An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.

The location of N-acetylgalactosamine in the walls of Bacillus subtilis 168.

The N-acetylgalactosamine in the walls of Bacillus subtilis 168 occurs in two polymers. One of these contains N-acetylgalactosamine, glucose and phosphorus and is attached to the peptidoglycan through an alkali-labile bond; preliminary studies indicate that a repeating unit of this polymer is glucosyl-N-acetylgalactosamine 1-phosphate. N-Acetylgalactosamine is also associated with the peptidoglycan in a component that is not converted into the free sugar or other soluble compounds on treatment of the walls with alkali. The two polymers containing N-acetylgalactosamine are released on autolysis of the walls and can be separated by ion-exchange chromatography. As glucose 6-phosphate is produced by gentle hydrolysis of the wall with acid a third phosphate polymer, poly(glucose 1-phosphate), may occur in this wall. However, as no polymer with this structure could be separated from that containing galactosamine, its existence has not been established unequivocally. The methods described permit the study of variations in N-acetylgalactosamine content with respect to growth conditions.