Assembly of D-alanyl-lipoteichoic acid in Lactobacillus casei: mutants deficient in the D-alanyl ester content of this amphiphile.

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 compared with the parent, strains AN-1 and AN-4 incorporated less than 10% of D-[14C]alanine into LTA, whereas AN-2, AN-3, and AN-5 incorporated 50%. The synthesis of D-[14C]alanyl-lipophilic LTA was virtually absent in the first group and was approximately 30% in the second group. The mutant strains synthesized and selected the glycolipid anchor for LTA assembly. In addition, all of the strains synthesized the poly(glycerophosphate) moiety of LTA to the same extent as did the parent or to a greater extent. It was concluded that the membranes from the mutant strains AN-1 and AN-4 are defective for D-alanylation of LTA even though acceptor LTA is present. Mutant strains AN-2 and AN-3 appear to be partially deficient in the amount of the D-alanine-activating enzyme. Aberrant morphology and defective cell separation appear to result from this deficiency in D-alanyl ester content.     

D-alanylation of lipoteichoic acid: role of the D-alanyl carrier protein in acylation

The D-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-positive organisms requires the D-alanine-D-alanyl carrier protein ligase (AMP) (Dcl) and the D-alanyl carrier protein (Dcp). The dlt operon encoding these proteins (dltA and dltC) also includes dltB and dltD. dltB encodes a putative transport system, while dltD encodes a protein which facilitates the binding of Dcp and Dcl for ligation with D-alanine and has thioesterase activity for mischarged D-alanyl-acyl carrier proteins (ACPs). In previous results it was shown that D-alanyl-Dcp donates its ester residue to membrane-associated LTA (M. P. Heaton and F. C. Neuhaus, J. Bacteriol. 176: 681-690, 1994). However, all efforts to identify an enzyme which catalyzes this D-alanylation process were unsuccessful. It was discovered that incubation of D-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolysis of this D-alanyl thioester. D-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated D-alanyl LTA, a time and concentration-dependent formation of D-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of D-alanyl-Dcp and stimulated the hydrolysis of D-alanyl-Dcp. Since these reactions are specific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the D-alanylation process. The reversibility of this process provides a mechanism for the transacylation of the D-alanyl ester residues between LTA and wall teichoic acid.     

Biosynthesis of D-alanyl-lipoteichoic acid: the tertiary structure of apo-D-alanyl carrier protein.

The D-alanylation of lipoteichoic acid (LTA) allows the Gram-positive organism to modulate its surface charge, regulate ligand binding, and control the electromechanical properties of the cell wall. The incorporation of D-alanine into LTA requires the D-alanine:D-alanyl carrier protein ligase (AMP-forming) (Dcl) and the carrier protein (Dcp). The high-resolution solution structure of the 81-residue (8.9 kDa) Dcp has been determined by multidimensional heteronuclear NMR. An ensemble of 30 structures was calculated using the torsion angle dynamics approach of DYANA. These calculations utilized 3288 NOEs containing 1582 unique nontrivial NOE distance constraints. Superposition of residues 4-81 on the mean structure yields average atomic rmsd values of 0.43 +/- 0.08 and 0.86 +/- 0.09 A for backbone and non-hydrogen atoms, respectively. The solution structure is composed of three alpha-helices in a bundle with additional short 3(10)- and alpha-helices in intervening loops. Comparisons of the three-dimensional structure with the acyl carrier proteins involved in fatty acid, polyketide, and nonribosomal peptide syntheses support the conclusion that Dcp is a homologue in this family. While there is conservation of the three-helix bundle fold, Dcp has a higher enthalpy of unfolding and no apparent divalent metal binding site(s), features that distinguish it from the fatty acid synthase acyl carrier protein of Escherichia coli. This three-dimensional structure also provides insights into the D-alanine ligation site recognized by Dcl, as well as the site which may bind the poly(glycerophosphate) acceptor moiety of membrane-associated LTA.     

Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and alpha-glucose substitution of poly(glycerophosphate) backbones

Lipoteichoic acids (LTAs) have been shown to act as bacterial counterparts to the receptor binding proteins of LL-H, LL-H host range mutant LL-H-a21, and JCL1032. Here we have used LTAs purified by hydrophobic interaction chromatography from different phage-resistant and -sensitive strains of Lactobacillus delbrueckii subsp. lactis. Nuclear magnetic resonance analyses revealed variation in the degree of alpha-glucosyl and D-alanyl substitution of the 1,3-linked poly(glycerophosphate) LTAs between the phage-sensitive and phage-resistant strains. Inactivation of phages was less effective if there was a high level of D-alanine residues in the LTA backbones. Prior incubation of the LTAs with alpha-glucose-specific lectin inhibited the LL-H phage inactivation. The overall level of decoration or the specific spatial combination of alpha-glucosyl-substituted, D-alanyl-substituted, and nonsubstituted glycerol residues may also affect phage adsorption.     

Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid.

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) is a widespread macroamphiphile which plays a vital role in the growth and development of gram-positive organisms. The biosynthesis of this polymer requires the enzymic activation of D-alanine for its transfer to the membrane-associated LTA (mLTA). A small, heat-stable, and acidic protein that is required for this transfer was purified to greater than 98% homogeneity from Lactobacillus casei ATCC 7469. This protein, previously named the D-alanine-membrane acceptor ligase (V. M. Reusch, Jr., and F. C. Neuhaus, J. Biol. Chem. 246:6136-6143, 1971), functions as the D-alanyl carrier protein (Dcp). The amino acid composition, beta-alanine content, and N-terminal sequence of this protein are similar to those of the acyl carrier proteins (ACPs) of fatty acid biosynthesis. The isolation of Dcp and its derivative, D-alanyl approximately Dcp, has allowed the characterization of two novel reactions in the pathway for D-alanyl-mLTA biosynthesis: (i) the ligation of Dcp with D-alanine and (ii) the transfer of D-alanine from D-alanyl approximately Dcp to a membrane acceptor. It has not been established whether the membrane acceptor is mLTA or another intermediate in the pathway for D-alanyl-mLTA biosynthesis. Since the D-alanine-activating enzyme (EC 6.1.1.13) catalyzes the ligation reaction, this enzyme functions as the D-alanine-Dcp ligase (Dcl). Dcl also ligated the ACPs from Escherichia coli, Vibrio harveyi, and Saccharopolyspora erythraea with D-alanine. In contrast to the relaxed specificity of Dcl in the ligation reaction, the transfer of D-alanine to the membrane acceptor was highly specific for Dcp and did not occur with other ACPs. This transfer was observed by using only D-[14C]alanyl approximately Dcp and purified L. casei membranes. Thus, D-alanyl approximately Dcp is an essential intermediate in the transfer of D-alanine from Dcl to the membrane acceptor. The formation of D-alanine esters of mLTA provides a mechanism for modulating the net anionic charge in the cell wall.     

Biosynthesis of lipoteichoic acid in Lactobacillus rhamnosus: role of DltD in D-alanylation

The dlt operon (dltA to dltD) of Lactobacillus rhamnosus 7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by D-alanine. These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA. dltA and dltC encode the D-alanine-D-alanyl carrier protein ligase (Dcl) and D-alanyl carrier protein (Dcp), respectively. Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown. To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of dltD from Lactobacillus casei 102S. Permeabilized cells of a dltD::erm mutant lacked the ability to incorporate D-alanine into LTA. This defect was complemented by the expression of DltD from pNZ123/dlt. In in vitro assays, DltD bound Dcp for ligation with D-alanine by Dcl in the presence of ATP. In contrast, the homologue of Dcp, the Escherichia coli acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with D-alanine. DltD also catalyzed the hydrolysis of the mischarged D-alanyl-ACP. The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane. It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine and that the resulting D-alanyl-Dcp is translocated to the primary site of D-alanylation.     

Effect of alanine ester substitution and other structural features of lipoteichoic acids on their inhibitory activity against autolysins of Staphylococcus aureus.

Native substitution with the D-alanine ester of lipoteichoic acids (LTAs) affects their immunological properties, the capacity to bind divalent cations, and LTA carrier activity. In this study we tested the influence of the D-alanine ester on anti-autolytic activity, using extracellular autolysin from Staphylococcus aureus and nine LTAs with alanine/phosphorus molar ratios of between 0.23 and 0.71. The inhibitory activity, highest with alanine-free LTA, exponentially decreased with increasing alanine content, approaching zero at substitutions of greater than 0.6. Correspondingly, dipolar ionic phospholipids were not inhibitory, in contrast to negatively charged ones. Glycosylation of LTA up to an extent of 0.5 did not depress inhibitory activity, and even at a degree of 0.8 the effect was comparatively small. On comparison of LTAs from various sources, differences in lipid structures and chain lengths were without effect. The inhibitory activity drastically decreased when the glycolipid carried a single glycerophosphate residue or the hydrophilic chain had the unusual structure [6 leads to Gal(alpha 1--6)Gal(alpha 1--3)Gro-(2 comes from 1 alpha Gal)-P]n, in which digalactosyl moieties connect the alpha-galactosylated glycerophosphate units. Principally, the same results were obtained with the more complex system of autolysis of S. aureus cells. We hypothesize that the anti-autolytic activity of LTA resides in a sequence of glycerophosphate units and that the negative charges of appropriately spaced phosphodiester groups play a crucial role. The alanine ester effect is discussed with respect to the putative in vivo regulation of autolysins by LTA.     

