Structural studies on the amino-acid-linked teichuronic acid of Bacillus subtilis AHU 1219 cell walls

Structural studies were carried out on a teichuronic acid isolated from a mild acid extract of Bacillus subtilis AHU 1219 cell walls. The teichuronic acid contained D-glucuronic acid, D-glucose, D-galactose, L-serine and L-threonine in a molar ratio of 1:1:1:0.5:0.5. Results of analyses of the polysaccharide by Smith degradation, methylation and 1H-NMR and 13C-NMR spectroscopy, in combination with data on analyses of oligosaccharides obtained by partial acid hydrolysis and alkaline hydrolysis of the polymer, led to the most likely structure for the repeating unit, ----4)(L-Ser/L-Thr)-D-GlcA(beta 1----3)-D-Glc(beta 1----4)-D-Gal(alpha 1----. In each unit, either amino acid is linked to the glucuronic acid residue through an amide bond.     

Structural and immunochemical studies of teichoic acid of Listeria monocytogenes

An immunologically active teichoic acid component was isolated from the cell wall of Listeria monocytogenes strain EGD. The teichoic acid component, accounting for about 20% of the weight of cell wall, contained N-acetylglucosamine, rhamnose, ribitol, and phosphorus in a molar ratio of 0.95 : 1.0 : 0.97 : 0.98. The molecular weight of the teichoic acid chain was about 120,000 as analyzed by gel filtration. The probable structure was deduced from the results of methylation analysis, Smith degradation, and proton magnetic resonance spectrometry of the teichoic acid, together with the characterization of fragments obtained by treatment with hydrofluoric acid, as follows: (formula; see text) Inhibition testing with monosaccharide and fragments obtained from HF treatment of Listeria teichoic acid in the quantitative precipitin reaction suggested that the rhamnose residue is a major antigenic determinant.     

Solubilization and properties of UDP-D-glucose:N-acetylglucosaminyl pyrophosphorylundecaprenol glucosyltransferase from Bacillus coagulans AHU 1366 membranes

The glucosyltransferase which catalyzes the conversion of GlcNAc-PP-undecaprenol into Glc(beta 1----4)GlcNAc-PP-undecaprenol in the presence of UDP-glucose was solubilized from Bacillus coagulans AHU 1366 membranes by treatment with 0.1% Triton X-100 and partially purified by means of column chromatography on Sephacryl S-300 and DEAE-Sephacel. The final preparation was virtually free from other enzymes involved in the de novo synthesis of teichoic acid. The enzyme had a pH optimum of 6.6-8.0 and a Km value for UDP-glucose of 21 microM. The enzyme required 40 mM MgCl2, 0.6 M KCl, and 0.1% Nonidet P-40 for full activity.     

Structural studies on N-acetylmannosaminuronic acid-containing and glucuronic acid-containing teichuronic acids in the cell wall of Bacillus megaterium AHU 1375

Structural studies were carried out on two kinds of teichuronic acid-glycopeptide complexes (designated as TU-GP-I and TU-GP-II) isolated from lysozyme digest of N-acetylated cell walls of Bacillus megaterium AHU 1375 by ion-exchange chromatography and gel chromatography. TU-GP-I, accounting for about 25% of the cell walls, contained N-acetylmannosaminuronic acid, N-acetylglucosamine, glucose, galactose, glycerol, and phosphorus in an approximate molar ratio of 1:1:2:1:0.5:0.5, together with small amounts of glycopeptide components. TU-GP-II, accounting for about 9% of the cell walls, contained glucuronic acid, glucose, and fucose in a molar ratio of about 2:1.5:1, together with small amounts of glycopeptide components. The results of analyses involving Smith degradation, chromium oxidation, methylation, acetolysis, and H-NMR measurement led to the conclusion that the polysaccharide chain of TU-GP-I comprised repeating units,----6) Glc(alpha 1----3)-ManNAcUA(beta 1----4)[Gal(alpha 1----3)][Glc(beta 1----6)]GlcNAc(beta 1----. About half of the repeating units were substituted by glycerophosphoryl residues at C-6 of the beta-glucosyl residues linked to the N-acetylglucosamine residues. By means of a similar procedure, the polysaccharide chain of TU-GP-II was shown to comprise repeating units,----4)GlcUA(alpha 1----3)GlcUA(alpha 1----3)Glc(alpha 1----3)Fuc(alpha 1----, of which about half were substituted by alpha-glucosyl residues at C-3 of the 4-substituted glucuronosyl residues.     

