Permeability changes associated with osmotic swelling of bacterial protoplasts.

Bacterial protoplasts, released from confinement within the rigid wall of whole cells, imbibe water, swell, and burst unless their suspending medium contains high concentrations of solutes which have difficulty crossing the osmotic barrier in the protoplast surface membrane (C. Weibull, Exptl. Cell Res. 9:139, 1955; P. Mitchell and J. Moyle, J. Gen. Microbiol. 20:434, 1959). Hence, protoplast permeability to a particular compound can be evaluated from the capacity of that compound to serve as an osmotic stabilizer. Recently (R. E. Marquis, Biochem. Biophys. Res. Commun. 20:580, 1965) it was found that the stabilizing capacities of lipophobic sugars for nonrespiring protoplasts were directly related to their molecular sizes; larger sugars were better stabilizers. This finding led to the conclusion that the protoplast membrane is perforated with small aqueous channels through which lipophobic molecules can diffuse. However, it could not be decided whether these channels are of fixed size, or whether they may become dilated in conjunction with the membrane extension occurring during osmotic swelling of protoplasts. We have approached this question experimentally by directly measuring the average size of surviving protoplasts in populations undergoing osmotic bursting in solutions containing glucose, sucrose, or raffinose. Procedures for growing cells of Bacillus megaterium strain KM and for releasing protoplasts are described elsewhere (R. E. Marquis, Arch. Biochem. Biophys., in press). Protoplast suspensions were chilled, and then samples were diluted 40-fold over a period of about 45 min with appropriate chilled solutions to lower the osmolality of the suspending medium. Slow, dropwise addition of diluent solution resulted in relatively little bursting in the protoplast population until some critical osmolality was reached; then bursting commenced and increased progressively as the extraceilular osmolality was lowered further. The number of protoplasts per milliliter was determined by use of a Petroff-Hausser counting chamber. Individual cells were measured with a calibrated ocular micrometer, and all the cells in a particular volume of suspension were measured to obtain average (of 150 measurements) values for diameter. Volumes were calculated from diameters on the assumption that the protoplasts were essentially spheres. Osmotic coefficients were determined from published data for glucose (Earl of Berkeley and E. G. J. Hartley, Phil. Trans. Royal Soc. London Ser. A 206:481, 1906) and sucrose (G. Scatchard, W. J. Hamer, and S. E. Wood, J. Am. Chem. Soc 60:3061, 1938) solutions; coefficients for raffinose solutions were calculated from osmotic-pressure values obtained by use of a Mechrolab vaporpressure osmometer. The values used for effective hydrodynamic radii are those reported by Schultz and Solomon (J. Gen. Physiol. 44:1189, 1961). An example of the results obtained is presented in Fig. 1; both the extracellular osmolalities at which 25% bursting occurred and the average volumes of the surviving protoplasts are plotted against the effective hydrodynamic radii of the sugars used. It is apparent that the osmolality of the suspending medium was lower when 25% bursting took place in sucrose solution compared with glucose solution, and it was still lower when 25% bursting occurred in raffinose solution. Moreover, the surviving protoplasts were markedly more swollen in sucrose solution than in glucose solution, and they were even more swollen in raffinose solution. In these experiments, osmotically unstable protoplasts burst rapidly during the dilution period, whereas the swollen survivors appeared to remain effectively impermeable to the external sugar, since they were stable for 1 day or more after dilution and initially contracted rather than swelled. We have chosen to present values obtained with protoplast populations in which only 25%, rather then 50%, bursting occurred, because it was found that protoplasts which had swollen to