Separation of the poly(glycerophosphate) lipoteichoic acids of Enterococcus faecalis Kiel 27738, Enterococcus hirae ATCC 9790 and Leuconostoc mesenteroides DSM 20343 into molecular species by affinity chromatography on concanavalin A

This study shows for the first time microheterogeneity of 1,3-linked poly(glycerophosphate) lipoteichoic acids. The lipoteichoic acids investigated were those of Enterococcus faecalis Kiel 27738 (I), Enterococcus hirae (Streptococcus faecium) ATCC 9790 (II), and Leuconostoc mesenteroides DMS 20343 (III). Lipoteichoic acids II and III are partially substituted by mono-, di-, tri-, and tetra-alpha-D-glucopyranosyl residues with (1----2) interglycosidic linkages. Lipoteichoic acid I is substituted with alpha-kojibiosyl residues only. Lipoteichoic acids I and III additionally carry D-alanine ester. Lipoteichoic acids were separated on columns of concanavalin-A-Sepharose according to their increasing number of glycosyl substituents per chain. It was evident that all molecular species are usually glycosylated and that alanine ester and glycosyl residues occur on the same chains. The chain lengths of lipoteichoic acid I and II vary between 9-40 glycerophosphate residues, whereas those of lipoteichoic acid III appear to be uniform (33 +/- 2 residues). Molecular species differ in the extent of glycosylation but their content of alanyl residues is fairly constant. All lipoteichoic acids contain a small fraction (5-15%) different in composition from the bulk and most likely reflecting an early stage of biosynthesis. Two procedures for chain length determination of poly(glycerophosphate) lipoteichoic acids are described.     

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.     

The D-Alanyl carrier protein in Lactobacillus casei: cloning, sequencing, and expression of dltC.

The incorporation of D-alanine into membrane-associated D-alanyl-lipoteichoic acid in Lactobacillus casei requires the 56-kDa D-alanine-D-alanyl carrier protein ligase (Dcl) and the 8.9-kDa D-alanyl carrier protein (Dcp). To identify and isolate the gene encoding Dcp, we have cloned and sequenced a 4.3-kb chromosomal fragment that contains dcl (dltA). In addition to this gene, the fragment contains three other genes, dltB, d1tC, and a partial dltD gene. dltC (246 nucleotides) was subcloned from this region and expressed in Escherichia coli. The product was identified as apo-Dcp lacking the N-terminal methionine (8,787.9 Da). The in vitro conversion of the recombinant apo-Dcp to holo-Dcp by recombinant E. coli holo-ACP synthase provided Dcp which accepts activated D-alanine in the reaction catalyzed by Bcl. The recombinant D-alanyl-Dcp was functionally identical to native D-alanyl-Dcp in the incorporation of D-alanine into lipoteichoic acid. L. casei Dcp is 46% identical to the putative product of dltC in the Bacillus subtilis dlt operon (M. Perego, P. Glaser, A. Minutello, M. A. Strauch, K. Leopold, and W. Fischer, J. Biol. Chem. 270:15598-15606, 1995), and therefore, this gene also encodes Dcp. Comparisons of the primary sequences and predicted secondary structures of the L. casei and B. subtilis Dcps with that of the E. coli acyl carrier protein (ACP) were undertaken together with homology modeling to identify the functional determinants of the donor and acceptor specificities of Dcp. In the region of the phospho-pantetheine attachment site, significant similarity between Dcps and ACPs was observed. This similarity may account for the relaxed acceptor specificity of the Dcps and ACPs in the ligation Of D-alanine catalyzed by Dcl. In contrast, two Dcp consensus sequences, KXXVLDXLA and DXVKXNXD, share little identity with the rest of the ACP family and, thus, may determine the donor specificity of D-alanyl-Dcp in the D-alanylation of membrane-associated D-alanyl-lipoteichoic acid.     

