Physical Properties of Escherichia coli Spheroplast Membranes

Abstract

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.

Cytoplasmic sensing by the inner membrane histidine kinase EnvZ

“…Recent studies of bacterial cytoplasmic membranes indicate that their basic properties are significantly different from red cell membranes and lipid vesicles. For example, the spheroplast membranes are tensionless and the surface area appears to be controlled by a membrane reservoir equivalent to membrane folds (Sun et al, 2014). This finding may challenge our existing notion that mechanosensitive channels play an important role during volume changes in E. coli (Martinac et al, 1987;Perozo and Rees, 2003).…”

“…Previous studies have measured the concentration of the cytoplasm up to 1.8 osmoles (Cayley et al, 2000) in response to osmolytes. This enormous change in osmolality is also accompanied by large changes in volume; estimates of changes in spheroplast volumes suggest that this can be up to 3-fold (Sun et al, 2014). Perhaps when E. coli undergoes this large volume change, it transmits mechanical stress to the TM domains of EnvZ, altering its signaling properties.…”

Bacterial Spheroplasts as a Model for Visualizing Membrane Translocation of Antimicrobial Peptides

“…However, if signal on the membrane is sufficiently strong it can "contaminate" slices ostensibly taken "inside" the cell, as we have observed in measurements of the membrane-localized dye di-8-ANEPPS (Fig. 1).In order to overcome these resolution limits, we have employed bacterial spheroplasts (12)(13)(14). Spheroplasts are produced by culturing bacteria in the presence of an antibiotic, such as cephalexin, that prevents division while still allowing cells to grow.…”

“…In order to overcome these resolution limits, we have employed bacterial spheroplasts (12)(13)(14). Spheroplasts are produced by culturing bacteria in the presence of an antibiotic, such as cephalexin, that prevents division while still allowing cells to grow.…”

“…wall may have a "sieving" effect with some larger peptides that would be lost in spheroplasts, requiring additional controls comparing spheroplasts and "normal" cells in other assays (18). However, even with these caveats we believe that spheroplasts provide an excellent model system compared to other alternatives to overcome size and shape limitations, such as giant unilamellar vesicles (19)(20)(21), as spheroplasts preserve a physiological bacterial membrane composition and are viable if returned to growth conditions (13,22). Moreover, although spheroplasts have generally been produced from E. coli, protocols can be adjusted to make them from strains of other species (23).…”