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When cells of a marine pseudomonad were washed and suspended in 0.5m sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5m NaCl and then suspended in 0.5m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5m NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up α-aminoisobutyric acid by a Na+ -dependent process.

Nutrition and Metabolism of Marine Bacteria XVI. Formation of Protoplasts, Spheroplasts, and Related Forms from a Gram-negative Marine Bacterium