S. Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S. Sawada, T. Kawamura, Y. Masuho, and K. Tomibe, J. Infec. Dis. 152:1290-1299, 1985). In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P. aeruginosa IID 1008 (S. Yokota, S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito, Eur. J. Biochem. 167:203-209, 1987). Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P. aeruginosa strains reactive against MAb E87. The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P. aeruginosa isolates.

The distribution and substrate specificities of enzymes involved in the formation of linkage units which contain N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) or glucose and join teichoic acid chains to peptidoglycan were studied among membrane systems obtained from the following two groups of gram-positive bacteria: group A, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, and Lactobacillus plantarum; group B, Bacillus coagulans. All the membrane preparations tested catalyzed the synthesis of N-acetylglucosaminyl pyrophosphorylpolyprenol (GlcNAc-PP-polyprenol). The enzymes transferring glycosyl residues to GlcNAc-PP-polyprenol were specific to either UDP-ManNAc (group A strains) or UDP-glucose (group B strains). In the synthesis of the disaccharide-bound lipids, GlcNAc-PP-dolichol could substitute for GlcNAc-PP-undecaprenol. ManNAc-GlcNAc-PP-undecaprenol, ManNAc-GlcNAc-PP-dolichol, Glc-GlcNAc-PP-undecaprenol, Glc-GlcNAc-PP-dolichol, and GlcNAc-GlcNAc-PP-undecaprenol were more or less efficiently converted to glycerol phosphate-containing lipid intermediates and polymers in the membrane systems of B. subtilis W23 and B. coagulans AHU 1366. However, GlcNAc-GlcNAc-PP-dolichol could not serve as an intermediate in either of these membrane systems. Further studies on the exchangeability of ManNAc-GlcNAc-PP-undecaprenol and Glc-GlcNAc-PP-undecaprenol revealed that in the membrane systems of S. aureus strains and other B. coagulans strains both disaccharide-inked lipids served almost equally as intermediates in the synthesis of polymers. In the membrane systems of other B. subtilis strains as well as B. licheniformis and B. pumilus strains, however, the replacement of ManNAc-GlcNAc-PP-undecaprenol by Glc-GlcNAc-PP-undecaprenol led to a great accumulation of (glycerol phosphate)-Glc-GlcNAc-PP-undecaprenol accompanied by a decrease in the formation of polymers.

A neutral and an acidic polysaccharide with molecular masses of about 22 kDa and 45 kDa, respectively, were isolated from the N-acetylated cell walls of Bacillus subtilis AHU 1219 by heating at pH 2.5, followed by separation of the water-soluble product by ion-exchange chromatography and gel chromatography. The neutral polysaccharide, accounting for 40% of the mass of the cell walls, contained glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylmannosamine in a molar ratio of 1:2:1:1. The minor, acidic polysaccharide contained glucuronic acid, glucose, galactose, L-serine and L-threonine in an approximate molar ratio of 1:1:1:0.5:0.5. Lysozyme digestion of the N-acetylated cell walls gave a polymer containing the neutral polysaccharide and glycopeptide components and another polymer which contained the acidic polysaccharide components together with small proportions of the neutral polysaccharide and glycopeptide components. Thus, the neutral and acidic polysaccharide chains seem to be attached to peptidoglycan through acid-labile linkages in the cell walls of this strain. Structural analysis of the neutral-polysaccharide preparation, involving 1H-NMR and 13C-NMR measurement, methylation and Smith degradation, led to the most likely structure, ----6)[Glc(beta 1----3)]GalNAc(alpha 1----4)-[GlcNAc(beta 1----3)]ManNAc(beta 1----4)GlcNAc(beta 1----, for the repeating units of this polysaccharide.

Structural studies were carried out on the O-polysaccharide fraction obtained from the lipopolysaccharide of Pseudomonas aeruginosa IID 1012, the standard strain of Homma serogroup K, by mild acid treatment. The O-polysaccharide was composed of L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-D-galactosaminuronic acid. The results from analysis of fragments obtained by acid hydrolysis and Smith degradation of the O-polysaccharide, together with data on methylation analysis and nuclear magnetic resonance spectroscopic measurement of the polysaccharide, led to the most likely structure of the repeating units of the polymer chain, ----4)D-GalNAcA(alpha 1----3)D-QuiNAc(beta 1----2)L-Rha(alpha 1----3)L-Rha(alpha 1----, in which about 20% of the N-acetylgalactosaminuronic acid residues were in an amide form and about 75% of the same residues were O-acetylated at C-3.