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.     

Function of alpha-D-glucosyl monophosphorylpolyprenol in biosynthesis of cell wall teichoic acids in Bacillus coagulans.

D-[alpha-14C]]glucosyl phosphorylpolyprenol ([ 14C]Glc-P-prenol) was formed from UDP-D-[14C]glucose in each of the membrane systems obtained from Bacillus coagulans AHU 1631 and AHU 1634 and two Bacillus megaterium strains. Membranes of these B. coagulans strains, which possess beta-D-glucosyl branches on the repeating units in their major cell wall teichoic acids, were shown to catalyze the transfer of the glucose residue from [14C]Glc-P-prenol to endogenous polymer. On the other hand, membranes of B. coagulans AHU 1366, which has no glucose substituents in the cell wall teichoic acid, exhibited neither [14C]Glc-P-prenol synthetase activity nor the activity of transferring glucose from [14C]Glc-P-prenol to endogenous acceptor. The enzyme which catalyzes the polymer glycosylation in the former two B. coagulans strains was most active at pH 5.5 and in the presence of the Mg2+ ion. The apparent Km for [14C]Glc-P-prenol was 0.6 microM. Hydrogen fluoride hydrolysis of the [14C]glucose-linked polymer product yielded a major fragment identical to D-galactosyl-alpha(1----2)(D-glucosyl-beta(1----1/3)) glycerol, the dephosphorylated repeating unit in the major cell wall teichoic acids of these B. coagulans strains. This result, together with the behavior of the radioactive polymer in chromatography on Sepharose CL-6B, DEAE-Sephacel, and Octyl-Sepharose CL-4B, led to the conclusion that [14C]Glc-P-prenol serves as an intermediate in the formation of beta-D-glucosyl branches on the polymer chains of cell wall teichoic acids in B. coagulans.     

Synthesis of teichoic acid by Bacillus subtilis protoplasts

Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane. During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane. No evidence could be obtained for a transphosphorylation mechanism for the translocation process. It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle.     

Possible inhibitory effect of teichoic acid on Bacillus subtilis transfer ribonucleic acid

1. tRNA of Bacillus subtilis was found to be variably contaminated with membrane teichoic acid. 2. Samples with high contents of teichoic acid showed no accepting activity for tRNA(Phe) and tRNA(Tyr). 3. Removal of teichoic acid restored accepting activity and fractions containing teichoic acid, separated on Sephadex G-150, inhibited the charging of tRNA(Tyr). 4. The presence of teichoic acid did not inhibit the charging of tRNA(His).     

The primary structure of teichuronic acid in Bacillus subtilis AHU 1031

Structural studies were carried out on the acidic polysaccharide fraction obtained from lysozyme digest of the cell walls of Bacillus subtilis AHU 1031. The polysaccharide fraction contained N- acetylmannosaminuronic acid ( ManNAcA ), N-acetylglucosamine (GlcNAc), glucose, glycerol and phosphorus in a molar ratio of 2:2:4:1:1, together with glycopeptide components. The results of analyses involving Smith degradation, chromium trioxide oxidation, methylation and proton magnetic resonance spectroscopy led to the conclusion that the backbone chain of the polysaccharide has the repeating unit----6)Glc(alpha 1----3/4) ManNAcA (beta 1----4)GlcNAc(beta 1----. About 50% of the N-acetylglucosamine residues in the backbone chain seem to be substituted at C-3 by the glycosidic branches, glycerol phospho-6-glucose, while the other half seem to be substituted by glucose.     

