Increase In External Concentration Research Articles

Origins of Escherichia coli Growth Rate and Cell Shape Changes at High External Osmolality

In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope, followed by an active recovery. After recovery, and if the external osmolality remains high, cells have been shown to grow more slowly, smaller, and at reduced turgor pressure. Despite the fact that the active recovery is a key stress response, the nature of these changes and how they relate to each other is not understood. Here, we use fluorescence imaging of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their volume to the initial, preshock value and continue to grow at a slower rate immediately after the recovery. We show that the cell envelope material properties do not change after hyperosmotic shock, and that cell shape recovers along with cell volume. Taken together, these observations indicate that the turgor pressure recovers to its initial value so that reduced turgor is not responsible for the reduced growth rate observed immediately after recovery. To determine the point at which the reduction in cell size and turgor pressure occurs after shock, we measured the volume of E. coli cells at different stages of growth in bulk cultures. We show that cell volume reaches the same maximal level irrespective of the osmolality of the media. Based on these measurements, we propose that turgor pressure is used as a feedback variable for osmoregulatory pumps instead of being directly responsible for the reduction in growth rates. Reestablishment of turgor to its initial value might ensure correct attachment of the inner membrane and cell wall needed for cell wall biosynthesis.

Plasmolysis and Cell Shape Depend on Solute Outer-Membrane Permeability during Hyperosmotic Shock in E. coli

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.

Distribution of electrolytes between membraneous and bulk phases, and the dielectric properties of membraneous water, studied by impedance spectroscopy measurements on dense cellulose acetate membranes

The dielectric properties of dense cellulose acetate (CA) membranes have been determined using impedance spectroscopy. The impedance of the membranes in equilibrium with external salt solutions (10–4–1 mol dm–3) and at varying pH as a function of the frequency of the applied field was measured. From the data, and using classical Donnan theory together with the Nernst–Planck electrodiffusion equations and known dielectric theory, it is possible to find the concentration ratios of the ions between the membranes and the surrounding solution as a function of the external salt concentration, as well as their diffusion coefficients. In addition, the relative permittivities of the membrane matrix and of the solution present in the membrane alveoles, as well as the size of the latter, can be estimated. The results show (1) that there is no qualitative difference between NaCl and KCl. (2) The concentration ratios of the cations and the anions differ very much from unity at low (10–4 mol dm–3) external salt concentration. The cation ratios attain values of several hundred, decreasing rapidly as the external concentration increases. (3) The (relative) permittivity of the solvent inside the membrane is ca. 20, indicating either that the rotational degrees of freedom of the water in the alveoles are, to a considerable extent, hindered compared with normal water, or that the temporary H-bond clusters in pore water are less extended than in bulk water, with a resulting lower effective dipole moment; and (4) the average radius of the alveoles is ca. 6.5 ± 1.0 nm. Our results are at variance with measurements on similar membranes reported a few years ago by Wiggins and van Ryn, J. Macromol. Sci., Part A, Chem., 1986, 23, 875, who found considerable qualitative differences in the behaviour of the two alkali-metal salts. On the other hand, the low relative permittivity estimated for the water in the membrane supports the idea of anomalous water in the membrane alveoles, however, this seems to be caused by the properties and size of the alveoles, rather than by the presence of a particular cation.

Intestinal absorption of vitamin B6. I. Pyridoxol uptake by rat intestinal tissue.

The uptake of 3H-pyridoxol by everted intestinal rings of the rat was studied mainly at concentrations of 0.1-4μM of the vitamin in the medium. Incubation of the tissue at 37°C for 60 min in Krebs-Ringer bicarbonate medium containing glucose and labelled pyridoxol resulted in intracellular accumulation of the labelled compounds. The total intracellular concentration of the compounds after the incubation was always higher than the extracellular concentration, and this accumu-lation was especially appreciable at relatively low external concentra-tions of the 3H-vitamin. The uptake under the same conditions was partially saturated with increasing concentration of 3H-pyridoxol in the medium, whereas the uptake during the initial 5 min of incubation increased in a linear manner with an increase of external concentration. The uptake was markedly depressed by lowering the temperature of incubation or addition of 4-deoxypyridoxol. The inhibition by 4-deoxypyridoxol affected the time course of uptake mainly in its later period, and also depressed both the linear and saturable components contained in the relation of uptake to the external concentration of the 3H-vitamin. However, a considerable uptake occurred even at the maximum inhibition with 4-deoxypyridoxol. Examination of the intracellular forms of the 3H-vitamin taken up revealed that the major components were three B6 phosphates. This phosphorylating tendency was particularly extreme when the tissue was incubated under conditions allowing marked accumulation of the 3H-vitamin. Of these phosphate esters, 3H-pyridoxamine 5'-phosphate appeared most highly responsible for the accumulation. 4-Deoxypyridoxol remark-ably diminished the 3H-vitamin B6 phosphates with a consequent de-crease of total intracellular 3H-vitamin B6 compounds, although it permitted free 3H-pyridoxol increase. From these results it is con-cluded that pyridoxol enters the intracellular space by simple diffusion, is accumulated by metabolic conversion to the phosphate forms of the vitamin, and the converion is inhibited by 4-deoxypyridoxol.

