Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme

Abstract

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...

A Continuum of Anionic Charge: Structures and Functions ofd-Alanyl-Teichoic Acids in Gram-Positive Bacteria

“…This complex regulatory system reflects, in part, the sporulation capability of this organism and the fact that spore LTA contains no detectable D-alanyl ester. In contrast, the L. rhamnosus dlt operon contains a single putative promoter region (Ϫ10 and Ϫ35 with a 20-bp spacer) similar to those reported for Lactobacillus species (86,209).…”

“…Isolation of the gene encoding the activating enzyme (dltA) (Fig. 9) from L. rhamnosus ATCC 7469 provided the key for identifying the role of this enzyme in D-alanine incorporation (209,367). The enzyme is a member of a large protein on May 10, 2018 by guest http://mmbr.asm.org/ family that both activates and transfers amino or fatty acids via a 4Ј-phosphopantetheine prosthetic group of a carrier protein or coenzyme A CoA (262).…”

“…However, the functions of TAs within the wall matrix have been more difficult to define. The D-alanyl esters of these polymers, resulting from a single D-alanine incorporation system encoded by the dlt (for "D-alanyl-LTA") operon (209,367,383), constitute important substituents for modulating the properties of the envelope in many species. For this reason, knowledge of these ester residues is essential for understanding the functions of TAs in bacterial physiology as well as in host-mediated responses.…”

Lantibiotic Resistance

“…The dlt operon has been characterized across many species and contains four genes required for functionality: dltABCD (43). The role of DltA is to act as a D-alanine-D-alanyl carrier protein ligase (Dcl), which activates D-alanine by hydrolysis of ATP and transfers it to the phosphopantetheine cofactor of a specified alanine carrier protein (Dcp), encoded by dltC (44). The hydrophobic DltB protein, which may have a transmembrane location, is required for D-alanine incorporation into teichoic acids, possibly through the transfer of activated D-alanine across the cytoplasmic membrane (42).…”

Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus

“…Staphylococci produce polyglycerol phosphate polymers even in the absence of glycolipids, because mutant strains that cannot synthesize Glc 2 -DAG can still anchor LTA via diacylglycerol (19,20). Mutations in the dlt operon abolish D-alanyl esterification of LTA without affecting the synthesis of polyglycerol phosphate (21)(22)(23)(24). Both dlt and glycolipid anchor mutants continue to multiply, thereby revealing that these nonessential genes are not ideal targets for antibiotic development (20,23).…”