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

The metabolism of D-alanyl substituents of lipoteichoic acid (LTA) and teichoic acid was studied in Staphylococcus aureus. Double labelling with [3H]glycerol and o-[14C]alanine revealed that during the chase LTA was stable whereas its 14C label rapidly decreased. Half-time comparison indicated an enzyme-rather than a base-catalyzed process. Correlated with the loss of [14C]alanine from LTA was an increase of the radioactivity in wall-linked alanine ester which, after hydrolysis with HF, proved to be linked to teichoic acid. These results suggest that LTA-alanine is the donor for alanine esterification of teichoic acid. In connection with previous data we hypothesize that the loss of alanine from LTA is compensated by de novo incorporation. o-alanyl ester [1,3] which greatly modifies the biological activities in particular of lipoteichoic acid [4][5][6]. In Staphylococcus aureus, the alanine ester content of teichoic acid [7] and LTA [8] varies with [NaC1] of the growth medium, suggesting that alanine incorporation into both polymers is under the same control [8]. As shown with Lactobacillus casei, the incorporation of D-alanine into LTA is accomplished by activation to D-alanyl AMP and subsequent transfer to the polymer chain [9]. The incorporation of D-alanine into teichoic acid has not yet been elucidated. So far no attention has been paid to the possibility of a turnover of alanyl substituents although it might supply the cell with LTA species of different biological activities.In this communication we report turnover of alanyl substituents of LTA and provide evidence that they are the precursor of the alanyl ester of teichoic acid.

Spinach chloroplasts were purified on gradients of Percoll which preserved envelope impermeability and CO2-dependent oxygen evolution in the light. Application of (35)SO4″ to purified chloroplasts resulted in a light-dependent labeling of a lipid component which was indentified as sulfoquinovosyl diacylglycerol. Fractionation of chloroplasts showed that after 5 min of labeling most of the newly synthesized sulfolipid was present in thylakoids. Only a small percentage was recovered from the envelopes. Molecular species from envelopes and thylakoids were identical. The molecular species did not change during incubation times ranging from 5 min up to 4.5 h. Mesophyll protoplasts from (35)SO4″-labeled oat primary leaves were gently disrupted and separated into organelles by sucrose gradient centrifugation. Labeled sulfolipid was located almost exclusively in the chloroplasts. This, in combination with the experiments carried out with isolated chloroplasts, indicates that the final assembly steps in the biosynthesis of sulfolipid are confined to the chloroplasts.

Vacuoles were released from oat (Avena sativa) mesophyll protoplasts and purified by sedi­mentation and flotation. Disruption of isolated vacuoles followed by density gradient centri­fugation gave two membrane bands which after combination were further purified on sucrose gradients. A significant contamination by microbodies, thylakoids, mitochondria, endoplasmic reticulum and Golgi membranes can be excluded, whereas markers for plasma membrane and chloroplast envelope were present in the final membrane preparation.In the purified membrane fraction the following enzymatic activities were detected: NADH- cytochrome C reductase E.C. 1.6.2.2, ATPase E.C. 3.6.1.3. UDPG: sterol glucosyltransferase, UDP-Gal: diacylglycerol galactosyltransferase E.C. 2.4.1.46, glucan synthetase II, CDP-choline: diacylglycerol phosphocholinetransferase E.C. 2.7.8.2, formation of acylgalactosyl diacylglycerol and acyl-CoA thioesterase E.C. 3.1.2.2. None of these can be considered to be specific for the tonoplast. Acid phosphatase E.C. 3.1.3.2 was present in the cell sap.The vacuolar membranes contain phospholipids and glycolipids of the most complex composi­tion found so far in a membrane system isolated from mesophyll protoplasts. About half of the glycolipids were accounted for by glycosyl diacylglycerols usually considered to be confined to plastids. Steryl glycosides and acyl steryl glycosides were other prominent glycolipids. A cerebroside was the predominating lipid component of this membrane preparation.