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.     

Occurrence of ribitol-containing lipoteichoic acid in Staphylococcus aureus H and its glycosylation

A ribitol-containing lipoteichoic acid was obtained from the 20,000 x g supernatant fraction of Staphylococcus aureus H by extraction with Triton X-100 followed by fractionation on Sepharose 6B and DEAE-cellulose columns. The purified lipoteichoic acid was composed of phosphate, glycerol, glucose, glucosamine, ribitol, and fatty acids in a molar ratio of 1 : 0.9 : 0.06 : 0.03 : 0.09 : 0.07. Based on the structural analysis of fragments from alkali and HF hydrolysis, the lipoteichoic acid appears to consist of three moieties, namely a ribitol phosphate oligomer, poly(glycerol phosphate) which has about 30 glycerol phosphate units, and beta-glucosyl-beta-glucosyl(1 leads to 1)diacylglycerol. N-Acetylglucosamine was linked to the ribitol residues. The lipoteichoic acid serves as an acceptor of glycosyl moieties from UDP-glucose and UDP-N-acetylglucosamine in the enzyme reaction catalyzed by the membrane preparation. The rate of enzymatic glycosylation was increased by prior treatment of the lipoteichoic acid with N-acetyl-beta-D-glucosaminidase. The glycosylation seems to occur at the ribitol residues of the lipoteichoic acid.     

Comparative studies of lipoteichoic acids from several Bacillus strains.

Structural studies were carried out on lipoteichoic acids obtained from defatted cells of 10 Bacillus strains by phenol-water partition followed by chromatography on DEAE-Sephacel and Octyl-Sepharose columns. A group of the tested bacteria (group A), Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus, was shown to have a diacyl form of lipoteichoic acids which contained D-alanine, D-glucose, D-glucosamine, fatty acids, and glycerol in molar ratios to phosphorus of 0.35 to 0.69, 0.07 to 0.15 to 0.43, 0.06 to 0.11, and 0.95 to 1.18, respectively, whereas the other group (group B), Bacillus coagulans and Bacillus megaterium, had diacyl lipoteichoic acids which contained D-galactose, fatty acids, and glycerol in molar ratios to phosphorus of 0.05 to 0.42, 0.06 to 0.12, and 0.96 to 1.07, respectively. After treatment with 47% hydrogen fluoride, the lipoteichoic acids obtained from group A strains commonly gave a hydrophobic fragment, gentiobiosyl-beta (1----1 or 3)diacylglycerol, in addition to dephosphorylated repeating units, glycerol, 2-D-alanylglycerol, N-acetyl-D-glucosaminyl-alpha (1----2)glycerol, and D-alanyl-N-acetyl-D-glucosaminyl-alpha (1----2)glycerol, whereas the lipoteichoic acids from group B strains yielded diacylglycerol in addition to glycerol and D-galactosyl-alpha (1----2)glycerol. The results together with data from Smith degradations indicate that in the lipoteichoic acids of group A strains the polymer chains, made up of partially alanylated glycerol phosphate and glycosylglycerol phosphate units, are joined to the acylglycerol anchors through gentiobiose. However, in the lipoteichoic acids of group B strains, the partially galactosylated poly(glycerolphosphate) chains are believed to be directly linked to the acylglycerol anchors.     

Structure of the lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum

The lipoteichoic acids from Bifidobacterium bifidum spp. pennsylvanicum were extracted from cytoplasmic membranes or from disintegrated bacteria with aqueous phenol and purified by gel chromatography. The lipoteichoic acid preparations contained phosphate, glycerol, galactose, glucose and fatty acids in a molar ratio of 1.0:1.0:1.3:1.2:0.3. Chemical analysis and NMR studies of the native preparations and of products from various acid and alkaline hydrolysis procedures gave evidence for the structure of two lipoteichoic acids. The lipid anchor appeared to be 3-O-(6'-(sn-glycero-1-phosphoryl)diacyl-beta-D-galactofuranosyl)-sn-1, 2-diacylglycerol. The polar part showed two structural features not previously described for lipoteichoic acids. A 1,2-(instead of the usual 1,3-) phosphodiester-linked sn-glycerol phosphate chain is only used substituted at the terminal glycerol unit with a linear polysaccharide, containing either beta(1----5)-linked D-galactofuranosyl groups or beta(1----6)-linked D-glucopyranosyl groups.     

