Structure of anionic carbohydrate-containing cell wall polymers in several representatives of the order actinomycetales

A new teichoic acid was identified in the cell walls of Streptomyces griseoviridis VKM Ac-622T, Streptomyces sp. VKM Ac-2091, and Actinoplanes campanulata VKM Ac-1319T. The polymer is poly(glycosylglycerol phosphate). The repeating units of the polymer, alpha-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-galactopyran+ ++ osyl-(1-->1)-glycerols, are in phosphodiester linkage at C-3 of glycerol and C-6 of galactose. The structures of cell wall teichoic acids in the strains Streptomyces chryseus VKM Ac-200T and "Streptomyces subflavus" VKM Ac-484 similar in morphology and growth characteristics are also identical: 1,5-poly(ribitol phosphate) substituted at C-4(2) by 2-acetamido-2-deoxy-beta-glucopyranosyl residues and 1,3-poly(glycerol phosphate). The taxonomic aspects of these results are discussed.     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23

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.     

Carbohydrate-containing polymers of the cell wall of the thermophilic streptomycete Streptomyces thermoviolaceus subsp. thermoviolaceus VKM Ac-1857T

Anionic polymers of the cell surface of a thermophilic streptomycete were investigated. The cell wall of Streptomyces thermoviolaceus subsp. thermoviolaceus VKM Ac-1857(T) was found to contain polymers with different structure: teichoic acid--1,3-poly(glycerol phosphate), disaccharide-1-phosphate polymer with repeating unit -6)-alpha-Galp-(1-->6)-alpha-GlcpNAc-P-, and polysaccharide without phosphate with repeating unit -->6)-alpha-GalpNAc-(1-->3)-beta-GalpNAc-(1-->. Disaccharide-1-phosphate and polysaccharide without phosphate have not been described earlier in prokaryotic cell walls.     

The cell wall teichoic acids of streptomycetes from the "Streptomyces cyaneus" cluster

The cell wall anionic polymers of the 13 species of the "Streptomyces cyaneus" cluster have a similar structure and contain beta-glucosylated 1,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate). In the degree of glucosylation of the ribitol phosphate units of their teichoic acids, the cluster members can be divided into two groups. The streptomycetes of the first group (S. afghaniensis, S. janthinus, S. purpurascens, S. roseoviolaceus, and S. violatus) are characterized by a very similar structure of their cell walls, completely glucosylated 1,5-poly(ribitol phosphate) chains, and a high degree of DNA homology (67-88%). The cell wall teichoic acids of the second group (S. azureus, S. bellus, S. caelestis, S. coeruleorubidus, S. curacoi, and S. violarus) differ in the degree of beta-glucosylation of their 1,5-poly(ribitol phosphate) chains and have a lower level of DNA homology (54-76%). Two streptomycetes of the cluster (S. cyaneus and S. hawaiiensis) are genetically distant from the other cluster members but have the same composition and structure of the cell wall teichoic acids as the second-group streptomycetes. The data obtained confirm the genetic relatedness of the "S. cyaneus" cluster members and suggest that the structure of the cell wall teichoic acids may serve as one of the taxonomic criteria of the species-level status of streptomycetes.     

Teichoic acids in the cell walls of Microbispora mesophila Ac-1953t and Thermobifida fusca Ac-1952t

The cell walls of Microbispora mesophila strain Ac-1953T (the family Streptosporangiaceae) and Thermobifida fusca Ac-1952T (the family Nocardiopsiceae) were found to contain teichoic acids of a poly(glycerol phosphate) nature. The teichoic acid of M. mesophila (formerly Thermomonospora mesophila) represents a poly(glycerol phosphate) containing 5% of substituent 2-acetamido-2-deoxy-alpha-galactosaminyl residues. The teichoic acid of such kind was found in actinomycetes for the first time. The cell wall of T. fusca (formerly Thermonospora fusca) contains two teichoic acids, namely, unsubstituted 1,3-poly(glycerol phosphate) and beta-glucosylated 1,3-poly(glycerol phosphate).     

Studies on the linkage between teichoic acid and peptidoglycan in a bacteriophage-resistant mutant of Staphylococcus aureus H.

