PHASE AND ELECTRON-MICROSCOPIC OBSERVATIONS OF OSMOTICALLY INDUCED WRINKLING AND THE ROLE OF ENDOCYTOTIC VESICLES IN THE PLASMOLYSIS OF THEGRAM-NEGATIVE CELL-WALL

When a Cram-negative bacterium is challenged with a sufficient concent ration of a non-penetrating solute such as sucrose, water is sucked ou t of the cell. Plasmolysis spaces may form if the cell's cytoplasmic m embrane (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, free ze-substitution electron microscope studies used here are much too slo w to study the kinetics of shrinkage, wrinkling and plasmolysis. Howev er, they are consistent with faster phase microscope studies and previ ous 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 associ ated with the formation of small membrane-bound vesicles in the nearby cytoplasm. Such vesicles formed by osmotic challenge are called 'endo cytotic' in plant cell systems. They had been recorded in earlier plas molysis studies in bacteria, but not interpreted as a concomitant part of plasmolysis space formation in certain locations of the cell. We s uggest that the endocytotic vesicles form because the phospholipid mem branes are capable of very little contraction so extra membrane must b e 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 ex cess membrane. We suggest that it is this biophysical property of lipi d membranes that leads to the frequent formation of plasmolysis spaces at a pole and at the site of future division. We also observed a nove l structure, this is only seen under mild osmotic up-shock, and consis ts of very thin, straight, uniform and long plasmolysis spaces which w ere called 'lamellar spaces'; these commonly formed inside the sidewal ls and were usually associated with the formation of endocytotic vesic les. Since lipoprotein links the M to the OM layers and thus could aff ect plasmolysis, we examined both wild-type and deficient strains. Som e effects were observed, but they were minimal. The volume of the peri plasmic space of growing unshocked cells was determined to be about 7% .