Structural studies were carried out on the O-polysaccharide fraction obtained by mild acid treatment of the lipopolysaccharide from Pseudomonas aeruginosa IID 1009 (ATCC 27585). The O-polysaccharide was composed of L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-L-galactosaminuronic acid in a molar ratio of 1:1:1. The results from analysis of fragments obtained by hydrogen fluoride hydrolysis of O-polysaccharide, together with data on methylation analysis and nuclear magnetic resonance spectroscopic analysis, led to the most likely structure of the repeating units of the polymer chain ----4)L-GalNAcA(alpha 1----3)D-QuiNAc(alpha 1----3)L-Rha(alpha 1----, in which about 70% of the rhamnose residues were O-acetylated at C-2. This structure coincides with that of the repeating unit of Lanyi 02 a,b polysaccharides.

Article de synthese sur la structure chimique de la jonction entre le peptidoglycane et les polyosides (acides teichoiques, acides teichuroniques et autres polyosides) dans la paroi des bacteries Gram positif

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.

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.

Incubation of UDP-[14C]galactose with membranes of Bacillus coagulans led to the formation of a radioactive glycolipid, which was tentatively characterized as beta-galactosyl phosphorylpolyprenol (Gal-P-prenol) on the basis of its chromatographic behavior and data from structural analysis of its sugar 1-phosphate moiety. The sugar moiety of [14C]Gal-P-prenol was shown to be incorporated into a membrane-bound polymer, which coincided with the diacyl form of lipoteichoic acid in its chromatographic behavior on columns of Sephacryl S-300, DEAE-Sephacel and octyl-Sepharose. Hydrogen fluoride hydrolysis of the polymer afforded an alpha-galactoside identical with Gal(alpha 1----2)Gro obtained from lipoteichoic acids. The incorporation of galactose residues from [14C]Gal-P-prenol into the polymer was greatly enhanced by exogenous lipoteichoic acids, especially of the diacyl and monoacyl forms. The optimal pH and metal concentration for the Gal-P-prenol formation, respectively, were found to be 8.4 and 10 mM (MgCl2), whereas those for the transfer of galactose from this lipid intermediate to polymer were 4.5 and 16 mM (CaCl2). The above results lead to the conclusion that Gal-P-prenol serves as the direct galactosyl donor in the synthesis of lipoteichoic acids in B. coagulans.

An enzyme which catalyzes the conversion of GlcNAc-PP-undecaprenol into ManNAc(beta 1----4)GlcNAc-PP-undecaprenol, a key lipid intermediate in the de novo synthesis of various teichoic acids, was partially purified from the 20,000 x g supernatant fraction of Bacillus subtilis AHU 1035 cell homogenate. By means of ammonium sulfate precipitation, gel chromatography, and ion-exchange chromatography, the enzyme was purified about 70-fold, giving a preparation virtually free from substances obstructive to measurement of the N-acetylmannosaminyltransferase reaction. The enzyme was shown to be specific to UDP-ManNAc. The Km value for UDP-ManNAc was 4.4 microM, and the optimum pH was 7.3. The enzyme required 10 mM MgCl2, 0.3 M KCl, 25% glycerol, and 0.1% Nonidet P-40 to function at full activity.

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.

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacillus polymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HC1 buffer, pH 2.5, at 100 degrees C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I-III, which contained different amounts of pyruvic acid (0,0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-II, PS-III and polysaccharide-linked glycopeptides by treatment with 10 mM HC1 at 100 degrees C for 1 h. Results of analyses of these polysaccharide preparations by 1H-NMR and 13C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, ----3)[4,6-O-(1-carboxyethylidene)]ManNAc(beta 1----4)GlcNac(beta 1----, for the acidic polysaccharide of this strain.

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.

The pathway for the de novo synthesis of a teichoic acid, poly(galactosylglycerol phosphate), in Bacillus coagulans AHU 1366 was studied by means of characterization and stepwise conversion of lipid-linked intermediates. Incubation of membranes with UDP-N-acetylglucosamine and UDP-glucose yielded a disaccharide-linked polyprenylpyrophosphate, whose sugar moiety was characterized as glucosyl(beta 1----4)N-acetylglucosamine (Glc-GlcNAc). By incubation with membranes and CDP-glycerol, Glc-GlcNAc-PP-prenol was converted to a series of glycolipids characterized as (Gro-P)1-6-Glc-GlcNAc-PP-prenol (Gro = glycerol). Glc-[14C]GlcNAc-PP-prenol was converted to polymer by incubation with membranes, CDP-glycerol and UDP-galactose. Smith degradation of the polymer gave two radioactive fragments corresponding to (Gro-P)3-Glc-GlcNAc and (Gro-P)4-Glc-GlcNAc. These results, together with data on gel chromatography of radioactive polymer synthesized from UDP-[3H]galactose, CDP-glycerol and Glc-[14C]GlcNAc-PP-prenol, led to the conclusion that in this strain poly(galactosylglycerol phosphate) is probably synthesized through the following pathway: GlcNAc-PP-prenol----Glc-GlcNAc-PP-prenol----(Gro-P)3-4 -Glc-GlcNAc-PP-prenol----(Gro-P-Gal)n- (Gro-P)3-4-Glc-GlcNAc-PP-prenol----(Gro-P-Gal)n- (Gro-P)3-4-Glc-GlcNAc-P-peptidoglycan complex.

