Chemical, biological, and structural properties of stable Proteus L forms and their parent bacteria.

Proteus L forms were disrupted by osmotic shock, and the sedimentable material present in the homogenate was further fragmented in a Sorvall pressure cell. The pressure cell was also used for disrupting normal Proteus cells. The homogenates obtained were fractionated by differential centrifugation. Purified endotoxins were isolated from the major fractions by phenol extraction. Material extracted with phenol from the membrane fraction of the L forms was about as toxic and pyrogenic on a weight basis as the typical enterobacterial endotoxins isolated from cell walls of normal bacteria. The yield of extract from L forms was about one-third of that from an equal weight of normal bacteria. No differences in the gross chemical composition of the phenol extracts from the L forms and the normal cells could be ascertained. A close serological relationship existed between extracts obtained from two L forms and their respective parent bacteria, but no such relationship was found in the case of the third L form studied and its parent bacterium. Diaminopimelic acid was not detected in the membranes of the L forms, but these membranes contained most of the succinic dehydrogenase of the organisms. Only small amounts of this enzyme were present in the wall fraction of normal bacteria. The data obtained suggest that precursors of the Proteus endotoxins are formed either in the soluble protoplasm of normal cells and L forms or at sites on the membrane from which they are readily liberated into the protoplasm, whereas the final steps of the synthesis of these toxins take place at the cytoplasmic membrane. In normal cells, much endotoxin is transported to and concentrated in the walls.