4.0 and 1-2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca(2+) and Mg(2+) for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca(2+) and Mg(2+) concentrations were increased. Third, the apparent affinity for Ca(2+) and Mg(2+) was significantly diminished at elevated external H(+) concentrations. Fourth, the anomalous-mole fraction behavior of H(+) permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca(2+) and Mg(2+) bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H(+) concentrations, the affinity of TRPM7 for Ca(2+) and Mg(2+) is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg(2+)-inhibitable cation current (MIC) or Mg nucleotide-regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.     

The mutation F227I increases the coupling of metal ion transport in DCT1

Metal ion transport by DCT1, a member of the natural resistance-associated macrophage protein family, is driven by protons. The stoichiometry of the proton to metal ion is variable, and under optimal transport conditions, more than 10 protons are co-transported with a single metal ion. To understand this phenomenon better, we used site-directed mutagenesis of DCT1 and analyzed the mutants by complementation of yeast suppressor of mitochondria import function-null mutants and electrophysiology with Xenopus oocytes. The mutation F227I resulted in an increase of up to 14-fold in the ratio between metal ions to protons transported. This observation suggests that low metal ion to proton transport of DCT1 resulting from a proton slippage is not a necessity of the transport mechanism in which positively charged protons are driving two positive charges of the metal ion in the same direction. It supports the idea that the proton slippage has a physiological advantage, and the proton slip was positively selected during the evolution of DCT1.     

Intracellular pH regulation in a nonmalignant and a derived malignant human breast cell line

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.     

The Characteristics of the Adjustment in the pH of the Xylem Exudate of Segments of Vigna Hypocotyls during Xylem Perfusion

The xylem vessels of segments of Vigna hypocotyl were perfusedwith solutions of various pH values. With both acid and alkalineperfusion solutions, the pH of the xylem exudate tended to equilibrateat approximately 6 during the course of xylem perfusion. Thisphenomenon was observed both in air and under anoxia, and alsoin segments pretreated with methanol or preheated in a microwaveoven. Therefore, the adjustment in pH does not depend on therespiratory metabolism but originates in the buffering actionof the xylem wall apoplast. High concentrations of protons inducedthe release of other cations into the xylem exudate under anoxia,and high concentrations of K⁺ or Ca²⁺ ions induced the releaseof protons into the xylem under anoxia. These results indicatethat a cation-exchange reaction on the xylem cell wall was responsiblefor the buffering effect. The physiological role of the highbuffering capacity of the xylem wall apoplast is discussed inlight of the role of the proton pump of the plasmalemma in elongationgrowth. (Received September 25, 1990; Accepted January 21, 1991)     

Characterization of the Bacillus subtilis motile system driven by an artificially created proton motive force.

Transient swimming was induced in energy-depleted cells of Bacillus subtilis by an artificial proton motive force, which was created by valinomycin addition and a pH reduction. This system did not require any ions except protons in the medium. The size of the induced motility was strongly influenced by changes in the size of either the K+ diffusion potential or the pH gradient. A rough estimation indicated that a proton motive force higher than -100 mV was required for induction of translational swimming of the cell. Corresponding with the transient appearance of swimming, a rapid but transient efflux of K+ and influx of H+ were observed. With decreases in the rate of H+ influx, the amount of motility decreased. A rate of H+ influx higher than 0.2 mumol/s per ml of cell water gave translational swimming. These results suggest direct coupling of H+ influx to rotation of bacterial flagella.