Anionic carbohydrate-containing cell wall polymers of Streptomyces melanosporofaciens and related species

The structures of cell wall anionic carbohydrate-containing polymers in Streptomyces melanosporofaciens VKM Ac-1864T and phylogenetically close organisms-S. hygroscopicus subsp. hygroscopicus VKM Ac-831T, S. violaceusniger VKM Ac-583T, S. endus VKM Ac-1331, S. endus VKM Ac-129, and S. rutgersensis subsp. castelarensis VKM Ac-832T--have been comparatively studied by chemical and NMR spectroscopic methods. The natural polymer of a new, previously unknown structure, Kdn (3-deoxy-D-glycero-Dgalacto-non-2-ulopyranosonic acid) with beta-galactose residues at C-9, has been found in the cell walls of all the strains under study. The cell walls of all the studied organisms contain three teichoic acids (TA): a predominant TA (1,3-poly(glycerol phosphate) with N-acetylated alpha-glucosaminyl substitutes by C-2 of glycerol, and minor TAs, 1,3- and 2,3-poly(glycerol phosphate) polymers without substitution. Their chains have O-acetyl and O-lysyl groups. Microorganisms of the above-mentioned species differ in the number of alpha-glucosaminyl substitutes and in the degree of their acetylation in the predominant teichoic acid.     

NMR-based identification of cell wall anionic polymers of Spirilliplanes yamanashiensis VKM Ac-1993(T).

The cell wall of Spirilliplanes yamanashiensis VKM Ac-1993(T) contains four anionic polymers, viz., three teichoic acids and a sugar-1-phosphate polymer. The following are the structures of the teichoic acids: poly[-6-beta-D-glucopyranosyl-(1-->2)-glycerol phosphate] (PI), 1,3-poly(glycerol phosphate) bearing N-acetyl-alpha-D-glucosamine residues at O-2 (70%) (PII), and poly[-6-N-acetyl-alpha-D-glucosaminyl-(1-->2)-glycerol phosphate] (PIII). The repeating unit of the fourth polymer (PIV) has the structure of -6-alpha-D-GlcpNAc-(1-->6)-alpha-D-GlcpNAc-1-P- with a 3-O-methyl-alpha-D-mannopyranosyl residues at position 3 of some 6-phosphorylated N-acetylglucosamine residues (50%). Polymers PI, PIII and PIV have not hitherto been found in prokaryotic cell walls.     

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.     

A new neutral polymer from the cell wall of actinomycete Kineosporia aurantiaca VKM Ac-702T

The major cell wall polymer of Kineosporia aurantiaca VKM Ac-702T a representative of the suborder Frankineae, is a galactomannan with a repeating unit of the following structure: -->3)-beta-D-Galp-(1-->6)-beta-D-Manp-(1-->4)-beta-D-Manp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-beta-D-Manp-(1--> that has not been reported so far. This was established using chemical degradation methods and NMR spectroscopy. The polysaccharide identified in the present study provides the second example of neutral galactomannans in actinomycete cell walls. The cell wall of K. aurantiaca VKM Ac-702T also contains a minor teichoic acid, viz., 1,3-poly(glycerol phosphate) partially substituted with alpha-glucosamine residues, only part of which are N-acetylated.     

Teichoic acid from the cell wall of Actinomadura carminata--a producer of the antibiotic carminomycin

The cell walls of Actinomadura carminata, producing the antibiotic carminomycin, contain a poly(glycerol phosphate) teichoic acid. The polymer belongs to 1,3-type and consists of about 8 glycerol phosphate units, two of them have 2-acetamido-2-deoxy-alpha-D-galactopyranosyl substituent and one--3-O-methyl-beta-D-galactopyranosyl-(1----3)-2- acetamido-2-deoxy-alpha-D-galactopyranosyl residue at C2 of glycerol. The structure of the polymer was established by chemical analysis and 13C-NMR spectroscopy. The teichoic acid accounted for about 10% of the cell wall dry weight. 3-O-methylgalactose in the structure of the teichoic acid was found for the first time.     

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).     

