The isolation of protoplasts from Bacillus megaterium by controlled treatment with lysozyme.

Certain bacteria are killed and rapidly lysed following exposure to the enzyme lysozyme. Lysozyme sensitivity resulting in extensive lysis occurs in a relatively restricted number of species: notably Micrococcus lysodeikticus, Sarcina lutea, and certain Bacillus spp. It was shown some years ago (Epstein and Chain, 1940; Meyer and Hahnel, 1946) that a mucopolysaccharide extractable from the cells of M. lysodeikticus could be depolymerized by the enzyme with liberation of reducing sugars. More recently, Salton (1952) has found that the isolated cell walls of M. lysodeikticus can be rapidly and completely dissolved by treatment with lysozyme. Tomesik and Guex-Holzer (1952), working with a Bacillus sp, have given cytological evidence for the specific action of lysozyme on the bacterial cell wall. It appears, therefore, that lysozyme is a polysaccharase and that its action on susceptible bacteria consists of a specific destruction of the fabric of the cell wall. This interpretation is supported by recent chemical analyses (Salton, 1953) on the isolated cell walls of a number of bacteria; the walls of lysozyme sensitive organisms are closely similar in composition and markedly different from the walls of lysozyme resistant species. Hence the treatment of susceptible bacteria with lysozyme should constitute a method of unrivalled specificity and gentleness for the isolation of the structural elements within the cells-a method which avoids the wholesale disorganization of cellular components which results from cell breakage by mechanical abrasion or sonic treatment. It will be shown in this paper that it is possible to isolate the bacterial protoplast as an entity