Osmotic Pressurein Escherichia Coli As Rendered Detectable By

JouRNAL or BAcmOLoGY, May 1973, p. 549-555 Vol. 114, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Osmotic Pressure in Escherichia coli as Rendered Detectable by Lysozyme Attack PAUL SCHEIE Department of Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 8 January 1973 The enhanced susceptibility of plasmolyzed Escherichia coli to lysozyme attack was used to estimate the internal osmotic pressure of these cells under various conditions. Differences were detected between strains, culture media, stages in the growth cycle, and the osmotically active material used to produce plasmolysis. Lysozyme also was found to attack unplasmolyzed cells at 0 C and between 50 and 70 C. The osmotic pressure of bacteria is believed more, several of the studies on plasmolysis to afford a measure of the concentration of have involved washed cells from which many small, free solute molecules contained within osmotically active particles probably had been the semipermeable plasma membrane. This removed. Hence, the osmotic pressure ofE. coli concentration helps establish the internal envi- should not be considered a well-determined ronment in which the chemistry of the cell parameter. takes place. Moreover, the differential osmotic Osmotic injury in the form of plasmolysis pressure, that is, the difference between the renders E. coli susceptible to lysozyme attack osmotic pressure in the cell and that of the (3). Subsequent dilution of such cells in a medium, will determine how firmly the plasma hypotonic medium can result in the formation membrane is pressed against the rigid pep- of spheroplasts or cell lysis. Lysis, in turn, is tidoglycan layer. This differential osmotic easily monitored by turbidity measurements. pressure thereby may affect one or more of the While this method is commonly utilized in the several membrane-related activities. preparation of protoplasts or gently lysed cells, Studies on the osmotic properties of Esche- little attention has been given to the possibility richia coli generally have involved varying the of using the same technique to measure the osmotic pressure of the medium and observing internal osmotic pressure of E. coli or as an the response of the cells. Envelopes of E. coli assay for certain injuries, osmotic or other, to cells are subject to alteration by osmotic stress. the cell envelope. Thermal damage, for in- Hypotonicity leads to the release of small stance, would seem to be a likely candidate for metabolites (11) and several envelope-bound detection. Thermal inactivation of cells is be- enzymes (8) as well as to the formation of lieved by some to involve a leaky plasma finger-like protrusions (2) and, perhaps, lysis membrane (12), and microscopic observations (12). Hypertonicity leads to plasmolysis during have shown heat-induced alterations in the which the plasma membrane shrinks away appearance of the outer membrane (6). More- from the rigid peptidoglycan layer (5, 13). The over, one might anticipate a role for internal osmotic pressure of the medium at which osmotic pressure in thermal inactivation to the plasmolysis first becomes microscopically evi- extent that outward pressure on a thermally dent generally is cited as the internal osmotic weakened envelope effects irreversible changes pressure of the cell. It is tacitly assumed that Osmotic pressure in E. coli cells subjected to the plasma membrane is free to shrink away various conditions and assayed by susceptibil- from the cell envelope, at least in some regions. ity to lysozyme attack are reported here. Differ- Values so obtained range from 2 atm for resting ences were found between strains, culture me- cells to 15 atm for rapidly growing cells (10). It dia, stages in the growth cycle, and osmotically is, of course, difficult and tedious to quantitate active materials used in the assay. Investiga- the prevalence of plasmolysis for large numbers tion of the effect of lysozyme on heated cells of cells by microscope examination. Further- indicated an increased susceptibility to attack 549 550 SCHEIE J. BACrERIOL. between 50 and 70 C, in addition to which a concentration of 1.5 to 2 x 108 cells/ml (absorbance evidence was found for damage to the en- -0.4). A 2-ml amount of this suspension was added velopes of some cells when placed at 0 C. to a preincubated test tube containing 8 ml of C-minimal medium lacking the MgCl2 and Na2SO4 but with MATERIALS AND METHODS lysozyme and ethylenediaminetetracetic acid (EDTA) added to give final concentrations of 56 E. coli strains B/r, B.