Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA. Phosphatidylglycerol (PG) has been proposed to be the donor of sn-glycerol 1-phosphate (GroP) units of the poly(glycerophosphate) moiety of lipoteichoic acid (LTA) (8,9,13,14) according to the following reaction: tion would result in a cycling of the diglyceride moiety of this phospholipid.The goals of these experiments with Lactobacillus casei were to characterize further the effect of phosphate on the assembly of the LTA, PG + LTA-poly(glycerophosphate)" -* LTA-poly(glycerophosphate),+1 + 1,2-diglyceride Inorganic phosphate stimulates both the synthesis of PG and the elongation of D-alanyl-lipophilic LTA in vitro (4). These observations supported the proposed role for PG in the elongation of LTA.During the elongation of LTA, one of the reaction products is 1,2-diglyceride. Significant amounts of this diglyceride might be expected to accumulate during chain elongation. Since large amounts of diglyceride are not commonly found in bacteria (25), it is proposed that the diglyceride is either degraded or reutilized for phospholipid synthesis. A diglyceride kinase similar to that found in Escherichia coli (24) could phosphorylate the diglyceride to phosphatidic acid, a known precursor of PG (25). This phosphorylat Present address: College of Dentistry, University of Illinois at the Medical Center, Chicago, IL 60612. to determine the site of the addition of GroP units to the growing polymer, and to suggest a fate for the 1,2-diglyceride. Toluene-treated cells were used to demonstrate the synthesis and elongation of D-alanyl-LTA as well as the synthesis of various phospholipids. These cells, which are permeable to GroP, ATP, and Dalanine, synthesized LTA and phospholipids in significant amounts. For detecting diglyceride kinase, membranes were u...