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.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.     

N-acetylmannosaminyl(1----4)N-acetylglucosamine, a linkage unit between glycerol teichoic acid and peptidoglycan in cell walls of several Bacillus strains.

The structure of teichoic acid-glycopeptide complexes isolated from lysozyme digests of cell walls of Bacillus subtilis (four strains) and Bacillus licheniformis (one strain) was studied to obtain information on the structural relationship between glycerol teichoic acids and their linkage saccharides. Each preparation of the complexes contained equimolar amounts of muramic acid 6-phosphate and mannosamine in addition to glycopeptide components and glycerol teichoic acid components characteristic of the strain. Upon treatment with 47% hydrogen fluoride, these preparations gave, in common, a hexosamine-containing disaccharide, which was identified as N- acetylmannosaminyl (1----4) N-acetylglucosamine, along with large amounts of glycosylglycerols presumed to be the dephosphorylated repeating units of teichoic acid chains. The glycosylglycerol obtained from each bacterial strain was identified as follows: B. subtilis AHU 1392, glucosyl alpha (1----2)glycerol; B. subtilis AHU 1235, glucosyl beta(1----2) glycerol; B. subtilis AHU 1035 and AHU 1037, glucosyl alpha (1----6)galactosyl alpha (1----1 or 3)glycerol; B. licheniformis AHU 1371, galactosyl alpha (1----2)glycerol. By means of Smith degradation, the galactose residues in the teichoic acid-glycopeptide complexes from B. subtilis AHU 1035 and AHU 1037 and B. licheniformis AHU 1371 were shown to be involved in the backbone chains of the teichoic acid moieties. Thus, the glycerol teichoic acids in the cell walls of five bacterial strains seem to be joined to peptidoglycan through a common linkage disaccharide, N- acetylmannosaminyl (1----4)N-acetylglucosamine, irrespective of the structural diversity in the glycosidic branches and backbone chains.     

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.     

Biosynthesis of the wall acidic polysaccharide in Bacillus cereus AHU 1356

Biosynthetic studies on an acidic polysaccharide, comprising galactose, rhamnose, N-acetylglucosamine and sn-glycerol 1-phosphate, were carried out with a membrane system obtained from Bacillus cereus AHU 1356. Incubation of the membranes with UDP-[14C]Gal, TDP-[14C]Rha and UDP-[14C]GlcNAc resulted in the formation of four or more labeled-sugar-linked lipids and a labeled polysaccharide. Data on structural analysis of the sugar moieties released from the glycolipids, together with results of enzymatic conversion of [14C]galactose-linked lipid and [14C]Rha-Gal-linked lipid to higher-oligosaccharide-linked lipids and polysaccharide, led to the conclusion that the acidic polysaccharide is probably synthesized through the following pathway: (sequence in text) The glycerophosphate residues seem to be derived from phosphatidylglycerol.     

Antigenic polysaccharides of bacteria. 27. Structure of the O-specific polysaccharide chain of lipopolysaccharides from Pseudomonas syringae pv. atrofaciens 2399, phaesolica 120a and Pseudomonas holci 8299 belonging to serotype VI

Lipopolysaccharides from Pseudomonas syringae pvs atrofaciens 2399. phaseolicola 120a and Pseudomonas holci 8299, belonging to serogroup VI. possess an identical polysaccharide chain composed of D-rhamnose and D-fucose. On the hasis of methylation, partial acid hydrolysis, 1H- and 13C-NMR data, it was concluded that the backbone of the polysaccharide represents D-rhamnan built up of tetrasaccharide repeating units and alpha-D-fucofuranose residues are attached to the backbone as the monosaccharide branches. The following structure of the repeating unit is established: (Formula: see text).     

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.     

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.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

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.     

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.     

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.     

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.     

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.     

Structural analysis of the O-antigen polysaccharide from the Shiga toxin-producing Escherichia coli O172.