Lipoteichoic Acid Localization in Mesosomal Vesicles of Staphylococcus aureus

Mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P have been prepared and examined for the presence of lipoteichoic acid. Lipids were first removed by treatment with pyridine-acetic acid-butanol (22:31:100, vol/vol/vol) and chloroform-methanol (2:1, vol/vol). Subsequently, lipoteichoic acid was removed with 40% phenol in water. The lipoteichoic acid from mesosomal vesicles was characterized by (i) equimolar glycerol and phosphate, (ii) alanine upon hydrolysis (2 N NH₄OH, 18 h, 22 C), and (iii) fatty acids, diglycerol triphosphate, glycerol monophosphate, and glycerol diphosphate upon alkaline hydrolysis (1 N NaOH, 3h, 100 C). The plasma membranes contained no lipoteichoic acid. The presence in mesosomal vesicles of 18% of the dry weight as lipoteichoic acid and its absence from plasma membranes provide the first major chemical differences between these organelles. A study of the lipoteichoic acid content in various fractions of the cell showed that the mesosomal vesicles were the major and probably the sole site for the localization of the lipoteichoic acid in these organisms. A new method for the preparation of mesosomes in increased yields is reported. A theory for the control of cell division involving lipoteichoic acid and the mesosome is proposed.     

Immunochemical studies on the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum

Antisera to lipoteichoic acid of Bifidobacterium bifidum subsp. pennsylvanicum were obtained by injecting lipoteichoic acid/methylated BSA complexes into rabbits. Precipitin tests showed that the glycerol phosphate backbone is primarily responsible for serological specificity while the polysaccharide part of the molecule plays a minor role. Whole cells of B. bifidum subsp. pennsylvanicum were capable of absorbing antibodies, indicating the presence of lipoteichoic acid (14% of the total content) at or near the bacterial surface. Cross-reactivity with strains of the genera Bifidobacterium and Lactobacillus was tested using absorption of antiserum by whole bacteria and reactivity of phenol extracts. The results indicated that lipoteichoic acid is a common antigen within the genus Bifidobacterium. The cross-reactivity with the lactobacilli tested was very low.     

An inositol containing lipomannan from Propionibacterium freudenreichii

Abstract A lipoglycan has been extracted from cells of Propionibacterium freudenreichii by the standard procedures used to isolate lipoteichoic acids from Gram-positive bacteria. The polymer was purified by chromatography and shown to contain mannose, inositol, glycerol, fatty acids and phosphate. The presence of the components of phosphatidylinositol suggests the lipoglycan may be a mannan anchored to the membrane by a covalently linked phosphatidylinositol although alternative structures cannot be excluded.     

D-alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin.

Vesicles containing lipoteichoic acid (LTA) have been isolated from Lactobacillus casei ATCC 7469 grown in the presence of either benzylpenicillin or D-cycloserine. These cell wall antibiotics enhanced the rate of LTA and lipid secretion 6.7 times, whereas chloramphenicol inhibited their release. The formation of these vesicles from peripheral and septal wall regions did not appear to be the result of bacteriolysis. The vesicle composition of LTA and lipid was similar to that of the cytoplasmic membrane whereas the protein composition was dissimilar. The size of these vesicles ranged from 20 to 40 nm and the length of LTA ranged from 5 to 50 glycerol phosphate residues. The isolation of these vesicles provides a potential in vitro acceptor system for studying the D-alanylation of lipoteichoic acid.     

Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

The function of beta-N-acetyl-D-glucosaminyl monophosphorylundecaprenol in biosynthesis of lipoteichoic acids in a group of Bacillus strains

Membrane preparations, obtained from Bacillus strains which have N-acetylglucosamine-linked lipoteichoic acids in their membranes, were shown to catalyze the transfer of N-[14C]acetylglucosamine (GlcNAc) from beta-[14C]GlcNAc-P-undecaprenol to endogenous polymer. In this reaction, alpha-GlcNAc-P-undecaprenol or alpha-GlcNAc-PP-undecaprenol could not substitute for beta-GlcNAc-P-undecaprenol as the N-acetylglucosamine donor. This enzyme was most active at pH 6.0 and in the presence of 40 mM MgCl2. The apparent Km for beta-GlcNAc-P-undecaprenol was 2 microM. The radioactive polymer products, solubilized by hot phenol treatment, coincided with lipoteichoic acids in chromatographic behavior. Hydrogen fluoride treatment of the polymer products gave a major fragment identical with GlcNAc(alpha 1----2)glycerol, which corresponded to the dephosphorylated repeating units of the lipoteichoic acids in the examined strains. Thus it is concluded that beta-GlcNAc-P-undecaprenol serves as the donor of N-acetylglucosamine in the biosynthesis of lipoteichoic acids in a group of Bacillus strains.     

The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus

Pulse-chase experiments with [2-3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol leads to (glycerophospho)25-diglucosyldiacylglycerol + 25 diacylglycerol. Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor. Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative. In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.     

Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells

In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp. strain A007. When cells of B. subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol. Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity. The [32P]phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B. subtilis W23. A unique metabolism of phosphatidylglycerol was found in Bacillus sp. strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B. subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate. These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover.     

Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid.

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.     