A novel enzyme which specifically hydrolyses N‐acetylglucosaminyl pyrophosphorylpolyprenol to liberate N‐acetylglucosamine was found in membranes of Bacillus polymyxa AHU 1385. The enzyme seems to be inactive toward α‐N‐acetylglucosaminyl phosphorylundecaprenol, β‐N‐acetylglucosaminyl phosphorylundecaprenol, N‐acetylglucosamine 1‐phosphate, N‐acetylglucosamine 1‐pyrophosphate, or UDP‐N‐acetyl‐glucosamine. Much lower activities of the same enzyme were also found in membranes of several other strains of Bacilli.

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.

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.

The stepwise formation and characterization of linkage unit intermediates and their functions in ribitol teichoic acid biosynthesis were studied with membranes obtained from Staphylococcus aureus H and Bacillus subtilis W23. The formation of labeled polymer from CDP-[14C]ribitol and CDP-glycerol in each membrane system was markedly stimulated by the addition of N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine (ManNAc-GlcNAc) linked to pyrophosphorylyisoprenol. Whereas incubation of S. aureus membranes with CDP-glycerol and ManNAc-[14C]GlcNAc-PP-prenol led to synthesis of (glycerol phosphate) 1-3-ManNAc-[14C]GlcNAc-PP-prenol, incubation of B. subtilis membranes with the same substrates yielded (glycerol phosphate)1-2-ManNAc-[14C]GlcNAc-PP-prenol. In S. aureus membranes, (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol as well as (glycerol phosphate)3-ManNAc-[14C]GlcNAc-PP-prenol served as an acceptor for ribitol phosphate units, but (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol did not. In B. subtilis W23 membranes, (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol served as a better acceptor for ribitol phosphate units than (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol. In this membrane system (ribitol phosphate)-(glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol was formed from ManNAc-[14C]GlcNAc-PP-prenol, CDP-glycerol and CDP-ribitol. The results indicate that (glycerol phosphate)1-3-ManNAc-GlcNAc-PP-prenol and (glycerol phosphate)1-2-ManNac-GlcNAc-PP-prenol are involved in the pathway for the synthesis of wall ribitol teichoic acids in S. aureus H and B. subtilis W23 respectively.

Structural studies were carried out on the linkage unit which joins ribitol teichoic acid to peptidoglycan in the cell walls of Lactobacillus plantarum AHU 1413. The heating of the cell walls at pH 2.5 led to release of only 5% of ribitol teichoic acid components as water-soluble material. In contrast, the same treatment of the cell walls after N-acetylation led to release of about 80% of the teichoic acid moiety, giving a teichoic-acid-linked sugar preparation which contained about equimolar amounts of mannosamine, glucosamine and glycerol as minor components. The teichoic-acid-linked sugar was hydrolyzed by mild alkaline treatment into a disaccharide, N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine and ribitol teichoic acid linked to glycerol. The Smith degradation of the N-acetylated cell walls gave a characteristic fragment, 1,2-ethylenediol-phospho-glycerol-phospho-N-acetylmannosaminyl(beta 1----4) N-acetylglucosamine. Furthermore, when the intact cell walls were subjected to the NaNO2 treatment followed by NaBH4 reduction, the ribitol teichoic acid moiety was recovered for the most part in the water-soluble polymer fraction, from which a sugar, N-acetylmannosaminyl-2,5-anhydromannitol, was released by mild alkaline treatment. These results lead to the conclusion that the ribitol teichoic acid chain in the intact cell walls of this organism is linked to peptidoglycan through a unique linkage unit, glycerol-phospho-N-acetylmannosaminyl(beta 1----4)-glucosamine. The anomalous stability of the linkage between the teichoic acid moiety and peptidoglycan against acid hydrolysis seems to be accounted for by the involvement of the N-substituted glucosamine residue in the phosphodiester bridge that joins the two polymers.

Structural studies were carried out on the acidic polymer fraction isolated from lysozyme digests of the N-acetylated cell walls of Bacillus cereus AHU 1356. The acidic polymer fraction contained glucosamine, galactose, rhamnose, glycerol and phosphorus in a molar ratio of 1:1:2:1:1, together with small amounts of glycopeptide components and muramic acid 6-phosphate. The hydrogen fluoride treatment led to removal of glycerol and phosphorus from the polymer without loss of other components. Results of the NaIO4 oxidation, methylation and proton magnetic resonance spectroscopy of the native and dephosphorylated preparations, in combination with data of the analysis of oligosaccharides obtained from partial hydrolysis of polysaccharide, led to the most likely structure of the repeating units of the acidic polysaccharide chain, ----4)N-acetylglucosaminyl-(alpha 1----3)rhamnosyl(alpha 1----3)galactosyl(alpha 1----4)[sn-glycerol 1-phospho-2]rhamnosyl(alpha 1----.