Accumulation of 3-(N-morpholino)propanesulfonate by osmotically stressed Escherichia coli K-12.

We found that exogenous morpholinopropanesulfonate (MOPS) is concentrated approximately fivefold in the free volume of the cytoplasm of Escherichia coli K-12 (strain MG1665) when grown at high osmolarity (1.1 OsM) in two different media containing 40 mM MOPS. MOPS was not accumulated by E. coli grown in low-osmolarity MOPS-buffered medium or in 1.1 OsM MOPS-buffered medium containing the osmoprotectant glycine betaine. Salmonella typhimurium LT2 did not accumulate MOPS under any condition examined. We infer that accumulation of MOPS by E. coli K-12 is not due to passive equilibration but rather to transport, possibly involving an as yet uncharacterized porter not present in S. typhimurium. Glutamate and MOPS were the only anionic osmolytes we observed by 13C nuclear magnetic resonance in E. coli K-12 grown in MOPS-buffered medium. The increase in positive charge accompanying the increase in the steady-state amount of K+ in cells shifted from low to high external osmolarity appeared to be compensated for by changes in the amounts of putrescine, glutamate, and MOPS. MOPS is not an osmoprotectant, because its accumulation did not increase cell growth rate.     

Effect of proline and K+ on the stimulation of cellular activities in Escherichia coli K-12 under high salinity

Growth of Escherichia coli K-12 in a modified Davis minimal medium was inhibited under high osmolarity, but it recovered remarkably with the addition of 1 mM proline. The co-existence of K+ with proline enhanced the recovery of growth under high osmolarity more than that in the presence of proline alone. The same was true for the activities of respiration and glucose uptake. A similar supplementary effect of K+ was observed for the activities of proline uptake under high osmolarity. These results suggest that K+ and proline support not only growth but respiration and uptake of the respiratory substrate glucose in the cell cytoplasm when exposed to high osmolarity. External K+ almost disappeared with 1 h of incubation at low osmolarity, indicating that active accumulation of K+ in the cells occurred. On the other hand, a gradual accumulation of K+ was recognized at high osmolarity in the presence of 1 M NaCl, especially at > 2 h of incubation. This study of L-[5-3H]proline uptake in the cell cytoplasm indicates that proline was incorporated as a substrate of protein synthesis in the absence of NaCl, but was efficiently utilized as a compatible solute in the presence of high concentrations of NaCl.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae.

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.     

Characteristics of K+ uptake in S. enteritidis bacteria

Bacterial Salmonella enteritidis var. Issatchenko in media without exogenic energy source uptakes K+ in one step with Km 2.1 mM and Vmax 0.08 mM min-1/10(12) cells. This K+ uptake does not depend on pH and osmotic shock and is not inhibited by DCC. Endogenic energy source (glucose) leads to K+ uptake with Km 2.8 mM and Vmax 0.10 mM min-1/10(12) cells, and secretion of H+. The ratio of the DCC-sensitive fluxes of H+ to K+ equals 2. Arsenate and protonophores depress the K+ uptake. Valinomycin decreases the rate of K+ uptake. It is assumed that K+ uptake takes place via the Trk-like system, which works as a separate system as supercomplex with the H+-ATPase complex.     

The capacity of anaerobically grown bacteria to exchange the 2H+ of the cell for the K+ of the medium and to maintain a high K+ distribution between cell and medium

The N,N'-dicyclohexylcarbodiimide sensitive exchange of 2H+ of a cell for K+ of medium stable to pH, K+ activity and temperature changes has been discovered in anaerobically grown gram-negative Escherichia coli, Salmonella typhimurium. S. enteritidis, Proteus mirabilis, P. vulgaris, anaerobic gram-positive bacteria Streptococcus faecalis, Lactobacillus salivarius, L. lactis in the presence of exogenic energy source. This exchange in gram-negative bacteria is operating only at increase of medium osmolarity. The high K+ distribution between cell and medium has been reached during the exchange of 2H+ for one K+ and the corresponding potassium equilibrium potential is much more than the measured delta psi. In aerobically grown E. coli, S. typhimurium, Brevibacterium flavum and aerobic Micrococcus luteus exchange of 2H+ for K+ does not take place, the K+ distribution is lower and in good conformity with the measured delta psi. It is assumed that exchange of 2H+ for K+ in anaerobic bacteria is carried out by the H+-ATPase complex and the Trk (or Trk-like) system of K+ absorption united into the same membrane supercomplex which functions as the H+-K+-pump and supports the high K+ distribution between cell and medium.     

