Streptococcus mutans Ingbritt was grown in a chemostat at defined dilution rates and pH values and under carbohydrate limitation. At a constant dilution rate of D = 0.1 h-' and with either 0.5% glucose or 0.5% sucrose, the amounts of both cellular and extracellular lipoteichoic acid increased as the culture pH increased from 5.0 to 7.5. At a constant pH of 6.0, the amount of cellular lipoteichoic acid formed by cultures growing in 0.2% or 0.5% glucose was relatively

The lipoteichoic acids (LTA) of gram-positive bacteria are known to bind spontaneously to a variety of animal cell membranes. We investigated the biological and biochemical characteristics of the binding of LTA of Streptococcus pyogenes and S. faecalis to human and sheep erythrocytes. The kinetics of the binding of the radiolabeled LTA ([ 3 H]LTA) from each of these organisms to erythrocytes was similar. The dissociation constants for sheep and adult human erythrocytes were 1.6 μM and 4.5 μM, respectively, whereas that of human cord blood erythrocytes was approximately 10-fold higher, 31 μM. The number of binding sites for sheep erythrocytes was calculated to be 7.2 × × 10 6 per cell, and that of human erythrocytes, 29 × 10 6 per cell. Binding was reversible. More than 50% of bound [ 3 H]LTA was displaced from erythrocytes by a 50-fold excess of unlabeled LTA. LTA prepared from heterologous species of gram-positive bacteria were all inhibitory to the binding of [ 3 H]LTA whether derived from S. pyogenes or from S. faecalis . Among a number of potential receptor analogues and other inhibitors tested, including serum albumin, gangliosides Gm 2 and Gm 3 , lipopolysaccharide of gram-negative bacteria, and various sugars, only albumin and the gangliosides significantly inhibited LTA binding. Trypsin or neuraminidase treatment of erythrocytes had no effect on LTA binding. Deacylation of [ 3 H]LTA abolished binding ability and binding was restored by esterification of the deacylated material with stearoyl chloride, indicating that ester-linked lipids are necessary for membrane binding.

Purified lipoteichoic acids (LTAs) from several gram-positive organisms have been shown, by methods involving spectral changes of an added merocyanine dye probe, to have critical micelie concentrations in the range of 1 to 10 jig/ml, suggesting that acylated LTAs in their monomer forms may represent the major configuration of extracellular LTAs in bacterial culture fluids. The critical micelle concentrations obtained did not differ markedly with degree of carbohydrate substitution of the polymers. The significance of these findings in relation to the biological properties of LTA is discussed.Lipoteichoic acid (LTA) is the generic name given to a group of structurally related amphipathic molecules or amphiphiles found as cell membrane components in a wide range of gram-positive bacteria. The hydrophilic portion of the molecule is typically a 1,3-phosphodiester-linked polymer of glycerophosphate, variously substituted in the C-2 position of the glycerol residues with sugars in glycosidic linkage and D-alanine in ester linkage. The hydrophobic region of the molecule, linked covalently through the phosphomonoester end of the polymer, is generally either a glycolipid or phosphatidylglycolipid. This lipid "end" is intercalated with the upper half of the bilayer of the cell membrane and provides a membrane anchor for the molecule. LTA is also found as a soluble excreted product in cultures of gram-positive bacteria (29, 35).In aqueous solution, amphipathic molecules or amphiphiles tend to form micellar aggregates to occlude water from the hydrophobic regions of their molecular structure. LTAs similarly form high-molecular-weight aggregates or presumptive micelles in aqueous solution, as judged by their behavior on gel chromatography (9,29,35). Treatment with detergents reduces the high-molecular-weight aggregates to what is presumed to be a monomeric form (9). In any solution of an amphiphile, the micellar form must coexist with monomer. environment (10,25,26). Merocyanine dyes are among the more thoroughly characterized of the various types of compounds used as indicators of solvent polarities. With change in solvent polarities, their electronic spectra can show very large shifts in their absorption bands or intensity of fluorescence which in turn are presumed to be due to changes in the balance of benzenoid and quinonoid valence structure of the probe (4). The merocyanine dye most commonly used in such studies is 4-[2-(1-methyl-1,4,dihydropyridin-4-ylidene)ethylidene] cyclohexa-2-5-dien-1-one, which shows a shift in the UV-visible absorption spectrum towards longer wavelength as the solvent polarity decreases (5). More recently, an amphipathic merocyanine dye has been synthesized in which the methyl group has been replaced by a hexadecyl chain (8), thereby increasing its hydrophobic character and favoring its solubilization by micelles. Studies with this probe showed upward wavelength shifts in its absorption spectrum in solutions of various detergents as the concentrations of the latter were increased from below to ...

Streptococcus sanguis G9B was grown in continu6tis culture at different generation times and pH values in media containing either glucose or fructose and differing in the concentrations of Na+ and K'. The growth pH, carbohydrate, and cation concentration each affected the yield of organisms, their ability to adhere to saliva-coated hydroxyapatite beads, and their hydrophobicity, as measured by adhesion to hexadecane. There was no correlation between adhesion to saliva-coated hydroxyapatite beads and hydrophobicity, the values for hydrophobicity varying between 44 and 83% for organisms that adhered poorly and between 24 and 75% for those that adhered effectively. For organisms grown in batch culture at pH 6.0 or 7.0 there was similarly no correlation between adhesion and hydrophobicity. The growth conditions also had a considerable influence on the productibn of extracellular protein. The total amount was greater at pH 7.5 than at other pH values, and there were also differences in the individual components in response to changes in generation time, pH, carbohydrate source, and catidn concentration. Two protein bands were identified, namely, glucosyltransferase and protein P1 (also called antigen B or I/II). However, there was no correlation between a particular protein component and adhesion.

We examined the effect of growth conditions in chemostat culture on the quantity and composition of the cell wall teichoic acids of Streptococcus mutans BHT and Lactobacillus plantarum NCIB 7220 and the membrane lipoteichoic acid from S. mutans Ingbritt. With the cell wall teichoic acids, which are covalently linked to peptidoglycan, the amount of teichoic acid is independent of the growth conditions employed. However, the extent of glucosyl substitution of the polymer from L. plantarum was dependent on growth conditions. S. mutans Ingbritt lipoteichoic acid, on the other hand, was little affected by growth conditions in terms of composition or serological activity, but the amount produced was markedly affected by changes in growth conditions.