The Formation of Protoplasts and Quasi-spheroplasts in Normal and Chloramphenicol-pretreated Bacillus subtilis

SUMMARY The lytic action of lysozyme upon Bacillus subtilis walls was studied by following the disappearance of bacillary-colony-forming units and the appearance of L-colony-forming-units. The rapidity of cell wall removal by lysozyme fluctuated markedly during growth in a chemically defined medium, presumably because subtle changes in the cell wall were constantly occurring. When lysozyme-sensitive bacilli were grown with chloram-phenicol 10 μg./ml. for 3 hr they showed a notable increase in lysozyme resistance; at the same time, their walls almost doubled in thickness. As lysozyme attack proceeded in a given culture, the bacilli passed first through a rod-shaped osmotically sensitive stage, and then a spherical stage characterized by incomplete removal of cell wall before finally reaching the naked protoplast stage. The spherical forms with adherent wall residues formed L colonies on a medium containing the reversion inhibitor D-methionine and bacillary colonies on the same medium without D-methionine. Under the latter conditions, the cell wall residue served as a starting point for rebuilding of complete wall, much as residual wall permits reversion of Gram-negative spheroplasts to the bacillary state. In the presence of d-methionine, the feedback sequence required for wall formation was severed, resulting in heritable propagation of the protoplast state.