Ultrastructural and Chemical Studies on Wall-deficient Forms, Spheroplasts and Membrane Vesicles from Mycobacterium aurum

Abstract

Wall-deficient forms of M. aurum were prepared by agitating the cells during exponential growth with D-cycloserine, glycine, lysozyme, EDTA and LiCl for approximately the time of 3 cell divisions (18 h). Wall-deficient forms were then converted to spheroplasts by gentle stirring with lysozyme and EDTA in a Tris/HCl buffer containing sucrose until all the cells appeared spherical by phase contrast microscopy. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles. Ultrastructural and chemical properties of the spheroplasts and membrane vesicles are described. The spheroplasts were susceptible to lysis by 0.25% (wt/vol) sodium dodecyl sulfate and were permeable to certain enzyme substrates.