1. In addition to poly(ribitol phosphate) the walls of a bacteriophage-resistant mutant of Staphylococcus aureus H contain glycerol phosphate residues that are not removed on digestion with trypsin or extraction with phenol. 2. The glycerol phosphate is present in a chain, containing three or four glycerol phosphate residues, which is covalently attached to the peptidoglycan through a phosphodiester linkage to muramic acid; this linkage is readily hydrolysed by dilute alkali. 3. The degradative studies described suggest that the poly(ribitol phosphate) chains of the wall teichoic acid may be attached to the wall by linkage to this glycerol phosphate oligomer.     

Structure of a new ribitol teichoic acid-like O-polysaccharide of a serologically separate Proteus vulgaris strain, TG 276-1, classified into a new Proteus serogroup O53

An unusual ribitol teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide from a previously non-classified Proteus vulgaris strain TG 276-1. Structural studies using chemical analyses and 2D (1)H and (13)C NMR spectroscopy showed that the polysaccharide is a zwitterionic polymer with a repeating unit containing 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and two D-ribitol phosphate (D-Rib-ol-5-P) residues and having the following structure:[formula: see text] where the non-glycosylated ribitol residue is randomly mono-O-acetylated. Based on the unique O-polysaccharide structure and the finding that the strain studied is serologically separate among Proteus bacteria, we propose to classify P. vulgaris strain TG 276-1 into a new Proteus serogroup, O53.     

Teichoic acids from the cell wall of Nocardiopsis prasina BKM Ac-1880T

The cell wall of Nocardiopsis prasina VKM Ac-1880T was found to contain two structurally different teichoic acids: unsubstituted 3,5-poly(ribitol phosphate) and 1,3-poly(glycerol phosphate), substituted at position 2 by 10% with alpha-N-acetylglucosamine and by 5% with O-acetyl groups. The structure of the polymers was studied by chemical analysis and NMR spectroscopy. The results obtained correlate well with 16S rRNA sequence data and confirm the species-specificity of teichoic acids in the genus Nocardiopsis.     

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.     

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.     

Influence of alanyl ester residues on the binding of magnesium ions to teichoic acids.

The binding of Mg2+ to the ribitol teichoic acid of Staphylococcus aureus H walls was examined by equilibrium dialysis in solution and in the intact wall; the influence of alanyl ester groups on binding was determined. In solution the ribitol polymer had a lower affinity than did a glycerol teichoic acid and bound Mg2+ in the ratio Mg2+/P of 1:1. The presence of alanyl ester residues caused a decrease in the amount of cations bound in stoicheiometric proportion to the ratio Ala/P, but the affinity constant was unaltered. It is concluded that in solution the ribitol teichoic acid binds Mg2+ univalently to phosphate groups and univalently to a counter-ion. In the intact wall the binding of Mg2+ was different. The affinity constant was higher and resembled that of a glycerol teichoic acid. It is concluded that Mg2+ forms bridges across phosphate groups in teichoic acid chains lying adjacent to each other in the wall. The effect of alanyl esters was similar to that in solution, but Scatchard plots were not linear at low concentrations of Mg2+ where it was shown that the difference in affinities between walls with and without alanyl ester residues was much greater than it was at higher concentrations of Mg2+. Thus at very low concentrations of Mg2+ effective binding to the wall is markedly improved by loss of alanyl ester residues.     

Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139

The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a 4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, obtained by condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide and 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, was converted to 8-azido-3,6-dioxaoctyl 3-O-benzyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-6-O-benzyl-2-deoxy-beta-D-glucopyranoside (6) by reductive opening of the acetal, followed by deacetylation and selective benzylation. Phosphorylation of 6 furnished two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. Glycosylation of the (S)-phosphate with 2,4-di-O-benzyl-3,6-dideoxy-alpha-L-xylo-hexopyranosyl bromide under halide-assisted conditions gave the desired tetrasaccharide, together with a trisaccharide. Global deprotection and reduction of the azide to an amine was effected by catalytic hydrogenation/hydrogenolysis to give the deprotected tetrasaccharide, which is functionalized for conjugation.     

Structure of the repeating unit of the O-specific polysaccharide of the lipopolysaccharide of Yersinia kristensenii strain 490 (O:12,25).

