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Teichoic acids are major wall components of most Gram-positive bacteria. Their discovery followed that of their nucleotide precursors. Lipoteichoic acids associated with the cell membrane were discovered at the same time. Events leading to these discoveries and the probable function of teichoic acids in cation control are described.
Teichoic acid (TA) isolated from the Gram-positive bacteria Staphylococcus aureus induces blue shifted metachromasia in the dye 1,9-dimethyl methyleneblue (DMMB). Results of conductometric titrations show that DMMB binds to TA with 1:1 stoichiometry. Unlike DNA, this TA does not induce a definite metachromatic band in the dye acridine orange. S. aureus TA also induces strong circular dichroism (CD) in this dye with molar ellipticity value of the order of 10(4). This induction of CD in DMMB supports our earlier inference that TA in dilute solution at neutral pH has a helical conformation, and is not a random coil polymer.
Membrane teichoic acids, sometimes described as lipoteichoic acids, are important but not major components of nearly all Gram-positive bacteria. They appear on the outer surface of the cytoplasmic membrane and possess antigenic properties. Several functions have been ascribed to these glycerol phosphate polymers, including the binding of divalent cations required for optimal activity of membrane-bound enzymes, and the control of certain lytic enzymes. A substance that is identical or closely similar to membrane teichoic acid, lipoteichoic acid carrier, plays an important part in the biosynthesis of wall teichoic acid; it accepts polyol phosphate residues from CDP-glycerol or CDP-ribitol to form a polyol phosphate chain which is then transferred after the incorporation of a tri(glycerol phosphate) linkage unit, to the growing glycan chain of peptidoglycan.
Cell wall and membrane teichoic acids from several bacteria formed soluble complexes with polysaccharides and bovine plasma in alkyl alcohol solutions. Polysaccharides which contain different monomeric units and anomeric configurations complexed with the teichoic acids, suggesting that the interaction is relatively nonspecific. Teichoic acids complexed glycogen or bovine plasma albumin in 50 to 97% ethanol solutions. The macromolecular association between teichoic acids and polysaccharides or proteins was independent of teichoic acid size over a threefold molecular weight range. Glycerol phosphates or an acid hydrolysate of teichoic acid would not complex to either glycogen or bovine plasma albumin in ethanol. The optimal interaction between glycogen and the Bacillus subtilis lipoteichoic acid occurred between pH 4.5 and 8.2. The ability of teichoic acids to bind polysaccharides and proteins in moderate dielectric constant solvents suggests that these polymers may serve as complexing agents for hydrophilic molecules found in membranes.
Teichoic acids of the cell wall of Staphylococcus aureus 209-P belong to the class of ribitol teichoic acids and can bind actively the molecules or acridine orange, changing its optical properties. The intravital fluorochromy of the cells with acridine orange may be caused by the sorption of its molecules by the teichoic acids of the cell wall.
The choline-containing teichoic acids of pneumococci can be modified by biosynthetic replacement of the choline residues with certain structural analogues, such as ethanolamine (EA) or the N-monomethyl-(MEA) and N-dimethyl-(DEA) amino derivatives of ethanolamine. Cells containing such analogues in their teichoic acids develop pleiomorphic alterations in several physiological properties, which include resistance to detergent-induced lysis and inhibition of cell separation (chain formation). We report here the results of physiological studies on the mechanism of these two phenomena. Our results are summarized in the following: (a) Pneumococci grown on various amino alcohols produce cell walls of identical amino sugar and amino acid composition. (b) Both choline- and EA-containing teichoic acids seem to follow the same conservative pattern of segregation during growth and cell division.(c)Lysis sensitivity of pneumococci requires the juxtaposition oflysissensitive (choline-containing) cell walls and endogenous autolysin at the cell wall growth zone. (d) Upon readdition of choline to ethanolamine-containing cells, lysis sensitivity and catalytically active (C-type) autolysin reappear in the bacteria with the same kinetics. (e) The chains of EA-grown pneumococci contain fully compartmentalized cells and normal cross walls.
