Abstract.-Six major oligosaccharides were released from the peptidoglycan of spores of Bacillus subtilis by lysozyme treatment. They were isolated and characterized as a disaccharide, tetrasaccharide, and hexasaccharide composed of equal amounts of muramic acid and glucosamine and containing two, three, and four acetyl groups, respectively. Three of the compounds were substituted by a single L-alanine residue, and the other three by a single tetrapeptide substituent on the acetylmuramic acid residue at the reducing end of each compound. The other muramic acid residue in the tetrasaccharides (and two of the three in the hexasaccharides) were shown to be present as muramic lactams, a sugar not previously found in nature and, hence, a unique spore constituent. Other features of the structure of spore peptidoglycan are discussed.The integument of bacterial spores contains peptidoglycan, in both a middle layer (the cortex) and in the innermost layer (the germ cell wall). The purpose of the present investigation was to elucidate the structure of the peptidoglycan in bacterial spore walls and to compare it to peptidoglycan in the walls of vegetative cells as a contribution to the problem of differentiation in the conversion of bacterial cells to spores. A number of important differences between the two structures has been found. The most interesting of these, the occurrence of the lactam of muramic acid (Fig. 1) in the peptidoglycan of bacterial spore walls, is described in the present paper. The occurrence of this sugar in natural materials has not previously been described, although N-acetylmuramic acid (Fig. 1) is an invariable constituent of the peptidoglycan of bacterial cell walls.Materials and Methods.-B. subtilis (Porton strain) was grown for 24 hr at 37°C on antibiotic medium no. 3 (Difco) supplemented with a mixture of salts. Spores were freed from sporangia and cells by autolysis at 4°C, washed with 0.2 M sodium phosphate, pH 6.5, and purified by two-phase centrifugation.I Autolytic enzymes in the spores were inactivated by autoclaving (120°C, 30 min). After disruption of the spores with glass beads in the Nossal disintegrator, spore integuments were sedimented at 20,000 X g for 20 min, washed with 0.2 M sodium phosphate buffer and water, and then digested with trypsin (0.5 mg/ml).Preparation of spore peptidoglycan: Integuments (from 50 to 100 mg spores/ml) were resuspended in 0.02 M Tris, pH 7.0, by brief sonication and then digested with lysozyme (0.3 mg/ml) for 16 hr at 37°C. Spore coats were sedimented at 20,000 X g and the soluble peptidoglycan was deproteinized with chloroform and amyl alcohol.Isolation of oligosaccharides from the lysozyme digest: The digest was fractionated on a small scale by gel filtration on a column (100 X 0.8 cm) of Sephadex 0-25. Individual fractions were subjected to preparative paper electrophoresis at pH 3.9. Oligosaccharides were detected on guide strips using the fluorescence procedure of Sharon and Seifter2 and eluted. The larger amounts required for nuclear magnetic res...

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Saccharomyces bailii grows in the presence of high concentrations of sorbic, benzoic and other short‐chain monocarboxylic acids commonly used as preservatives. Starved cells concentrate these acids intracellularly, approximately as expected from the pH of the ceil and the pKa of the acid. On addition of glucose, the intracellular content of preservative is considerably reduced. The glucose effect is sensitive to metabolic inhibitors, and anaerobic respiration is stimulated by the preservatives. The ability to maintain a low intracellular concentration of any of the preservatives tested is induced by growth in the presence of sorbic or benzoic acid and less effectively by butyric or acetic acid. Both induced and uninduced cells are permeable to benzoic and butyric acids. Benzoate and sorbate are not metabolized at a rate significant with respect to the permeation rate. Resistance to these preservatives apparently results primarily from an inducible, energy requiring system which transports preservative from the cell.

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