The glycerol teichoic acid from the cell wall of Bacillus stearothermophilus B65

1. A glycerol teichoic acid has been extracted from cell walls of Bacillus stearothermophilus B65 and its structure examined. 2. Trichloroacetic acid-extractable teichoic acid accounted for 68% of the total cell-wall phosphorus and residual material could be hydrolysed to a mixture of products including those characteristic of glycerol teichoic acids. 3. The extracted polymer is composed of glycerol, phosphoric acid, d-glucose and d-alanine. 4. Hydrolysis of the polymer with alkali gave glycerol, 1-O-alpha-d-glucopyranosylglycerol and its monophosphates, glycerol mono- and di-phosphate, as well as traces of a glucosyldiglycerol triphosphate and a glucosylglycerol diphosphate. 5. The teichoic acid is a polymer of 18 or 19 glycerol phosphate units having alpha-d-glucopyranosyl residues attached to position 1 of 14 or 15 of the glycerol residues. 6. The glycerol residues are joined by phosphodiester linkages involving positions 2 and 3 in each glycerol. 7. d-Alanine is in ester linkage to the hydroxyl group at position 6 of approximately half of the glucose residues. 8. One in every 13 or 12 polymer molecules bears a phosphomonoester group on position 3 of a glucose residue, the possible significance of which in linkage of the polymer to other wall constituents is discussed.     

Lipoteichoic Acids from Streptococcus sanguis

Two lipoteichoic acids, membrane (MLTA) and wall (WLTA), have been purified from Streptococcus sanguis by Sepharose and Ecteola-cellulose column chromatographies and concanavalin A-conjugated Sepharose affinity column chromatography. The teichoic acids were homogenous as judged by disc gel electrophoresis, column chromatography, and double diffusion tests. Both MLTA and WLTA consisted of glycerol, phosphate, glucose, and fatty acids in the ratios of 0.95:1:0.71:0.046 and 0.99:1:0.79:0.023, respectively. alpha-Glycerol-phosphate was obtained by the partial acid hydrolysis of the lipoteichoic acids suggesting that their backbone structure consists of the glycerol moieties linked by 1, 3-phosphodiester bonds. Both WLTA and MLTA form aggregates, perhaps due to micelle formation, in concentrated solution. The aggregate form of MLTA dissociates to a much greater extent than that of WLTA under similar conditions.     

Biosynthesis of the wall teichoic acid in Bacillus licheniformis

1. The biosynthesis of the wall teichoic acid, poly(glycerol phosphate glucose), has been studied with a particulate membrane preparation from Bacillus licheniformis A.T.C.C. 9945. The precursor CDP-glycerol supplies glycerol phosphate residues, whereas UDP-glucose supplies only glucose to the repeating structure of the polymer. 2. Synthesis proceeds through polyprenol phosphate derivatives, and chemical studies and pulse-labelling techniques show that the first intermediate is the phosphodiester, glucose polyprenol monophosphate. CDP-glycerol donates a glycerol phosphate residue to this to give a second intermediate, (glycerol phosphate glucose phosphate) polyprenol. 3. The glucose residue in the lipid intermediates has the beta configuration, and chain extension in the synthesis of polymer occurs by transglycosylation with inversion of anomeric configuration at two stages.     

Teichoic acid synthesis in Bacillus stearothermophilus

1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.     

Structural requirements for initiation of Limulus amebocyte lysate gelation by lipoteichoic acids

Abstract Lipoteichoic acids (LTAs) of varying chemical composition from five streptococcal species and one lactobacillus species initiated gelation of Limulus amebocyte lysate. Preincubation with antisera specific for the poly(glycerol phosphate) (PGP) chain or, when appropriate, with antisera, specific for a carbohydrate substituent inhibited gelation initiated by lipoteichoic acid, but did not inhibit lipopolysaccharide-initiated gelation. Higher specific activities were found for those LTAs with higher d -alanine or carbohydrate substitution or with a shortened PGP chain, which suggested that those structural features which decreased the relative hydrophilicity or charge to mass ratio appeared to increase the specific activity.     

In vitro synthesis of the unit that links teichoic acid to peptidoglycan.

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.     

Structural features of cell wall teichoic acid and peptidoglycan of Actinomadura cremea INA 292

The teichoic acid from the cell wall of Actinomadura cremea INA 292 has an unusual structure, being a poly(galactosylglycerol phosphate) chain with glycerol phosphate groups. Monomeric units of 1-O, beta-D-galactopyranosylglycerol monophosphate are joined in the polymer by phosphodiester links involving the glycerol C3 and the galactose C6 atoms. Approximately every second galactosyl substituent has a glycerol phosphate residue at its C3 atom. The teichoic acid structure was established by chemical analysis and 13C-NMR spectroscopy. There also is a peptidoglycan belonging to the A1 gamma type: as well as meso-2,6-diaminopimelic acid it contains small amounts of the LL form and glycine.