Autolytic Formation of Spheroplasts and Autolysis of Cell Walls in Clostridium botulinum Type A1

Abstract

Cells of Clostridium botulinum type A strain 190 harvested at logarithmic growth phase rapidly autolysed in phosphate buffer and most of the cells were converted auto-lytically into spheroplasts in 0.5 M sucrose-phosphate buffer within 2-3 hr at 37 C. Electron microscope observations on the process of autolysis and spheroplast formation revealed that lysis of the cell wall commenced at one end of the cell and the cytoplasmic contents were released through such lesion. The rod cell was thusly transformed into a fragile spherical form in the hypertonic sucrose-buffer. The lysis of the cell wall pro-ceeded centripetally and finally morphological integrity of the cell wall was completely lost. From these findings it is suggested that the autolysis of the organism is preceded by autodigestion of the cell wall at one end of the cell. A crude cell wall fraction isolated from log-phase cultures by sonication and fractionation rapidly autolysed in phosphate buffer. Reducing sugars and amino sugars of the wall were released from the autolysing wall fraction. Electron microscopy of the residues obtained from wall-autolysates demonstrated that the rigid structure of the wall completely disappeared and only fragile membranous or amorphous fragments remained after autolysis of the crude wall fraction. 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(s) may exist at or near the cell wall.