The distribution of a pulse of teichoic acid-specific radiolabel between wall and membrane teichoic acids in pneumococci was constant over a subsequent chase period, suggesting that wall and membrane teichoic acids are biosynthesized simultaneously and independently.
The use of counterimmunoelectrophoresis (CIE) for detection of serum antibodies to staphylococcal teichoic acids was evaluated against teichoic acids prepared by sonic treatment or lysostaphin extraction of Staphylococcus aureus (Lafferty strain). Of 54 patient sera from suspected cases of staphylococcal endocarditis, osteomyelitis, or septicemia, 33 (61.1%) were positive by CIE analysis; however, 128 of 291 sera (44.0%) from normal adult donors were also positive. Selected CIE-positive sera from patient and control groups were titered by Ouchterlony gel diffusion. In the control group of normal sera, 65% were also positive by gel diffusion, but only 15% had titers of >/=1:2. Of the patient sera, 44.4% had gel diffusion titers of >/=1:2. In addition to the specific teichoic acid band, a second precipitation band could be demonstrated with both patient or normal sera by CIE or gel diffusion. This second precipitin band was shown to involve interactions of test sera with staphylococcal protein A present in the teichoic acid extracts. The protein A precipitins were detected at high concentrations of the antigen extracts, whereas the anti-teichoic acid precipitins were optimally detected at lower antigen concentrations. The formation of protein A precipitin bands did not correlate with the presence of anti-teichoic acid antibodies, as most sera tested were positive for protein A regardless of anti-teichoic acid activity. This study suggests that a high incidence of normal people have levels of antibodies to teichoic acids which are detectable by the highly sensitive, but nonspecific, technique of CIE.
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.
The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.
Teichoic acid (TA) isolated from the gram-positive bacteria S. aureus binds cationic dyes like pinacyanol (PCYN), 1,9-dimethyl methylene blue, acridine orange, etc., depicting blue-shifted metachromasia, and they bind the cationic dye carbocyanine depicting the red-shifted J band. TAs do not show any uv absorption band, and exhibition of tailing CD in the short uv region hints at its chiral conformation. Chiral conformation of TA has been confirmed from the induction of strong biphasic CD in the TA-carbocyanine system. Relative affinities for Ca2+, Mg2+, and Na+ have been probed from the disruption of metachromasia of the TA-dye system by these ions. Results show Ca2+ and Mg2+ to be almost equally effective in destroying the metachromasia of the TA-PCYN system, thus not supporting the hypothesis of special affinity for Mg2+ ion.
The wall teichoic acid of Staphylococcus hyicus has been isolated and characterized. The teichoic acid is a glycerol phosphate polymer with glycosidically linked N-acetylglucosamine. Interaction with concanavalin A and susceptibility to alpha- but not to beta-N-acetylglucosaminidase showed that the sugar is in the alpha-configuration.
The location of the glucosylated teichoic acid in whole cells and isolated walls of Streptococcus faecalis 8191 has been investigated using ruthenium red, gold-labelled concanavalin A and concanavalin A-peroxidase-diaminobenzidine. Dense laminae were revealed in sections of osmium-fixed walls stained with ruthenium red which corresponded to similar regions stained by uranyl and lead. Such regions were not seen after teichoic acid had been extracted, suggesting that the uptake of stain was by teichoic acid. However, these regions were not labelled on exposure to gold concanavalin A or concanavalin A-peroxidase-diaminobenzidine; these stains indicated that teichoic acid was situated between the dense laminae, although the distribution of stain could have been due to the inability of the concanavalin A stains to penetrate deeply. Chemical binding studies showed that the teichoic acid was the major uranyl binding component in isolated walls, from which it might be inferred that teichoic acid was located in the densely staining regions. However, since osmification significantly increased the binding of uranyl (and lead stains) to non-teichoic acid material, such an inference was not necessarily valid. It is concluded that the presence of teichoic acid can be demonstrated in certain regions of the wall by concanavalin A, but its presence in densely staining regions has not been established. These experiments therefore suggest that teichoic acid may not be intimately associated with the mechanisms that generate contrast patterns in stained sections of cell walls of Streptococcus faecalis.
