OBSTACLES TO TRANSFORMATION PRESENTED BY BACTERIAL ANATOMY
ARMY BIOLOGICAL LABS FREDERICK MD
Pagination or Media Count:
Transformation is generally thought to be facilitated by removal or weakening of the cell wall. Newly developed techniques for rescuing and growing presumptive protoplasts were used to test this assumption with the Bacillus subtilis transforming system. DNA from an indole plus strain was mixed with competent cells of B. subtilis strain 168 indole minus in transforming medium containing 0.5 M sucrose and ten to the minus 4th power M EDTA. Lysozyme was added to samples from this transforming cell suspension at successive time intervals. Twenty to 40 minutes after lysozyme addition, when protoplasting in the samples was complete, the protoplasts and bacillary control suspensions were plated. In samples protoplasted soon after DNA addition, a sharp loss of transformants occurred 10- to 20-fold loss in the course of protoplast formation. Protoplasts formed prior to DNA addition were completely refractory to transformation. By contrast, when lysozyme was added 120 minutes after the DNA, protoplasting no longer caused a marked loss of transformants. Throughout these experiments, survival of protoplasts which emerge as L-colonies ranged from 60 to 100 per cent.