SUMMARY:Protoplasts were prepared from cells of Bacillus megaterium, strain M , by lysozyme treatment in the presence of sucrose. The protoplasts were shocked osmotically and the lysate thus obtained was fractionated into membranous 'ghosts ' and soluble protoplasm by means of differential centrifugations. The distribution of various enzymes between these two fractions was studied. The bulk of the cytochromes, the succinic dehydrogenase and the diphosphopyridine nucleotide oxidase of the lysate was recovered in the 'ghost' fraction. On the other hand, the soluble protoplasm contained most of the isocitric dehydrogenase, the catalase, the hexokinase and the acid phophatase of the lysed bacteria. Considerable amounts of malic dehydrogenase, lactic dehydrogenase and fumarase were found in both the 'ghosts ' and the soluble protoplasm. None of the enzymes studied was localized in the ribonucleic acid-containing particles of the bacterial protoplasm.Many of the enzymes of animal cells are localized in subcellular structures such as mitochondria and microsomes. It is doubtful whether a similar state of affairs prevails in bacterial cells. Numerous investigations (reviewed by Alexander, 1956) have made it clear that, in extracts obtained by a mechanical or sonic disintegration of bacterial cells, several enzymes are associated with the sedimentable particles. Without additional evidence it cannot be settled whether these particles exist as such in the intact cells or whether they represent the remnants of larger, subcellular structures. I n some cases at least, however, the 'differential release technique' devised by Marr & CotaRobles (1957) may be useful for discriminating between these two possibilities.Some of the hazards of the mechanical and sonic disintegration procedures are avoided when the bacterial cells are degraded enzymically or autolytic- Weibull & Bergstrom, 1958), it has been possible to bring into relation the enzymic and chemical properties of one and the same subcellular structure. METHODSOrganism, growth conditions and harvesting. Bacillus megaterium, strain M (Baumann-Grace & Tomcsik, 1957) was grown in the medium described by Gladstone & Fildes (1940). Fernbach flasks having a volume of 2-8 1. and containing 250-1000 ml. medium were inoculated with the organism and incubated for 16 hr. at 30" on a rotary shaker (speed of shaker 100 rev./min.). The bacteria, then being in the stationary growth phase, were harvested by centrifugation and suspended in 0.02 M-phosphate buffer (pH 7). The concentration of the bacteria in the suspension was made about 4 times that in the growth medium at harvesting.Preparation of bacterial fractions. To each 225 ml. of bacterial suspension, prepared as described above and brought to room temperature, 75 ml. 2 Msucrose and 6 ml. 1 yo (w/v) lysozyme were added. Protoplast formation was completed within about 30 min. The protoplasts were centrifuged for 20 min.at 20,000 g (Spinco model L preparative ultracentrifuge with the no. 30 rotor) and were then resuspended in 100 ml...

Numerous morphological, physiological, and biochemical studies have been carried out on bacterial forms exhibiting a pronounced osmotic fragility (for review see Weibull, 1958b). Naked bacterial protoplasts, obtained by means of a complete removal of the cell wall by lysozyme in a sucrose medium (Weibull, 1953) offer an example of such an osmotically fragile structure. Osmotically sensitive forms may also result when the cell wall is only partly degraded. Such structures have been named spheroplasts (Hurwitz et al., 1958; Michael and Braun, 1959). The so-called bacterial L forms, which are highly pleomorphic and which may arise when normal bacteria are subjected to an unfavorable environment, are also markedly sensitive to changes in the osmotic pressure of the surrounding medium (for reviews see Dienes and

SUMMARYThe microscopic elements which constituted cultures of a stable L form (Proteus L9) were found to be highly heterogeneous with respect to morphological, chemical and metabolic properties. The smallest elements found in the cultures studied (diameter < 0.3,~) contained more lipid-phosphorus (lipid-P), but less ribonucleic acid-phosphorus, deoxyribonucleic acid-phosphorus and protein-nitrogen, than whole cultures consisting predominantly of bodies of diameter > 1,u. The small elements respired a t about the same rate as whole cultures, but they showed low, if any, biosynthetic activity. The small elements probably possess a structural organization similar to that of L bodies of larger sizes, but most of them contained little, if any DNA.

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