Spheroplasts of Lactobacillus casei and the cellular distribution of bactoprenol

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Abstract
Spheroplasts of L. casei were prepared by the simultaneous action of trypsin and lysozyme in the presence of EDTA and 20% sucrose. Electron microscopy of thin sections of cells during the formation of spheroplasts showed that the spheroplasts emerged from the cell wall through a weak point at one end of the cell. Electron microscopy of negatively stained preparations showed that the structure of spheroplast membranes obtained by disruption in hypotonic medium varied according to the nature of the hypotonic medium used. Disruption in water gave small, delicate membrane fragments; disruption in 0.1 M tris-Cl gave large pieces with tubulo-vesicular attachments. The effects of Mg2+ and EDTA were also investigated. Bactoprenol, the C55 isoprenoid alcohol concerned in cell wall biosynthesis, was located by growing L. casei in [14C]mevalonic acid. Over 90% of the cellular bactoprenol was found in the spheroplast membrane when this was obtained with 0.1 M tris-Cl, but only 42% when the spheroplasts were burst in water. It is suggested that the missing bactoprenol may be solubilized with the membranes of mesosomal orgin, and that bactoprenol is therefore a component of both plasma and mesosomal membranes.