Synthesis of the lipoteichoic acid of the Streptococcus species DSM 8747

The lipoteichoic acid (LTA) of the Streptococcus species DSM 8747 consists of a β-d-galactofuranosyl diacylglycerol moiety (with different acyl groups) that is linked via 6-O to a poly(glycerophosphate) backbone; about 30% of the glycerophosphate moieties carry at 2-O hydrolytically labile d-alanyl residues. As typical LTA for this array of compounds LTA 1a was synthesized. To this end, from d-galactose the required galactofuranosyl building block 5 was obtained. The anomeric stereocontrol in the glycosylation step with 1,2-O-cyclohexylidene-sn-glycerol (4) was based on anchimeric assistance, thus finally leading to the unprotected core glycolipid 16. Regioselective protection and deprotection procedures permitted the defined attachment of the pentameric glycerophosphate 3 to the 6-hydroxy group of the galactose residue. Introduction of four d-alanyl residues led after global deprotection and purification to target molecule 1a possessing on average about two d-alanyl residues at 2-O of the pentameric glycerophosphate backbone, thus being in close accordance with the structure of the natural material.     

Heterogeneity of lipoteichoic acid detected by anion exchange chromatography

A complex polydispersity became apparent when the poly(glycerophosphate) lipoteichoic acid of Enterococcus faecalis was chromatographed on DEAE-sephadex. The chain length varied between 13 and 33 glycerophosphate residues per lipid anchor. In parallel, the extent of chain glycosylation increased from 0.2 to 0.4 diglucosyl residues per glycerophosphate unit. Substitution with D-alanine ester showed a reverse distribution dropping with increasing chain length from 0.53 to 0.23 mol D-alanine per mol phosphorus. Variations in the fatty acid composition were also observed. The results extent and modify the current picture of lipoteichoic acid biosynthesis. They further suggest that during infection the mammalian organism may be confronted particularly with long-chain less hydrophobic molecular species.     

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

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 1-phosphate 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.     

Structure and properties of lipoteichoic acid of group A Streptococcus type M 29

Lipoteichoic acid (LTA) of group A streptococci, type M 29 was studied. Chemical and 13C NMR spectroscopic analysis showed that the polymer contained poly(glycerophosphate) chain consisting of 12-14 glycerophosphate elements united by the 1----3 type phosphodiether bond and diglucosylglyceride. Oleic, stearic, palmitic and palmitoleic fatty acids predominated in the polymer composition. The content of the fatty acids amounted approximately to 2 per cent of LTA dry weight. The poly(glycerophosphate) chain contained 6-7 ether linked alanyl moieties. The results of the LTA biological study were analyzed in comparison to the data on a previous study of antitumor and cardiotoxic properties of teichoic acid from Streptomyces levoris K-3053 which is structurally close to the LTA hydrophilic moiety. It was assumed that the molecule negative charge had an effect on the cardiotoxic and antitumor activity.     

D-alanyl ester depletion of teichoic acids in Lactobacillus plantarum results in a major modification of lipoteichoic acid composition and cell wall perforations at the septum mediated by the Acm2 autolysin

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.     

Studies on glucosyltransferase and endogenous glucosyl acceptor in Bacillus cereus AHU 1030 membranes

A glucosyltransferase, extracted from the membranes of Bacillus cereus AHU 1030 with Tris-HCl buffer containing 0.1% Triton X-100 at pH 9.5, was separated from an endogenous glucosyl acceptor by chromatography on DEAE-Sepharose CL-6B subsequent to chromatography on Sepharose 6B. Structural analysis data showed that the glucosyl acceptor was a glycerol phosphate polymer linked to beta-gentiobiosyl diglyceride. The enzyme catalyzed the transfer of glucosyl residues from UDP-glucose to C-2 of the glycerol residues of repeating units of the acceptor. On the other hand, a lipoteichoic acid which contained 0.3 D-alanine residue per phosphorus was isolated from the cells by phenol treatment at pH 4.6. Except for the presence of D-alanine, this lipoteichoic acid had the same structure as the glucosyl acceptor. The rate of glucosylation observed with the D-alanine-containing lipoteichoic acid as the substrate was less than 40% of that observed with the D-alanine-free lipoteichoic acid, indicating that the ester-linked D-alanine in the lipoteichoic acid interferes with the action of the glucosyltransferase. The enzyme also catalyzed glucosylation of poly(glycerol phosphate) which was synthesized in the reaction of a separate enzyme fraction with CDP-glycerol. Thus, it is likely that the glucosyltransferase functions in the synthesis of cell wall teichoic acid.     