Glycerol teichoic acids were not detected immunochemically or chemically in phenol-water, hot saline (Rantz and Randall), or supernatant fluids of disrupted cells of Streptococcus mitis. Thus teichoic acids do not appear to be found in most Gram-positive bacteria, as has been suggested.
Purified S. aureus ribitol teichoic acid was covalently bound to carboxylated latex particles. The immunological properties of the polysaccharide antigen were preserved. The reagent obtained was used for the quantification of anti-ribitol teichoic acid antibodies by means of a direct and rapid agglutination test carried out on a slide. There was good correlation between the preliminary results of this test and those obtained with counter-immunoelectrophoresis (CIE). The method is faster and more sensitive than CIE.
Lipid analysis of several glycerol teichoic acid preparations strongly indicated that covalently bound lipid is not required for spontaneous adsorption of glycerol teichoic acid to erythrocyte membranes. Although fatty acids were detected in each of four batches, none were covalently bound. Chloroform-ether-extracted antigens retained potent erythrocyte membrane-binding activity as measured by passive hemagglutination, even though they were shown to contain less than one fatty acid residue per 4,869 teichoic acid chains. Mild ammonolysis abolished erythrocyte-sensitizing activity in passive hemagglutination, but further studies indicated the loss of activity was due to partial destruction of the polyglycerophosphate backbone and not to the removal of esterified lipid. The amount of hydrolyzed antigen required to produce 100% passive hemagglutination inhibition was between 170 and 330 times the amount required to produce the same result using unhydrolyzed glycerol teichoic acid. The average chain length was reduced from 19.1 to 9.7, 7.4, and 5.1 glycerophosphate residues for antigen samples hydrolyzed for 1, 5, and 16 h, respectively.
Bacillus stearothermophilus B65 and Bacillus subtilis var. niger WM both contain teichoic acids in their walls composed of glycerol, phosphate and glucose. The 13C nuclear magnetic resonance spectrum of B. stearothermophilus teichoic acid showed 13C-31P coupling on the signals from the C-5 and C-6 carbon atoms of the glucose molecule and an alpha-glucosidic linkage between glucose and the C-1 atom of the glycerol moiety. These data are consistent with a poly[glucosylglycerol phosphate] as the cell-wall teichoic acid in this organism. B. subtilis var. niger WM teichoic acid was oxidized by periodate and incubated in glycine buffer at pH 10.5. This treatment did not significantly increase the phosphomonoester content (by beta-elimination of the phosphate groups) of the teichoic acid molecule (7.1 to 9.5%), which is in accordance with earlier data derived from 13C nuclear magnetic resonance spectroscopy [De Boer et al. (1976) Eur. J. Biochem. 62, 1-6], that in this organism the glucose is not an integral part of the polymer chain. Similar treatment of B. stearothermophilus B65 teichoic acid increased the phosphomonoester content of the preparation from 0.15 to 68.1%.
An antigenic determinant isolated from a strain of the Gram-negative bacterium Butyrivibrio fibrisolvens reacted with specific antisera to the polyglycerophosphate backbone of membrane teichoic acids of lactobacilli. It gave a reaction of identity with membrane glycerol lipoteichoic acid and glycerol teichoic acid preparations from lactobacilli, and with phenol extracts of other Gram-positive bacteria. The antigen-antibody reactions was strongly inhibited by glycerol-phosphoryl-glycerol-phosphoryl-glycerol and the chemical composition was consistent with glycerol teichoic acid. It was concluded that this Gram-negative bacterium contained a glycerol teichoic acid whose polyglycerophospate backbone was acting as antigenic determinant. Extracts of 33 out of 52 other strains of butyrivibrios examined gave similar reactions.