The O-specific polysaccharide isolated by mild acid degradation of the lipopolysaccharide of Y. kristensenii strain 490 (O:12,25) contained D-glucose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2,6-dideoxy-L-galactose, glycerol, and phosphate in the ratios 2:2:1:1:1:1. On the basis of 31P- and 13C-n.m.r. data, methylation analysis, dephosphorylation, solvolysis with anhydrous hydrogen fluoride, and Smith degradation, it was concluded that the repeating unit of the polysaccharide was a branched hexaosylglycerol phosphate with the following structure. [formula: see text]     

Structural characterization of the O-chain polysaccharide of Aeromonas caviae ATCC 15468 lipopolysaccharide

The O-chain polysaccharide produced by a mild acid degradation of Aeromonas caviae ATCC 15468 lipopolysaccharide was found to be composed of L-rhamnose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and phosphoglycerol. Subsequent methylation and CE-ESIMS analyses and 1D/2D NMR ((1)H, (13)C and (31)P) spectroscopy showed that the O-chain polysaccharide is a high-molecular-mass acidic branched polymer of tetrasaccharide repeating units with a phosphoglycerol substituent having the following structure: [structure: see text] where Gro represents glycerol and P represents a phosphate group.     

A novel type of teichoic acid from the cell wall of Bacillus subtilis VKM B-762

The cell wall of Bacillus subtilis VKM B-762 contains, along with 1,5-poly[4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)ribitol phosphate], a novel type of glycopolymer involving three types of inter-monomeric bonds in the repeating unit, viz., amide, glycosidic and phosphodiester:Such a structural pattern of natural glycopolymers has been hitherto unknown. This polymer represents a novel type of teichoic acids.     

Structural analysis of the specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72)

The specific capsular polysaccharide of Streptococcus pneumoniae type 45 (American type 72) was found to be a high molecular weight polymer composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-fucose, L-rhamnose, glycerol, and phosphate (2:1:1:1:1:1:1). Partial hydrolysis, dephosphorylation, methylation analysis, periodate oxidation studies, and one- and two-dimensional 1H and 13C high-field nuclear magnetic resonance experiments showed the polysaccharide to be a branched polymer of a 1-phosphoglycerol-substituted hexasaccharide repeating unit having the structure: (formula; see text).     

Cell wall teichoic acids ofNocardiopsis prasina VKM Ac-1880T

The cell wall ofNocardiopsis prasina VKM Ac-1880^T was found to contain two structurally different teichoic acids: unsubstituted 3,5-poly(ribitol phosphate) and l,3-poly(glycerol phosphate) substituted at position 2 by 10% with α-N-acetylglucosamine and by 5% withO-acetyl groups. The structure of the polymers was studied by chemical analysis and NMR spectroscopy. The results obtained correlate wellwith 16S rRNA sequence data and confirm the species-specificity of teichoic acids in the genusNocardiopsis.     

Teichoic Acids in the Cell Walls of Microbispora mesophila Ac-1953T and Thermobifida fusca Ac-1952T

The cell walls of Microbispora mesophila strain Ac-1953^T (the family Streptosporangiaceae) and Thermobifida fusca Ac-1952^T (the family Nocardiopsaceae) were found to contain teichoic acids of a poly(glycerol phosphate) nature. The teichoic acid of M. mesophila (formerly Thermomonospora mesophila) represents a 1,3-poly(glycerol phosphate) containing 5% of substituent 2-acetamido-2-deoxy--D-galactosaminyl residues. Teichoic acid of such a kind was found in actinomycetes for the first time. The cell wall of T. fusca (formerly Thermonospora fusca) contains two teichoic acids, namely, unsubstituted 1,3-poly(glycerol phosphate) and -glucosylated 1,3-poly(glycerol phosphate).     

Structure of the glycerol phosphate-containing O-polysaccharides and serological studies of the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 classified into a new Proteus serogroup, O54

O-Polysaccharides were obtained from the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 and studied by chemical analyses and one- and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy, including rotating-frame nuclear Overhauser effect spectroscopy, H-detected (1)H,(13)C heteronuclear single-quantum spectroscopy and (1)H,(31)P heteronuclear multiple-quantum spectroscopy experiments. The Proteus mirabilis OE polysaccharide was found to have a trisaccharide repeating unit with a lateral glycerol phosphate group. The Proteus vulgaris TG 103 produces a similar O-polysaccharide, which differs in incomplete substitution with glycerol phosphate (c. 50% of the stoichiometric amount) and the presence of an O-acetyl group at position 6 of the 2-acetamido-2-deoxygalactose (GalNAc) residue. These structures are unique among the known bacterial polysaccharide structures. Based on the structural and serological data of the lipopolysaccharides, it is proposed to classify both strains studied into a new Proteus serogroup, O54, as two subgroups, O54a,54b and O54a,54c. The serological relatedness of the Proteus O54 and some other Proteus lipopolysaccharides is discussed.