Formation and Regeneration of Mycobacterial Protoplasts

A method has been developed for the large scale preparation of cell wall-deficient forms, mainly as protoplasts, from Mycobacterium smegmatis employing L-glycine, lysozyme and lytic enzyme no. 2, as the protoplasting agents. Regenerating processes of the protoplasts to the classical bacillary form were examined morphologically using both phasecontrast and electron microscopy. Most strains of mycobacteria, including tubercle bacilli, show resistance to cell-wall destroying enzymes. The cells were therefore previously treated with glycine, which has been known to inhibit the cell wall synthesis of Grampositive bacteria, making them sensitive to the enzymes. Optimum concentration of glycine varied according to the individual strains and their growth phase, but in the strain, M. smegmatis P53, used in the present experiments, it was 0.75 to 1.2%. Electron microscopy showed that, after the pretreatment of the cultures with glycine, the cell wall had effectively exfoliated from the cytoplasmic membrane. After the subsequent incubation with low concentrations of lysozyme (50 μg/ml) and lytic enzyme no.2 (30 μg/ml) in the presence of glycine, more than 90% of the cells had been converted to spherical forms. As for the morphological events, two apparent modes of regeneration to the classical bacillary forms of the protoplasts were observed. The first one was initiated by budding from the protoplasts. The buds gradually became mycerial forms accompanied by successive elongation, branching, and occasional septation. The second resulted from the intracellular formation of elementary bodies and their release from the protoplasts. It seemed likely that the elementary bodies appeared as the result of fragmentation and condensation of the cytoplasm after its irregular septations by the membrane system.