The structure of the O-antigen polysaccharide from Escherichia coli O172 has been determined. In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by mass spectrometry and two-dimensional NMR techniques. An O-acetyl group was present as 0.7 equivalent per repeating unit. Treatment of the O-deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized. Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O-polysaccharide from E. coli O172 as:-->P-4)-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D- GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp6Ac-(1-->     

A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model

Lipoteichoic acid (LTA) is an essential bacterial membrane polysaccharide (cell wall component) that is attached to the membrane via a lipid anchor. According to the currently accepted structure of pneumococcal LTA, the polysaccharide is comprised of several repeating units, each of which starts with glucose and ends with ribitol, with the lipid anchor predicted to be Glc(beta1-->3)AATGal(beta1-->3)Glc(alpha1-->3)-acyl(2)Gro, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose. However, this lipid anchor has not been detected in pneumococcal membranes. Furthermore, the currently accepted structure does not explain the Forssman antigen properties of LTA and predicts a molecular weight for LTA that is larger than its actual observed molecular weight. To resolve these problems, we used mass spectrometry to analyze the structure of LTA isolated from several pneumococcal strains. Our study found that the R36A pneumococcal strain produces LTA that is more representative of pneumococci than that previously characterized from the R6 strain. Analysis of LTA fragments obtained after hydrofluoric acid and nitrous treatments showed that the fragments were consistent with an LTA nonreducing terminus consisting of GalNAc(alpha1-->3)GalNAc(beta1-->, which is the minimal structure for the Forssman antigen. Based on these data, we propose a revised model of LTA structure: its polysaccharide repeating unit begins with GalNAc and ends with AATGal, and its lipid anchor is Glc(alpha1-->3)-acyl(2)Gro, a common lipid anchor found in pneumococcal membranes. This new model accurately predicts the observed molecular weights. The revised model should facilitate investigation of the relationship between LTA's structure and its function.     

Structural studies on the minor teichoic acid of Bacillus coagulans AHU 1631

The minor teichoic acid linked to glycopeptide was isolated from lysozyme digests of Bacillus coagulans AHU 1631 cell walls, and the structure of the teichoic acid moiety and its junction with the peptidoglycan were studied. Hydrolysis of the teichoic-acid--glycopeptide complex with hydrogen fluoride gave a nonreducing oligosaccharide composed of glucose, galactose and glycerol in a molar ratio of 3:1:1 which was presumed to be dephosphorylated repeating units of the polymer chain. From the results of structural analysis involving NaIO4 oxidation, methylation and acetolysis, the above fragment was characterized as glucosyl(beta 1----3)glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol. In addition, the Smith degradation of the complex yielded a phosphorus-containing fragment identified as glycerol-P-6-glucosyl(beta 1----1/3)glycerol. These results led to the most likely structure for the repeating units of the teichoic acid, -6[glucosyl(beta 1----3)]glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol-P-. The minor teichoic acid, just like the major teichoic acid bound to the linkage unit, was released by heating the cell walls at pH 2.5. The mild alkaline hydrolysis of the minor teichoic acid after reduction with NaB3H4 gave labeled saccharides characterized as glucosyl(beta 1----6)galactitol and glucosyl(beta 1----3)glucosyl(beta 1----6)galactitol, together with a large amount of the unlabeled repeating units of the teichoic acid chain. Thus, the minor teichoic acid chain is believed to be directly linked to peptidoglycan at the galactose residue of the terminal repeating unit without a special linkage sugar unit.     

Identification of a second lipopolysaccharide in Porphyromonas gingivalis W50

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.     

Elongation of teichuronic acid chains by a wall-membrane preparation from Micrococcus luteus.

A wall-plus-membrane preparation from Micrococcus luteus catalyzes the incorporation of [14C]glucose from UDP-[14C]glucose, into two fractions of teichuronic acid, which is the cell wall polysaccharide consisting of alternating residues of glucose and N-acetylmannosaminuronic acid (ManNAcUA). Membrane-associated teichuronic acid was extracted from the wall-membrane fraction of reaction mixtures by sodium dodecyl sulfate. The synthesis of membrane-associated teichuronic acid required UDP-glucose, UDP-ManNAcUA, and UDP-N-acetylglucosamine and was inhibited by tunicamycin. Glucose incorporated into wall-bound teichuronic acid remained in wall fragments after extraction with sodium dodecyl sulfate, and its incorporation required UDP-glucose and UDP-ManNAcUA (but not UDP-N-acetylglucosamine) and was insensitive to tunicamycin. Radioactive material incorporated into wall-bound teichuronic acid could be released by treatment with mild acid or by digestion with lysozyme, indicating that the wall-bound teichuronic acid was covalently linked to peptidoglycan. There were about 600 pmol of wall-bound teichuronic acid acceptor sites for glucose per mg of protein as measured in incorporation reaction mixtures lacking UDP-ManNAcUA. In the presence of both UDP-glucose and UDP-ManNAcUA, elongation of teichuronic acid acceptor sites occurred, with the addition of six to eight disaccharide units to each acceptor site.     

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.