-,, and W3110 obtained from ug/ml and 4%, respectively. The tubes were removed Stanley Person were grown at 37 C in 10, 15, or 25 ml at intervals from the bath and the absorbance was of medium bubbled with air. Growth media used measured at 425 nm. The first readings were made 20 were nutrient broth (Difco) at 8 g/liter and Roberts s after adding the cells to the hot medium and these C-minimal medium with 5% glucose (11). served as the values for initial absorbance. The Microscope observations were carried out with a measurement process required removing the tubes Bausch and Lomb bright-field phase-contrast micro- from the bath for about 15 s per reading. scope at a magnification of x900. Osmotic effects. Samples (5 ml) of cultures in exponential phase were placed in 12-ml heavy-wall RESULTS centrifuge tubes (Sorvall). The initial absorbance Osmotic pressure and differential pres- (-0.2) of the suspensions in these tubes was deter- sure. The decrease in absorbance of lysozyme- mined at 425 nm with a Bausch and Lomb spectro- E. coli after to various photometer, model 20, after which the cells were treated B/r subjection centrifuged at 8,000 x g at room temperature for 3 concentrations of sucrose at room temperature min. The supernatant fluid was decanted and the (-25 C) is indicated in Fig. 1. Figure 2 shows inside walls of the tubes were blotted to remove most the two positions in the growth cycle from of the remaining liquid. The tubes were than placed which samples were taken. Plasmolysis and in a bath at the appropriate temperature. Room lysis first became evident in exponential phase temperature was approximately 25 C; a stable 11 C cells at about 0.2 M sucrose and by 0.5 M su- bath was obtained with running cold tap water; and crose the maximum amount of lysis had been the 0 C bath consisted of an ice slurry. attained. These results agreed with those re- Two to three minutes were allowed for the pellet to who reach the desired temperature. The desired sucrose ported by Birdsell and Cota-Robles (3), or salt solution (0.1 ml), maintained at the same used a similar technique, as well as with those temperature as the pellet, was added to the tubes, of Scheie (13) who noted the percentage of and the pellets were suspended by a few seconds of cells plasmolyzed by counting under a micro- shaking on a Vortex mixer. A 0.1-ml sample of egg scope. The residual absorbance, even at high white lysozyme [56 Ag/ml in 0.0005 M tris (hydroxy- concentrations of sucrose, might be accounted methyl)aminomethane (Tris) at pH 7.2] was added for in one or more ways. Ghosts of either cells and the tube was reshaken. After an additional 30 to or spheroplasts contributed a small amount to 60 s in the bath the tubes were removed and the cells the absorbance. On the other hand, up to 20% were diluted. This dilution was accomplished with 5 ml of nutrient broth at 25 C for cells grown in of the cells may have been resistant to plasmol- nutrient broth and with 5 ml of double-distilled ysis; or, it is possible that some of the cells water for cells grown in the C-minimal medium. The were permeable to the sucrose after lysozyme absorbance was measured immediately following attack and did not lyse; or perhaps the outer dilution and again after 5 to 10 min. It was found that portions of the cell envelope on some of the little change occurred after 5 min. Results were cells, even if plasmolyzed, were impermeable expressed in terms of the ratio of final absorbance/ini- to lysozyme and, consequently, the lysozyme tial absorbance which, multiplied by 100, is per cent still could not reach its substrate. Microscope initial absorbance. The mean values of 3 to 8 separate of exponential-phase cells treated measurements were plotted as a function of solute observation concentration along with error bars indicating esti- with 0.8 M sucrose and lysozyme indicated the mates of the standard deviation of the mean (16). presence of both rods and spheres after dilu- One departure from this procedure began with tion into nutrient broth; hence, more than one cells grown in nutrient broth to an initial absorbance of the above must have been involved. of -0.65 when the osmotic pressure of cells from cul- E. coli B/r cells from cultures after exponen- tures after the exponential phase was measured. For tial phase were less susceptible to plasmolysis another set of experiments cells grown in C-minimal and lysozyme attack for a given concentration medium were washed twice with double-distilled of sucrose. Fifty percent lysis was not exhibited water before being subjected to sucrose and lysozyme. The initial absorbance was measured prior to until 1.0 M sucrose was used, at which point pelleting the cells after the second suspension in the maximum amount of lysis probably had water. The lysozyme applied to these washed cells not been reached. was dissolved in 0.01 M Tris at pH 7.2.

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