Effect of arsenate on inorganic phosphate transport in Escherichia coli.

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.     

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.     

Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli.

The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined. Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect. The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport. The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport. Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source. The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source. Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport. When membrane potential of E. coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential. These results suggest that Li+ stimulates proline transport by intact cells of E. coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline. It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport.     

Phosphate uptake by P-limited Oscillatoria agardhii

Abstract The cyanobacterium Oscillatoria agardhii was grown under phosphorus limitation. [³²P]PO³₄ uptake experiments showed a biphasic uptake rate vs. phosphate concentration curve. At low phosphate concentrations the initial phosphate uptake rate (V) was almost linearly related to the external phosphate concentration (S). At higher concentrations V was related to S according to Michaelis-Menten kinetics. Temperature, calcium concentration and arsenate showed hardly any effect on the initial slope of the V/S curve. The observed phosphate uptake was therefore regarded to comprise two mechanisms. The first step of transport is diffusion-mediated, whereas the next step must be enzymatically mediated.     

Trimethylamine oxide and the maintenance of volume of dogfish shark rectal gland cells

Determinants of the steady-state vol of the dogfish shark (Squalus acanthias) rectal gland cells were studied. The cellular levels of trimethylamine oxide (TMAO) in fresh tissue and slices incubated aerobically 60 min in standard (TMAO-free) elasmobranch saline were close to those in the plasma (71 +/- 5 mM S.E.M.); therefore, under these conditions, the cell membrane appears to be impermeable to this solute. However, depolarization of the cells in high-K+ media produced a rapid loss of TMAO. Thus, TMAO is a major, effectively impermeant solute in the rectal gland cells. The osmolarity of cell solutes in tissue water (fresh and incubated slices) did not differ significantly from values in the plasma or incubation medium, demonstrating the absence of an osmotic pressure gradient across the cell membrane. An analysis of a simple model of cell solutes under steady-state conditions shows that the presence of an (effectively) impermeant osmolyte decreases the cellular concentration of bulk cations. The analysis is consistent with available observations on the distribution of cell Na+ and K+ in tissues containing high concentrations of (nitrogeneous) osmolytes. One simplifying assumption of the model, i.e., identity (or closeness) of the respective reflection coefficients sigma for Na+ and K+ passage through the cell membranes could not be verified. Compared to available data on the steady-state cellular fluxes of 42K+ in slices of the rectal gland, the uptake of 22Na+ by the tissue was slow (the derived rate constant k' = 0.017 min-1, i.e., about one tenth of that for K+).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Changes of ATP, polyphosphate and K+ contents in Saccharomyces carlsbergensis during uptake of Mn2+ and glucose

The process of prolonged Mn2+ uptake by the yeast Saccharomyces carlsbergensis in the presence of 100 mM glucose and in the absence of phosphate can be divided into two steps. The first step (0-20 min) of Mn2+ uptake (4.3 mumol/g of wet cells) is characterized by an intense K+ efflux (23.8 mumol/g), synthesis of high molecular weight polyphosphate (HPP) (8.1 mumol/g) and decrease of ATP content (0.06 mumol/g). Simultaneously about 0.6 mumol of glucose is taken up and the level of low molecular weight polyphosphate (LPP) remains practically unchanged. The second step (20-120 min) of Mn2+ uptake (15.6 mumol/g) is characterized by a drop in HPP (16.6 mumol/g) and the synthesis of LPP (19.0 mumol/g). The ATP content decreases by 0.87 mumol/g as compared to the control, while that of K+ increases (5.7 mumol/g). During the first step of Mn2+ uptake the energy of the K+ concentration gradient may be used both for Mn2+ influx (2K+: 1Mn2+) and synthesis of HPP (1P:1.9K+). During the second step the Mn2+ accumulation is apparently driven by HPP conversion into LPP (1:1) and by ATPases serving the Mn2+/H+ exchange.     

Accumulation of K+ in E. coli grown in aerobic conditions

The character of K+ accumulation in E. coli grown aerobilcally in the salt medium with succinate was studied. K+ uptake via the Trk system has Km 3.4 mM and Vmax 0.45 mM X g+1 X min-1. The initial rates of K+ uptake were not changes at different pH from 6.0 to 8.3 and temperature 17-37 degrees C. DCC did not block, protonophores and arsenate blocked the operation of Trk system. Valinomycin increased (or had no effect) K+ accumulation. K+ distribution is in good conformity with the measured membrane potential. The Trk system works at the utilization of lactic acid and glucose as well as of succinate. The Trk system is described. K+ ionophore by using the membrane potential and ATP regulates functioning of this system.     

