A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from...

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca2+ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (greater than 98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cells, and in investigating envelope-related synthesis and assembly processes.