D-Alanyl-lipoteichoic acid (D-alanyl-LTA) from Lactobacillus casei ATCC 7469 contains a poly(glycerophosphate) moiety that is acylated with D-alanyl ester residues. The physiological function of these residues is not well understood. Five mutant strains of this organism that are deficient in the esters of this amphiphile were isolated and characterized. When (15,20). One of the components of LTA which has been proposed to modulate these functions is the substituent D-alanine. Evidence from in vitro experiments indicates that these D-alanyl esters alter the abilities of LTA to inhibit autolysins (19), chelate Mg2+ ions (a function shared with wall teichoic acid) (2,21,24,28), and function as a carrier for wall teichoic acid synthesis (17, 27). Fischer and co-workers (19) proposed that fluctuations of the D-alanyl ester content of LTA must be considered in its potential regulation of autolysins.Substitution of LTA by D-alanine is not a universal feature of all LTAs. For example, LTAs from Micrococcus varians, Streptococcus faecalis 9790, and S. lactis Kiel 42172 are devoid of D-alanine (16). However, 30 to 93% of the glycerol phosphate units of LTAs from most organisms are substituted with D-alanyl ester residues (16). Growth of Staphylococcus aureus in the presence of increasing concentrations of NaCl, 0.2% to 7.5%, decreases the degree of esterification by D-alanine from 78 to 36% (18). In addition, growth of this organism at pH 8.1 instead of pH 7.1 results in a decrease of D-alanine ester content from 45 to 5% (32). Thus, not only growth in NaCl but growth at higher pHs induces wide variations in the esterification of LTA by D-alanine.In Lactobacillus casei, incorporation of D-alanine into LTA is accomplished in the following two-step reaction sequence (4,7,10,30,36,39 It is our ultimate goal to determine the physiological function of the D-alanyl ester residues in the LTA of L. casei 7469. In this paper, we report the isolation and characterization of five mutant strains of this organism that are deficient in the ester content of this amphiphile. A study of these strains revealed defects in the D-alanine incorporation system. Aberrant morphology and defective cell separation appear to result from this deficiency in D-alanyl ester content.(A portion of this study was presented at the 85th Annual Meeting of the American Society for Microbiology, Las Vegas, Nev., 3-7 March, 1985.) MATERIALS AND METHODS Materials. We thank Werner Fischer for glycerophospho (Gro-P)-6-Glc(pl-6)Gal(al-2)Glc(al-3)diacylglycerol and lipid extract isolated from L. casei DSM 20021 (34, 35).

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...

Lipoteichoic acid (LTA) from Lactobacilus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D_[14C]alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. We propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.on July 16, 2020 by guest