Formation of inside-out vesicles of Bacillus licheniformis. Dependence on buffer composition and lysis procedure

1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.