NMR-based identification of cell wall galactomannan of Streptomyces sp. VKM Ac-2125

The major cell wall polymer of Streptomyces sp. VKM Ac-2125, the causative agent of potato scab, is galactomannan with the repeating unit of the following structure: [carbohydrate structure in text] The polysaccharide with such a structure is found in the bacterial cell wall for the first time. The cell wall also contains small amount of a teichoic acid of the poly(glycerol phosphate) type and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid.     

A new polymer of 8,9-di-O-glucosylated 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid from a cell wall of Brevibacterium casei AEI Ac-2114T

A polysaccharide containing the residues of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) was found in the cell wall of the Brevibacterium casei strain AEI Ac-2114T . The polymer structure was elucidated by analyzing one-dimensional spectra of 1H and 13C NMR and bidimentional experiments 1H/13C-COSY, TOCSY, 1H/13C-gHSQC, and 1H/13C-gHMBC. The polymer is built up of the 2--> 4-linked Kdn residues substituted by beta-D-Glcp residues at 8- and 9-hydroxyls; such a polymer with disubstituted Kdn residues was found for the first time. A glycosylated teichoic acid of the 1,3-poly(glycerophosphate) type was also identified among other anionic polymers of cell wall.     

The membrane teichoic acid of Staphylococcus lactis I3

1. Teichoic acid was isolated by extraction with trichloroacetic acid of the membrane fraction of disrupted cells of Staphylococcus lactis I3. 2. The purified material contains glycerol, phosphate and alanine, but little or no sugar or amino sugar. 3. A study of the products of hydrolysis with acid and alkali established that the membrane teichoic acid is a (1-->3)-linked poly(glycerol phosphate) that differs in structure from the glycerol teichoic acid in the wall of this organism. 4. The alanine ester residues show the characteristic high lability to alkali and are thus distinguishable from the more stable alanine ester residues of the wall teichoic acid. 5. The significance of these structural features and the possible function of teichoic acids 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.     

Biosynthesis of wall polymers in Bacillus subtilis.

Preparations of membrane plus wall derived from Bacillus subtilis W23 were used to study the in vitro synthesis of peptidoglycan and teichoic acid and their linkage to the preexisting cell wall. The teichoic acid synthesis showed an ordered requirement for the incorporation of N-acetylglucosamine from uridine 5'-diphosphate (UDP)-N-acetylglucosamine followed by addition of glycerol phosphate from cytidine 5'-diphosphate (CDP)-glycerol and finally by addition of ribitol phosphate from CDP-ribitol. UDP-N-acetylglucosamine was not only required for the synthesis of the teichoic acid, but N-acetylglucosamine residues formed an integral part of the linkage unit attaching polyribitol phosphate to the cell wall. Synthesis of the teichoic acid was exquisitely sensitive to the antibiotic tunicamycin, and this was shown to be due to the inhibition of incorporation of N-acetylglucosamine units from UDP-N-acetylglucosamine.     

The TagB protein in Bacillus subtilis 168 is an intracellular peripheral membrane protein that can incorporate glycerol phosphate onto a membrane-bound acceptor in vitro

Genes involved in the synthesis of poly(glycerol phosphate) wall teichoic acid have been identified in the tag locus of the model Gram-positive organism Bacillus subtilis 168. The functions of most of these gene products are predictable from sequence similarity to characterized proteins and have provided limited insight into the intracellular synthesis and translocation of wall teichoic acid. Nevertheless, critical steps of poly(glycerol phosphate) teichoic acid polymerization continue to be a puzzle. TagB and TagF, encoded in the tag locus, do not show sequence similarity to characterized proteins. We recently showed that recombinant TagF could catalyze glycerol phosphate polymerization in vitro. Based largely on homology to TagF, the TagB protein has been proposed to catalyze either an intracellular glycerophosphotransfer reaction or the extracellular teichoic acid/peptidoglycan ligation reaction. Here we have taken steps to characterize TagB, particularly through in vivo localization studies and in vitro biochemical assay, in order to make a case for either role in teichoic acid biogenesis. We have shown that TagB associates peripherally with the intracellular face of the cell membrane in vivo. We have also produced recombinant TagB and used it to demonstrate the enzymatic incorporation of labeled glycerol phosphate onto a membrane-bound acceptor. The data collected from this and the accompanying study are strongly supportive of a role for TagB in B. subtilis 168 teichoic acid biogenesis as the CDP-glycerol:N-acetyl-beta-d-mannosaminyl-1,4-N-acetyl-d-glucosaminyldiphosphoundecaprenyl glycerophosphotransferase. Here we use the trivial name "Tag primase."     

