The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacius casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound Upoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the doe gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the doe gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% ofthe total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.The D-alanyl-ester content of lipoteichoic acid (LTA) appears to play a role in determining cell shape. Mutants that are deficient in these esters are characterized by aberrant morphology and defective cell separation (52). To establish further the role of these esters, genetic methods were sought for modulating their content in LTA.In Lactobacillus casei, the incorporation of D-alanine into ester-linked D-alanyl-LTA is accomplished in the following two-step reaction sequence (6,9,11,42,51,54): enzyme + D-alanine + ATP enzyme * AMP-D-alanine + PPi(1) enzyme * AMP-D-alanine + mLTA D-alanyl-mLTA + enzyme + AMP (2) In reaction 1, enzyme-bound aminoacyl adenylate is synthesized by the D-alanine-activating enzyme (Dae; EC 6.3.2.4) in the presence of ATP. In reaction 2, the activated D-alanine is covalently linked to membrane-associated LTA (mLTA) in the presence of D-alanine:membrane acceptor ligase (EC 6.3.2.16). The mechanism of LTA acylation catalyzed by the ligase has not been established.The D-alanyl esters of LTA have been implicated in at least three functions. These include the regulation of autolytic activity (22), the binding of Mg2+ for enzyme function (5,25,30,37), and the synthesis of wall teichoic acid (20...