Electroosmosis in Ion‐Exchange Membranes

Application of an electric field to the system, electrolyte solution ⇌ membrane ⇌ electrolyte solution, gives rise to unidirectional flow of solvent phenomenologically called electroosmosis. The volume of this transference depends primarily on membrane water content and the concentration of the electrolyte solution bounding the membrane on either side. For the passage of 1F of electricity, the quantity of electroosmotic flow in dilute solutions is shown by some investigators to be dependent on current density, while others claim it to be independent of current density. Recent measurements indicate that membranes with low water contents (less than about 14%) produce water flows independent of current density, while those with higher water contents show current dependence. However, at external concentration greater than 0.1N, the current dependence of water flow disappears and the flow decreases with increase in external concentration. These results and others are discussed in terms of existing theories of electroosmosis in charged membranes.

The movement of thallium ions in muscle.

Measurements have been made of the fluxes of thallous ions (Tl(+)) across the membrane of frog sartorius muscle fibers. These show that at an external concentration of 74 microM the influx is about 270 x 10(-15) moles/cm.(2) sec., while the efflux from a muscle with an internal concentration equal to the above is 5 x 10(-15) moles/cm.(2) sec. The efflux is increased of the order of 300-fold during a muscle twitch, and Tl(+) reach a steady-state distribution between fiber water and Ringer solution that is very close to the corresponding ratio for K(+). High concentrations of Tl(+) depolarize the membrane about 58 mv. for a tenfold increase in external concentration. The results obtained are consistent with the view that the muscle fiber membrane cannot distinguish between the toxic heavy metal Tl(+) and K(+), provided that the concentrations of the former ion are kept low. High concentrations of Tl(+), if allowed to act for an appreciable period of time, lead to irreversible damage to muscle.

The mechanism of accumulation in living cells.

When a compound enters a living cell until its activity becomes greater inside than outside, it may be said to accumulate. Since it moves from a region where its activity is relatively low to a region where its activity is relatively high, it is evident that work must be done to bring this about. The following explanation is suggested to account for accumulation. The protoplasmic surface is covered with a non-aqueous layer which is permeable to molecules but almost impermeable to ions. Hence free ions cannot enter except in very small numbers. The experiments indicate that ions combine at the outer surface with organic molecules (carrier molecules) and are thus able to enter freely. If upon reaching the aqueous protoplasm these molecules are decomposed or altered so as to set the ions free, the ions must be trapped since they cannot pass out except in very small numbers. If we adopt this point of view we can suggest answers to some important questions. Among these are the following: 1. Why accumulation is confined to electrolytes. This is evident since only ions will be trapped. 2. Why ions appear to penetrate against a gradient. Actually there is no such penetration since the ions enter in combination with molecules. The energy needed to raise the activity of entering compounds is furnished by the reactions involved in the process of accumulation. 3. Why, in absence of injury, ions do not come out when the cell is placed in distilled water. Presumably the outgoing ions will combine at the outer surface with carrier molecules and then move inward in the same way as ions coming from without. 4. Why the relative rate of penetration falls off as the external concentration increases. This is because the entrance of ions is limited by the number of carrier molecules but no such limitation exists when ions move outward since they can do so without combining with carrier molecules. 5. Why accumulation is promoted by constructive metabolism which is needed to build up the organic molecules and by destructive metabolism which brings about their decomposition. 6. Why measuring the mobilities of ions in the outer protoplasmic surface does not enable us to predict the relative rate of entrance of ions. We find for example in Nitella that K(+) has a much higher mobility than Na(+) but the accumulation of these ions does not differ greatly. This is to be expected if they enter by combining with molecules at the surface. Only if K(+) is able to combine preferentially will it accumulate preferentially. 7. Why ions may come out in anoxia and at low temperatures. If these conditions depress the formation of carrier molecules and their decomposition in the protoplasm, the balance between intake and outgo of ions will be disturbed and relatively more may come out. 8. Why the excess of internal over external osmotic pressure is less in sea water than in fresh water. As the external concentration of ions increases the rate of intake does not increase in direct proportion since the number of carrier molecules does not increase and this slows down the relative rate of intake of ions. But it does not slow down the rate of exit of ions since they need not combine with carrier molecules in order to pass out. Hence the excess of ions inside will be relatively less as the concentration of external ions increases. 9. How water is pumped from solutions of higher to solutions of lower osmotic pressure. If metabolism and consequently accumulation is higher at one end of a cell than at the other, the internal osmotic pressure will be higher at the more active end and this makes it possible for the cell to pump water from solutions of higher osmotic pressure at the more active end to solutions of lower osmotic pressure at the less active, as shown experimentally for Nitella. This might help to explain the action of kidney cells and the production of root pressure in plants.