The glycerol teichoic acid from walls of Staphylococcus epidermidis I2

1. Walls of Staphylococcus epidermidis I2 contain 30% (w/w) of a glycerol teichoic acid containing phosphate, d-alanine and d-glucose in the molecular proportions 1:0.25:0.50. 2. The teichoic acid was isolated by extraction with trichloroacetic acid and with dilute aqueous NN-dimethylhydrazine at pH7, and was shown to be a (1-->3)-linked poly(glycerol phosphate) containing beta-d-glucopyranosyl and d-alanyl ester substituents. 3. 2-O-beta-d-Glucopyranosylglycerol was isolated and characterized as its crystalline hexa-O-acetate. 4. Unlike that of certain other bacteria, the peptidoglycan component of the wall is not solubilized by NN-dimethylhydrazine. 5. The membrane teichoic acid is also a (1-->3)-linked poly(glycerol phosphate) but contains a smaller proportion of glucosyl substituents.     

Phosphate-containing cell wall polymers of bacilli

Anionic phosphate-containing cell wall polymers of bacilli are represented by teichoic acids and poly(glycosyl 1-phosphates). Different locations of phosphodiester bonds in the main chain of teichoic acids as well as the nature and combination of the constituent structural elements underlie their structural diversity. Currently, the structures of teichoic acids of bacilli can be classified into three types, viz. poly(polyol phosphates) with glycerol or ribitol as the polyol; poly(glycosylpolyol phosphates), mainly glycerol-containing polymers; and poly(acylglycosylglycerol phosphate), in which the components are covalently linked through glycosidic, phosphodiester, and amide bonds. In addition to teichoic acids, poly(glycosyl 1-phosphates) with mono- and disaccharide residues in the repeating units have been detected in cell walls of several Bacillus subtilis and Bacillus pumilus strains. The known structures of teichoic acids and poly(glycosyl 1-phosphates) of B. subtilis, B. atrophaeus, B. licheniformis, B. pumilus, B. stearothermophilus, B. coagulans, B. cereus as well as oligomers that link the polymers to peptidoglycan are surveyed. The reported data on the structures of phosphate-containing polymers of different strains of B. subtilis suggest heterogeneity of the species and may be of interest for the taxonomy of bacilli to allow differentiation of closely related organisms according to the “structures and composition of cell wall polymers” criterion     

The function of teichoic acids in cation control in bacterial membranes

1. The effects of teichoic acids on the Mg(2+)-requirement of some membrane-bound enzymes in cell preparations from Bacillus licheniformis A.T.C.C. 9945 were examined. 2. The biosynthesis of the wall polymers poly(glycerol phosphate glucose) and poly(glycerol phosphate) by membrane-bound enzymes is strongly dependent on Mg(2+), showing maximum activity at 10-15mm-Mg(2+). 3. When the membrane is in close contact with the cell wall and membrane teichoic acid, the enzyme systems are insensitive to added Mg(2+). The membrane appears to interact preferentially with the constant concentration of Mg(2+) that is bound to the phosphate groups of teichoic acid in the wall and on the membrane. When the wall is removed by the action of lysozyme the enzymes again become dependent on an external supply of Mg(2+). 4. A membrane preparation that retained its membrane teichoic acid was still dependent on Mg(2+) in solution, but the dependence was damped so that the enzymes exhibited near-maximal activity over a much greater range of concentrations of added Mg(2+); this preparation contained Mg(2+) bound to the membrane teichoic acid. The behaviour of this preparation could be reproduced by binding membrane teichoic acid to membranes in the presence of Mg(2+). Addition of membrane teichoic acid to reaction mixtures also had a damping effect on the Mg(2+) requirement of the enzymes, since the added polymer interacted rapidly with the membrane. 5. Other phosphate polymers behaved in a qualitatively similar way to membrane teichoic acid on addition to reaction mixtures. 6. It is concluded that in whole cells the ordered array of anionic wall and membrane teichoic acids provides a constant reservoir of bound bivalent cations with which the membrane preferentially interacts. The membrane teichoic acid is the component of the system which mediates the interaction of bound cations with the membrane. The anionic polymers in the wall scavenge cations from the medium and maintain a constant environment for the membrane teichoic acid. Thus a function of wall and membrane teichoic acids is to maintain the correct ionic environment for cation-dependent membrane systems.     

