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

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.