Studies on Autolysis of Clostridium botulinum Type A

II. Autolytic Formation of Spheroplasts and Autolysis of Cell Walls

Abstract

Most of the cells of Clostridium botulinum type A strain 190 harvested at logarithmic growth phase in GYPT medium were converted autolytically to spheroplasts in 0.5M sucrose-phosphate buffer within 2-3 hours at 37°C. Electron microscopic observations on the formation of spheroplasts demonstrated that the cytoplasmic contents were extruded through partly dissolved walls at the end of the organism and formed spherical bodies.
Crude wall fraction, isolated from logarithmic phase cultures by sonication and fraction-ation, rapidly autolysed in phosphate buffer. The wall fraction isolated from the large colony type of the strain lysed more rapidly than those from the small one as reported on the whole cells. Reducing sugars and amino sugars being main constituents of the wall were released from the wall fraction as wall-autolysis occurred. Electron micorscopic study showed that the rigid structure of the wall was completely lost and only fragile membranous or amorphous components remained as residues. Heated wall preparations digested with trypsin and nagarse were dissolved by a soluble wall-autolysate, but not by a soluble cytoplasmic fraction. It seems likely that autolytic enzyme system may exist at or near the cell wall.