Biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei: D-alanyl-lipophilic compounds as intermediates

Abstract

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) from Lactobacillus casei contains a poly(glycerol phosphate) moiety that is selectively acylated with D-alanine ester residues. To characterize further the mechanism of D-alanine substitution, intermediates were sought that participate in the assembly of this LTA. From the incorporation system utilizing either toluene-treated cells or a combination of membrane fragments and supernatant fraction, a series of membrane-associated D-[14C]alanyl-lipophilic compounds was found. The assay of these compounds depended on their extractability into monophasic chloroform-methanol-water (0.8:3.2:1.0, vol/vol/vol) and subsequent partitioning into chloroform. Four lines of evidence suggested that the D-alanyl-lipophilic compounds are intermediates in the synthesis of D-alanyl-LTA. First, partial degradation of the poly(glycerol phosphate) moiety of D-alanyl-LTA by phosphodiesterase II/phosphatase from Aspergillus niger generated a series of D-alanyl-lipophilic compounds similar to those extracted from the toluene-treated cells during the incorporation of D-alanine. Second, enzymatic degradation of the D-alanyl-lipophilic compounds by the above procedure gave D-alanyl-glycerol, the same degradation product obtained from D-alanyl-LTA. Third, the incorporation of D-alanine into these compounds required the same components as the incorporation of D-alanine into membrane-associated D-alanyl-LTA. Fourth, the phosphate-induced loss of D-[14C]alanine-labeled lipophilic compounds could be correlated with the stimulation of phosphatidylglycerol synthesis in the presence of excess phosphate. We interpreted these experiments to indicate that the D-alanyl-lipophilic compounds are D-alanyl-LTA with short polymer chains and are most likely intermediates in the assembly of the completed polymer, D-alanyl-LTA.

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation

“…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.…”

“…In this study, two classes of LTA were defined on the basis of their solubility. Lipophilic LTA is short-chained lipoteichoic acid which partitions into the chloroform phase of the biphasic extraction system (3,4). Hydrophilic LTA is longer chained and partitions into the aqueous phase of this system.…”

“…1). It was previously suggested that the D-alanyl-lipophilic compounds are D-alanyl-LTA with short chains of poly(glycerophosphate) (4). Thus, the [14C]GroP-labeled compounds may represent lipophilic LTA of various chain lengths and degrees of D-alanine substitution.…”

“…This mixture was incubated for 50 min at 37°C. Isolation and purification of ['4C]glycerol [3H]glycerol-D-alanyl-LTA free of D-alanyl-lipophilic LTA, degradation with phosphodiesterase IIphosphatase, and assay for the degradation products were done as previously described (4,6).…”

“…Labeled chloroform-soluble compounds were isolated by a modification of the monophasic extraction procedure of Bligh and Dyer (3) described by Brautigan et al (4). The final chloroform-soluble fraction was transferred to a scintillation vial and assayed for radioactivity.…”