A procedure is described for measuring the extraction of lipoteichoic acids from gram-positive bacteria in absolute terms. Virtually complete extraction was achieved from various bacteria by hot phenol/water if the cells were disrupted. Extraction of whole and delipidated cells and of the membrane fraction gave considerably lower yields.Most of the nucleic acids co-extracted from disrupted cells was removed by treatment with nucleases. Nucleaseresistant nucleic acid, protein, polysaccharide, and teichoic acid were separated from lipoteichoic acid by anionexchange chromatography on DEAE-Sephacel or hydrophobic interaction chromatography on octyl-Sepharose. Purified preparations were essentially free of polymeric contaminants, retained their alanine ester substitution, and were in the sodium salt form.Hydrophobic interaction chromatography also made it possible to recognize contamination of lipoteichoic acid with its deacylated and lyso-form, and to discriminate molecular species containing two and three, or two and four acyl groups.

Pulse-chase experiments with [2-3H]glycerol and [ 14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90 % of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer :Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor.Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10 %was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative.In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.Characteristic lipid amphiphiles of the cytoplasmic membrane of gram-positive bacteria are glycolipids, glycerophosphoglycolipids and lipoteichoic acids which are related to each other (for review see [l]). Most lipoteichoic acids consist of a 1,3-linked poly(g1ycerophosphate) chain which is covalently linked to a particular membrane glycolipid, Glycerophosphoglycolipids are short-chain homologues of lipoteichoic acids as they carry a monoglycerophosphate or di(g1ycerophosphate) residue in place of the poly(g1ycerophosphate) chain on the same glycolipid species. This structural relationship, which also includes the stereochemical configuration of the glycerophosphate residues and the point of attachment at the glycolipid moiety, suggested glycerophosphoglycolipids to be either biosynthetic intermediates or enzymic breakdown products of lipoteichoic acids [l].Labelling experiments in vivo [2, 31, with membrane preparations [4-61 and toluenized cells [7] revealed that lipoteichoic acid biosynthesis occurs through transfer of glycerophosphate from phosphatidylglycerol with the concomitant formation of diacylglycerol. This together with the recent observation that the amount of glycerol in lipoteichoic acid approximately equals that in membrane lipids [l] suggested a substantial effect of lipoteichoic acid synthesis on membrane lipid metabolism. It was the aim of the present study to elucidate this interrelationship. Of particular interest in this context was: (a) the fraction and turnover rate of the phosphatidylglycerol involved, (b) the metabolic fate of the resulting

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.

The lipoteichoic acid of Streptococcus lactis Kiel 42172 was isolated. The lipid portions were released by HF and were established to be 3-O-[O-alpha-D-galactopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl]-2-O-alpha-D-galactopyranosyl-sn-glycero-1-phosphate, they are joined by phosphodiester bonds nosyl)]glycerol. The repeating units of the hydrophilic chain were established to be 3-O-[O-alpha-D-galactopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl]-2-O-alpha-D-galactopyranosyl-sn-glycero-1-phosphate; they are joined by phosphodiester bonds at carbon atom 6 of the galabiosyl residues. The innermost unit is linked to the glycolipid by a phosphodiester presumably at C-6 of the outer glucosyl moiety. The hydrophilic chain is 7.4--11.8 units in length, measuring 12--19 nm is extended conformation. The content of 2.7--2.96 acyl groups per 2 glucosyl residues indicates that 70--96% of the glycolipid consists of acyldiglucosyldiacylglycerol. The novel poly(glycosylgly-cerophosphate) structure provided for the first time the oplipoteichoic acids are the sn-1 isomer which has previously been suggested from biosynthetic studies (Glaser, L., & Lindsay, B. (1974) Biochem. Biophys. Res. Commun. 59, 1131--1136).

Native substitution with the D-alanine ester of lipoteichoic acids (LTAs) affects their immunological properties, the capacity to bind divalent cations, and LTA carrier activity. In this study we tested the influence of the D-alanine ester on anti-autolytic activity, using extracellular autolysin from Staphylococcus aureus and nine LTAs with alanine/phosphorus molar ratios of between 0.23 and 0.71. The inhibitory activity, highest with alanine-free LTA, exponentially decreased with increasing alanine content, approaching zero at substitutions of >0.6. Correspondingly, dipolar ionic phospholipids were not inhibitory, in contrast to negatively charged ones. Glycosylation of LTA up to an extent of 0.5 did not depress inhibitory activity, and even at a degree of0.8 the effect was comparatively small. On comparison of LTAs from various sources, differences in lipid structures and chain lengths were without effect. The inhibitory activity drastically decreased when the glycolipid carried a single glycerophosphate residue or the hydrophilic chain had the unusual structure [6-*Gal(a1-6)Gal(a1-3)Gro-(2*-1aGal)-P]., in which digalactosyl moieties connect the a-galactosylated glycerophosphate units. Principally, the same results were obtained with the more complex system of autolysis of S. aureus cells. We hypothesize that the antiautolytic activity of LTA resides in a sequence of glycerophosphate units and that the negative charges of appropriately spaced phosphodiester groups play a crucial role. The alanine ester effect is discussed with respect to the putative in vivo regulation of autolysins by LTA.