A common linkage saccharide unit between teichoic acids and peptidoglycan in cell walls of Bacillus coagulans

Teichoic acid-glycopeptide complexes were isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1631, AHU 1634, and AHU 1638, and the structure of the teichoic acid moieties and their linkage regions was studied. On treatment with hydrogen fluoride, each of the complexes gave a hexosamine-containing disaccharide, which was identified to be glucosyl(beta 1----4)N-acetylglucosamine, in addition to dephosphorylated repeating units of the teichoic acids, namely, galactosyl(alpha 1----2)glycerol and either galactosyl(alpha 1----2)[glucosyl(alpha 1----1/3)]glycerol (AHU 1638) or galactosyl(alpha 1----2)[glucosyl(beta 1----1/3)]glycerol (AHU 1631 and AHU 1634). From the results of Smith degradation, methylation analysis, and partial acid hydrolysis, the teichoic acids from these strains seem to have the same backbone chains composed of galactosyl(alpha 1----2)glycerol phosphate units joined by phosphodiester bonds at C-6 of the galactose residues. The presence of the disaccharide, glucosyl(beta 1----4)N-acetylglucosamine, in the linkage regions between teichoic acids and peptidoglycan was confirmed by the isolation of a disaccharide-linked glycopeptide fragment from each complex after treatment with mild alkali and of a teichoic acid-linked saccharide from each cell wall preparation after treatment with mild acid. Thus, it is concluded that despite structural differences in the glycosidic branches, the teichoic acids in the cell walls of the three strains are linked to peptidoglycan through a common linkage saccharide, glucosyl (beta 1----4) N-acetylglucosamine.     

Structural and biosynthetic studies on linkage region between poly(galactosylglycerol phosphate) and peptidoglycan in Bacillus coagulans

The HF treatment of teichoic acid-glycopeptide complexes isolated from lysozyme digests of Bacillus coagulans AHU 1366 cell walls gave a disaccharide, glucosyl beta (1 leads to 4)N-acetylglucosamine, along with dephosphorylated repeating units of the teichoic acid chain, galactosyl alpha (1 leads to 2) glycerol. Mild alkali treatment of the complexes yielded the disaccharide linked to glycopeptide, whereas direct heating of the cell walls at pH 2.5 yielded the same disaccharide linked to teichoic acid. The Smith degradation of the complexes revealed that the galactose residue is a component of backbone chain. Thus it is concluded that this disaccharide is involved in the linkage region between poly(galactosylglycerol phosphate) and peptidoglycan in cell walls. Membrane-catalyzed synthesis of this disaccharide on a lipid followed by transfer of glycerol phosphate from CDP-glycerol to the disaccharide-linked lipid in the absence or in the presence of UDP-galactose also supports this conclusion.     

Control of teichoic acid synthesis in Bacillus licheniformis ATCC 9945

Analysis of cell walls of Bacillus licheniformis ATCC 9945 grown under phosphate limitation showed that teichoic acid could be replaced by teichuronic acid under these conditions. Teichuronic acid, however, was always present in the walls to some extent irrespective of the growth conditions. The enzymes involved in teichoic acid synthesis were investigated and the synthesis of these was shown to be repressed when the intracellular Pi level fell. CDP-glycerol pyrophosphorylase was studied in some detail and evidence is presented to show that the enzyme is inactivated under phosphate-limited conditions. The mechanism of inactivation is unknown but it has been shown that it does not require protein synthesis de novo.     

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.     

Organization of teichoic acid in the cell wall of Bacillus subtilis.

The phytohemagglutinin, concanavalin A (Con A), interacts specifically and reversibly with the polyglucosyl glycerol phosphate teichoic acid of Bacillus subtilis 168 cell walls. Advantage has been taken of this interaction to examine the organization of the surface teichoic acid at the ultrastructural level. Con A-treated whole cells and cell walls contain an irregular, fluffy layer 25 to 60 nm thick which is absent in untreated or alpha-methyl glucoside-treated preparations. This discontinuous layer is present only on the outer profile of Con-A-treated cell walls. The surface teichoic acid is proposed to be oriented perpendicular to the long axis of the cell. Fixation and embedment for electron microscopy result in condensation of this layer which then contributes to the stainable portion of the wall. Con A treatment binds adjacent teichoic acid molecules in their native configuration producing the irregular, fluffy layer visualized.