Monocyte and macrophage activation by lipoteichoic Acid is independent of alanine and is potentiated by hemoglobin

Lipoteichoic acids (LTAs) are Gram-positive bacterial cell wall components that elicit mononuclear cell cytokine secretion. Cytokine-stimulating activity is thought to be dependent on retaining a high level of ester-linked D-alanine residues along the polyglycerol phosphate backbone. However, Streptococcus pyogenes LTA essentially devoid of D-alanine caused human and mouse cells to secrete as much IL-6 as LTA with a much higher D-alanine content. Furthermore, hemoglobin (Hb) markedly potentiates the stimulatory effect of various LTAs on mouse macrophages or human blood cells, regardless of their d-alanine content. LTA and Hb appear to form a molecular complex, based on the ability of each to affect the other's migration on native acrylamide gels, their comigration on these gels, and the ability of LTA to alter the absorption spectra of Hb. Because S. pyogenes is known to release LTA and secrete at least two potent hemolytic toxins, LTA-Hb interactions could occur during streptococcal infections and might result in a profound alteration of the local inflammatory response.     

A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria.

Teichoic acids (TAs) are major wall and membrane components of most gram-positive bacteria. With few exceptions, they are polymers of glycerol-phosphate or ribitol-phosphate to which are attached glycosyl and D-alanyl ester residues. Wall TA is attached to peptidoglycan via a linkage unit, whereas lipoteichoic acid is attached to glycolipid intercalated in the membrane. Together with peptidoglycan, these polymers make up a polyanionic matrix that functions in (i) cation homeostasis; (ii) trafficking of ions, nutrients, proteins, and antibiotics; (iii) regulation of autolysins; and (iv) presentation of envelope proteins. The esterification of TAs with D-alanyl esters provides a means of modulating the net anionic charge, determining the cationic binding capacity, and displaying cations in the wall. This review addresses the structures and functions of D-alanyl-TAs, the D-alanylation system encoded by the dlt operon, and the roles of TAs in cell growth. The importance of dlt in the physiology of many organisms is illustrated by the variety of mutant phenotypes. In addition, advances in our understanding of D-alanyl ester function in virulence and host-mediated responses have been made possible through targeted mutagenesis of dlt. Studies of the mechanism of D-alanylation have identified two potential targets of antibacterial action and provided possible screening reactions for designing novel agents targeted to D-alanyl-TA synthesis.     

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: A fundamental structure of the hydrophilic part

Previously, we showed that quantitatively minor several glycolipids only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97–112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779–86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.     

Coenzyme A-dependent transacylation system in rabbit liver microsomes

The activities of cofactor-independent and CoA-dependent transacylation were examined for various rabbit tissues. Liver microsomes were found to exhibit relatively high CoA-dependent transacylation activity, while the cofactor-independent transacylation activity was low. The apparent Km values for CoA were 1.4 microM (acceptor, 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC] and 3.8 microM (acceptor, 1-acyl-sn-glycero-3-phosphoethanolamine (1-acyl-GPE], respectively. The apparent Vmax values were 2.6 nmol/min/mg (1-acyl-GPC) and 1.2 nmol/min/mg (1-acyl-GPE), respectively. The CoA-dependent transacylation reaction shows a distinct fatty acid specificity. [14C]18:2 and [14C]20:4 at the 2-positions and [14C]18:0 at the 1-positions of donor phospholipids were transferred to lysophospholipids in the presence of CoA. We observed the formation of considerable amounts of acyl-CoA from these fatty acids during the reaction, without the participation of ATP. The transfer of other fatty acids between phospholipids was shown to be almost nil. The very low transfer of 18:1 was in marked contrast to the effective utilization of 18:1-CoA by acyl-CoA:1-acyl-GPC acyltransferase. The effects of several compounds and heat treatment on these two acylation reactions were also examined. The CoA-dependent transacylation reaction may be important for the selective acylation of certain lysophospholipids, such as 1-acyl-GPE, in living cells with the cooperation of acyl-CoA:lysophospholipid acyltransferase, which generates CoA for the former reaction.