Phase and electron microscopic observations of osmotically induced wrinkling and the role of endocytotic vesicles in the plasmolysis of the Gram-negative cell wall

When a Gram-negative bacterium is challenged with a sufficient concentration of a non-penetrating solute such as sucrose, water is sucked out of the cell. Plasmolysis spaces may form if the cell's cytoplasmic membrane (CM) separates from the murein wall (M) and the outer membrane (OM). However, we suggest that first wrinkling of the wall envelope, forced by dehydration of the cytoplasm, occurs. The cryofixation, freeze-substitution electron microscope studies used here are much too slow to study the kinetics of shrinkage, wrinkling and plasmolysis. However, they are consistent with faster phase microscope studies and previous stopflow experiments. For the electron microscopy studies reported here, only sucrose was used as the osmotic agent and under conditions that do not cause extreme plasmolysis. Plasmolysis spaces were associated with the formation of small membrane-bound vesicles in the nearby cytoplasm. Such vesicles formed by osmotic challenge are called 'endocytotic' in plant cell systems. They had been recorded in earlier plasmolysis studies in bacteria, but not interpreted as a concomitant part of plasmolysis space formation in certain locations of the cell. We suggest that the endocytotic vesicles form because the phospholipid membranes are capable of very little contraction so extra membrane must be disposed of when plasmolysis spaces form. In the case of plasmolysis spaces forming at poles and constriction sites, for geometric reasons the surface area of the CM may be conserved without disposition of excess membrane. We suggest that it is this biophysical property of lipid membranes that leads to the frequent formation of plasmolysis spaces at a pole and at the site of future division. We also observed a novel structure, this is seen only under mild osmotic up-shock, and consists of very thin, straight, uniform and long plasmolysis spaces which were called 'lamellar spaces'; these commonly formed inside the sidewalls and were usually associated with the formation of endocytotic vesicles. Since lipoprotein links the M to the OM layers and thus could affect plasmolysis, we examined both wild-type and deficient strains. Some effects were observed, but they were minimal. The volume of the periplasmic space of growing unshocked cells was determined to be about 7%.