Periplasmic space in Salmonella typhimurium and Escherichia coli.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.

Much of the contaminating material was glucose.
Since Salmonella typhLmurtum and E'scherichm coli are unable to absorb sucrose, but can rapidly accumulate glucose, it was necessary to purify the 1"CJsucrose. After paper electrophoresis in 1.0% borate, 1"Clsucrose was eluted with water, and the eluent was deionized with mixed bed resin (AG 50-H' and AG 3.OH-1. Just before use with bacterial cells, the labeled sucrose solution was incubated for 30 min with an aliquot of the same bacterial suspension. The cells were then removed, first by centrifugation (10 min at 10,000 x g), then by filtration (0.22 p Mlllipore filters, GSWP). Over 99% of the resulting radiolabeled material was found to be ["Cjsucrose and was susceptible to hydrolysis by invertase. 128,000 f 9,000 239,000 k 3,000 4,160 f 440 9,690 i 30 Radiolabeled solute pairs were added to suspensions containing 9.4 mg, dry weight, of cells in 2 ml of wash medium. After 30 min at room temperature these suspensions were centrifuged, and solute distributions were determined by the centrifugation method.
The radiolabeled solute pairs used were [3H]water or inulin-PHlmethoxy and one of the compounds listed under "Radiolabeled Solutes (S)." Values for V,,,, were obtained by summing the quantities (VW,,,, -V,) and (V, -V ,nu,,n ), for a particular S. Since the total is algebraically equivalent to (V,,,,, -Vinulin), its correspondence to V,,,, follows from the scheme outlined in Fig. 1 As long as X remains constant the osmotic pressure of a cell compartment or a whole cell will vary inversely with its volume. However, a solute will augment the osmotic strength of any compartment it enters. For instance, since sucrose equilibrates across the outer membrane it should contribute to the osmotic strength of the periplasm. If (sucrose) is the n PerI = R71XperrlV,,erl + (mmse)l (9) Changes in cell volume such as those shown in Table V  If, in Medium 63, the cell volume is V,,,, then using the derived value for X,,,i + X,,,, and Equation 8 gives: Ileell = 0.428 RTV',,,,IV,,,,. From Table  V, the osmotic strength can, therefore, be calculated as 270 mosM in Medium 63. This result agrees well with results obtained by independent methods (see below). Applying this result to Equation 5 gave a value of 3.3 atm as the net pressure on the cell walls of these bacteria at room temperature in Medium 63.
The intracellular osmotic strength can also be estimated by measuring changes in cell volume in response to extracellular sucrose concentrations.
Combining Equations 4, 7, and 9 gives: Since these experiments involved high concentrations of sucrose, where the solute contributed to the measured periplasmic volume, a correction for the volume of the sucrose examined.
These cut-outs were then trimmed so that only the cytoplasmic portions of the cells remained, and the photographic paper which constituted the trimmmg was weighed.
For each kind of cell, the ratio of the weight of the intact cell cut-outs to the weight of the corresponding periplasmic trimmings was taken as a measure of the fraction of total cell volume occupied by penplasm. Cell compartmentation was estimated from solute distribution data as outlined in  This information was then used in accord with Fig. 1 to estimate the total volume of cells in each suspension and to divide this volume into its cytoplasmic and periplasmic components.
These results are expressed as the periplasmic fraction of the total cell volume.

Bacterial strain
Nutrients present during growth were added. These labeled suspensions (plus unlabeled controls) were centrifuged to determine the distributions of the labeled solutes as described under "Measurement of Solute Distributions." The data so obtained were used in accord with the scheme in Fig. 1    The results were used to estimate cell volumes according to the scheme described in Fig. 1. Total cell (0) and cytoplasmic (0) volumes at various extracellular sucrose concentrations are shown in the inset.
These are expressed relative to the total volume of cells in Medium 63 without added sucrose (1.6 x 10m3 liter/g, dry weight, of cells). FIG. 7 (right).
Effects of sucrose on Salmonella typhimurium LT2 cells washed and suspended in water. Bacteria were grown in Medium 63 containing 0.2% D-glucose. After harvest, they were washed twice in water and then suspended in water. Aliquots of 0.50 ml (7.5 mg, dry weight, of cells) were added to 1.5.ml portions of water containing different amounts of sucrose, and cell volumes were determined as described in the legend to Fig. 6. Total (0) and cytoplasmic (0) volumes at various extracellular sucrose concentrations are shown in the znset. These are expressed relative to the total volume of cells in water with no addition of sucrose (3.0 ml/g, dry weight, of cells).
We emphasize that the difference in osmotic strength between cells suspended in water and in Medium 63 represented a volume change, not a significant change in X,,,,.
The V,,,, in water was about twice that in Medium 63 (the dry weight yields of cells harvested in water were not significantly different from those harvested in Medium 63). Apparently the cells increased in volume until the osmotically active material was diluted enough to be balanced by P,,,, n,a,l (see Equations 5 and 8). During this process, P,,,, wa,, increased only slightly, from about 3.5 atm in Medium 63 to slightly more than 4 atm in water.
The large change in cell volume and small change in P cell wall imply that the cell wall is not a rigid structure 8. Intracellular osmotic strength as determined by the vapor phase equilibrium method. Aliquots of 1 ml were removed from the cell suspension used in the experiment described in Fig. 7. These were placed on tared glass cover slips where they were frozen and lyophilized.
Each cover slip was then weighed and placed over the center well of a Conway microdiffusion dish containing a solution of sucrose in its outer well. After 1 day (similar results were obtained after 2 days) the cover slips were weighed again. W corresponds to the weight of water (in kg per g, dry weight, of cells) which had condensed on a cover slip. Details of the vapor phase equilibration method are given under "Experimental Procedures." The intracellular osmotic strength of cells in water was estimated to be 179 most from an X (equivalent to X,,,, + X,.,,,,) of 0.538 mosmol/g, dry weight, of cells and a I',,,, of 3.0 ml/g, dry weight, of cells.   in the legends to  Tables II, III, and IV (strain SL35551 for Experiments  1, 2, and 3,  respectively. lY2Na]NaC1 or [WllNaCl, and 1:'Hlwater or inulin-[3H]methoxy were added to these suspensions in the same way that ["C]sucrose was added (see corresponding tables). Final NaCl concentrations were: 10 rn~ in Experiment 1, and 1 mM in Experiments 2 and 3. I.j,,,, l/[.j<,, I was determined by the ratio CV, ~ VU,,,,,,,,)/ CK,,,,,,,, -vu,,,,,,, ) 'I The values used for v,,,,.,,,,,. -V ,,,,, ,,,, (= V,,,,.,  We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract.) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.