Several physicochemical properties of the teichoic acid of Bacillus subtilis 168 have been determined. The teichoic acid partial specific volume was found to be 0.57 ml/g. The apparent weight-average molecular weight of the polymer was 24,800. Sedimentation was strongly dependent on solvent. The sedimentation coefficient of the teichoic acid was found to have a value of s25w' = 1.90S. In

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of ["4C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of ["4C]concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage 025. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.The exact location and configuration of teichoic acids in the cell walls of gram-positive bacteria is still unknown. One important question that has remained unanswered is whether all of the cell wall teichoic acid molecules are surface exposed or whether some of the molecules are intercalated within the insoluble peptidoglycan network. Furthermore, with respect to bacilli, it is not known with certainty whether the teichoic acids are found in both hemispherical caps and rods.Several papers have appeared which deal peripherally with these problems. Burger (7) showed that by subjecting the cell walls of Bacillus subtilis 3610 and B. subtilis W-23 to lysozyme greater quantities of anti-teichoic acid antibody could be precipitated, suggesting that teichoic acids were arranged on both the surface and within the peptidoglycan matrix. Nermut (22) extracted the walls of B. megaterium M with hot formamide to remove the teichoic acid and found that the wall thickness decreased by approximately one-half. Nermut interpreted these results in terms of a two-layered cell wall structure, with the outer layer consisting of extractable teichoic acid and the inner layer consisting of nonextractable peptidoglycan. Birdsell et al. (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G212, p. 65) found that thin sections of concanavalin A-treated B. subtilis 168 cell walls contained an outer fluffy layer and a smooth inner wall face. The fluffy, discontinu-ous outer layer was absent when the walls were treated with the concanavalin A (Con A) inhibitor methyl-alpha-D-glucopyranoside or when walls containing non-glucosylated teichoic acid were employed. These findings suggested that in B. subtilis 168 at least a portion of the teichoic acid was located on the outer, but not the inner, surface of the cell wall. Recently, Weibull (26) showed that the cell wall of aldehyde-fixed, unstained B. subtilis ATCC 6051 possessed two electron-dense layers separated by an electron-transparent layer. One interpretation of these results is that the teichoic a...