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adaptive changes in phosphate uptake by the fungus Neurospora crassa in response to phosphate supply.

The phosphate uptake rate of Neurospora crassa germlings growing exponentially in media containing phosphate at concentrations between 10 mM and 50 micronM was virtually constant. The uptake characteristics of these germlings were studied in detail assuming the simultaneous operation of two uptake systems, one of low affinity and one of high affinity. The Km of the low-affinity system was constant after growth at phosphate concentrations greater than 1 mM but became progressively lower as the concentration was reduced below 1 mM. In contrast, the Km of the high-affinity system was independent of the phosphate concentration of the growth medium. The Vmax of each system was highest after growth at low phosphate concentrations. As the phosphate concentration was increased to a maximum of 100 mM, the Vmax of the low-affinity system fell gradually, whereas that of the high-affinity system at first fell rapidly but then reached a constant minimum value at concentrations of 2.5 mM and higher. The differences in the kinetic parameters fully account for the constancy of uptake rate shown by the germlings.     

Continuous culture of Rhodotorula rubra: kinetics of phosphate-arsenate uptake, inhibition, and phosphate-limited growth

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K(0.5) = 10.8 nm). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 nm, giving concentration gradients across the cell envelope of about 10(6). Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K(s) = 0.5 to 2.8 mum, V(max) = 55 mumoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 nm, whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K(i) for phosphate inhibition of arsenate uptake was 0.7 to 1.2 mum. Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of pH. Maximal affinity for phosphate occurred at pH 4 and at low concentrations of NaCl; however, V(max) for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 nm. Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux.     

Accumulation of arsenate, phosphate, and aspartate by Sreptococcus faecalis.

Uptake of arsenate and phosphate by Streptococcus faecalis 9790 is strictly dependent on concurrent energy metabolism and essentially unidirectional. targinine supports uptake only in presence of glycerol or related substances; glycerol is not directly involved in transport but depletes the cellular orthophosphate pool and thus relieves feedback inhibition of transport. Uptake of phosphate and arsenate is stimulated by K+ and by other permeant cations. The results suggest that electroneutrality is preserved by compensatory movement of either H+ or OH minus. Ionophores and N,N'-dicyclohexylcarbodiimide, which prevent establishment of a proton motive force, block the accumulation of thiomethylgalactoside and of threonine but not that of arsenate or phosphate. We conclude that arsenate accumulation requires adenosine 5'-triphosphate but is not driven by the proton-motive force. However, conditions and reagents that lower the cytoplasmic pH do inhibit accumulation of arsenate and phosphate, suggesting that uptake depends on the capacity of the cells to maintain a neutral or alkaline cytoplasm. We therefore propose that phosphate accumulation is an electroneutral exchange for OH driven by adenosine 5'-triphosphate or by a metabolite thereof. Accumulation of aspartate and glutamate also requires adenosine 5'-triphosphate but not the proton-motive force and may involve a similar mechanism.     

Osmotic stress,glucose transport capacity and consequences for glutamate overproduction in Corynebacterium glutamicum

Glucose uptake by Corynebacterium glutamicum is predominantly assured by a mannose phosphotransferase system (PTS) with a high affinity for glucose (Km=0.35 mM). Mutants selected for their resistance to 2-deoxyglucose (2DG) and lacking detectable PEP-dependent glucose-transporting activity, retained the capacity to grow on media in which glucose was the only carbon and energy source, albeit at significantly diminished rates, due to the presence of a low affinity (Ks=11 mM) non-PTS uptake system. During growth in media of different osmolarity, specific rates of glucose consumption and of growth of wild type cells were diminished. Cell samples from these cultures were shown to possess similar PTS activities when measured under standard conditions. However, when cells were resuspended in buffer solutions of different osmolarity measurable PTS activity was shown to be dependent upon osmolarity. This inhibition effect was sufficient to account for the decreased rates of both sugar uptake and growth observed in fermentation media of high osmolarity. The secondary glucose transporter was, however, not influenced by medium osmolarity. During industrial fermentation conditions with accumulation of glutamic acid and the corresponding increase in medium osmolarity, similar inhibition of the sugar transport capacity was observed. This phenomenon provokes a major process constraint since the decrease in specific rates leads to an increasing proportion of sugar catabolised for maintenance requirements with an associated decrease in product yields.