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.     

A polymer with a backbone of 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid, a teichuronic acid, and a beta-glucosylated ribitol teichoic acid in the cell wall of plant pathogenic Streptomyces sp. VKM Ac-2124.

Structures of cell wall anionic polymers of the strain Streptomyces sp. VKM Ac-2124, a causative agent of potato scab, which is phylogenetically the closest to plant pathogenic species S. setonii and S. caviscabies, were studied. The strain contains three anionic glycopolymers, viz., a teichuronic acid with a disaccharide repeating unit -->6)-alpha-d-Glcp-(1-->4)-beta-d-ManpNAc3NAcA-(1-->, a beta-glucosylated polymer of 3-deoxy-d-glycero-d-galacto-non-2-ulopyranosonic acid (Kdn), and a beta-glucosylated 1,5-poly(ribitol phosphate). The strain studied is the second representative of plant pathogenic streptomycetes inducing potato scab disease, the cell wall anionic polymers of which were shown to contain a Kdn-polymer. Presumably, the presence of Kdn-containing structures in the surface regions of pathogens is essential for their efficient attachment to host plant cells.     

Cell wall teichoic acids of Actinomadura viridis VKM Ac-1315T.

The cell walls of Actinomadura viridis contain poly(glycosylglycerol phosphate) chains of complex structure. On the basis of NMR spectroscopy of the polymer and glycosides thereof the following structural units were found: beta-D-Galp3Me-(1-->4)[beta-D-Glcp-(1-->6)]-beta-D-Galp-(1-->1)-++ +snGro (G1); beta-D-Galp-(1-->4)-beta-D-Galp-(1-->1)-snGro (G2); beta-D-Galp3Me-(1-->4)-beta-D-Galp-(1-->1)-snGro (G2a); beta-D-Galp-(1-->1)-snGro (G3); beta-D-Galp-(1-->1)[beta-D-Galp-(1-->2)]-snGro (G4); beta-D-Glcp-(1-->2)-snGro (G5). Glycosides G1, G2 and G3 were the predominant components of the teichoic acid: they formed the polymer chain via phosphodiester bonds involving C-3 of the glycerol residue and C-3 of the galactosyl residue which in turn glycosylates C-1 of the glycerol residue. Whether the different glycosides make up the one chain or whether there are several poly(glycosylglycerol phosphate) chains in the cell wall remains to be determined. It was suggested that the minor component G5 is located at the nonterminal end of the chains. Compound G4 which contains disubstituted glycerol residues (unusual for the teichoic acid) was also found as a minor component; this may be the glycoside of a new type of teichoic acid, or a glycoside on the terminal end of the above mentioned chains. In addition, small amounts of 1,3-poly(glycerol phosphate) chains were found in the cell wall.     

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 elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus epidermidis RP62A, a reference biofilm-positive strain

The ability to adhere to artificial surfaces and form biofilms is considered as a virulence factor of Staphylococcus epidermidis, one of the major causes of nocosomial infections, especially those related to implanted medical devices. Cell-wall teichoic acid is known to play an important role in biofilm formation of staphylococci. The structure of the cell wall and extracellular teichoic acids of S. epidermidis RP62A, a reference biofilm-positive strain, was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Their structures were found to be a (1-->3)-linked poly(glycerol phosphate), substituted at the 2-position of glycerol residues with alpha-Glc, alpha-GlcNAc, D-Ala and alpha-Glc6Ala. D-Alanyl acylation of a sugar hydroxyl group seems to be a novel structural feature of teichoic acids from staphylococci.