Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles.

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.     

Specific recognition of choline residues in the cell wall teichoic acid by the N-acetylmuramyl-L-alanine amidase of Pneumococcus.

Pneumococci growing on choline-containing medium are known to incorporate this amino alcohol into the wall teichoic acid and produce autolysin-sensitive cell walls. In contrast, bacteria grown on the choline analogue, ethanolamine, incorporate ethanolamine into the teichoic acid and synthesize cell walls that are resistant to the homologous autolysin. In this communication, we report experiments aimed at understanding the biochemical mechanism of this phenomenon. Ethanolamine-containing (autolysin-resistant) cell walls were methylated in vitro with methyl iodide. Under appropriate conditions, virtually all of the ethanolamine residues could be converted to choline. After methylation, the formerly autolysin-resistant walls could be quantitatively hydrolyzed by the pneumococcal autolysin. Methylated walls also recovered another property typical of cell walls isolated from choline-grown bacteria: they could induce the in vitro "conversion" of an inactive form of autolysin to the catalytically active form (Tomasz, A., and Westphal, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 2627-2630). The results suggest that the autolysin-catalyzed hydrolysis of amide bonds in the peptidoglycan requires an additional interaction between the enzyme protein and choline residues in the teichoric acid portion of the cell wall.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.     

Cell wall composition of Streptomyces roseoflavus var. roseofungini and its Nocardia-like variant

The composition of cell walls was comparatively studied in Streptomyces roseoflavus var. roseofungini 1128 and in its variant 1-68. In the logarithmic phase of growth, the content of teichoic acid in the cell wall of the parent culture was four times as high as in the cell wall of the variant. The cell walls of the parent culture contained 5 to 7 times more O-lysyl residues not only due to a higher content of teichoic acid in the walls but also owing to a lower content of lysyl groups in the teichoic acid of the variant. An additional polysaccharide comprising galactose and glucosamine was found in the cell wall of the variant but not in the parent strain. The peptidoglycan of the both cultures had a structure typical of Streptomyces spp.; its content in the cell walls of the two cultures was identical (ca. 50% of the dry cell wall biomass weight). The results are discussed in connection with the peculiarities of the variant hyphal septation.     

Teichoic acid-containing muropeptides from Streptococcus pneumoniae as substrates for the pneumococcal autolysin.

Pneumococcal cell walls in which the normal phosphorylcholine component of the wall teichoic acids is replaced with phosphorylethanolamine cannot absorb the homologous autolytic enzyme and are completely resistant to autolytic degradation (S. Giudicelli and A. Tomasz, J. Bacteriol. 158:1188-1190, 1984). We have now isolated and characterized soluble teichoic acid-containing muropeptides from such cell walls and tested them as substrates for the pneumococcal autolytic enzyme. Both choline- and ethanolamine-containing muropeptides were hydrolyzed to the same extent by the enzyme. Furthermore, free choline concentrations that totally inhibited the digestion of pneumococcal cell walls in vivo and in vitro were without effect when the soluble substrates were used.     

On the physiological functions of teichoic acids

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 of lysissensitive (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 molecular structure of bacterial walls. The size of ribitol teichoic acids and the nature of their linkage to glycosaminopeptides

1. Ribitol teichoic acids prepared by fractional precipitation of trichloroacetic acid extracts of bacterial cell walls are essentially undegraded and have similar chain length to the teichoic acid originally present in the walls. 2. The chain length of teichoic acid can be determined directly, without prior extraction from the wall. Accurate values have been obtained by measurement of the formaldehyde produced by oxidation of walls with periodate. Less accurate values have been derived from the amount of inorganic phosphate formed by heating walls at pH4. 3. The relative amounts of N-acetylglucosaminylribitol and its mono- and di-phosphates produced by heating walls of Staphylococcus aureus with alkali agree with the amounts calculated for the hydrolysis of teichoic acid having the chain length determined by other methods. 4. Chemical considerations indicate that the linkage between teichoic acid and the wall may involve a phosphoramidate bond between the terminal phosphate of the teichoic acid and one of the amino groups in the glycosaminopeptide.     

Nature and Origins of Phosphorus Compounds in Isolated Cell Walls of Staphylococcus aureus

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.     

Possible role of a choline-containing teichoic acid in the maintenance of normal cell shape and physiology in Streptococcus oralis.

Streptococcus oralis ATCC 35037 took up radioactively labeled choline from growth medium. Most of the choline (80 to 90%) was incorporated into the cell wall teichoic acid, and about 10% was localized in the plasma membrane. While cells grew in choline-free medium, they did so at slow rates and produced cell walls with greatly reduced amounts of phosphate and no detectable choline. Cells grown in choline-free medium had grossly abnormal shape and size. Both biochemical and morphological abnormalities were reversible by addition of choline to the medium.     

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.     

Identification of a lytic enzyme of Clostridium acetobutylicum that degrades choline-containing pneumococcal cell walls

Abstract A protein that degrades pneumococcal walls containing choline, but not ethanolamine, in the teichoic acids has been isolated and purified from supernatants obtained from cultures of Clostridium acetobutylicum . The analyses of the degradation products of [³H]choline-labeled cell walls treated with this enzyme indicated that the purified protein, showing an apparent M _r of 115 000, is an N-acetylmuramyl- l -alanine amidase. Our results also suggest that C. acetobutylicum contains choline in its cell wall.     

Asymmetric distribution of charge on the cell wall of Bacillus subtilis.

The cell wall of Bacillus subtilis is capable of binding different kinds of metal ions. The wall-ion complex appears to be dependent on both phosphoryl from teichoic acid and carboxylate from peptidoglycan. In the present study, cationized ferritin (CF) was used as a probe for charge distribution on the wall of B. subtilis 168. Detergent-extracted cell walls bound CF only on the outer wall face. Completed cell poles bound CF, but septa did not. When the walls were permitted to autolyze briefly, binding of CF occurred on both faces. In contrast, limited hydrolysis of the walls by egg white lysozyme resulted in the penetration of CF into the wall matrix. When walls were made teichoic acid-free, CF-binding asymmetry was preserved, suggesting that carboxyl groups were oriented toward the surface. Walls with carboxylates chemically neutralized also retained charge asymmetry. Phosphate-free and carboxyl-modified walls bound CF only poorly or not at all. These results indicate that negative charges contributed by both phosphate and carboxyl are responsible for the binding of CF and that the observed asymmetry in the distribution of the label is due to the orientation of teichoic acid and muramyl peptides toward the outside of the cell wall, above the plane of the glycan strands.     

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.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.     

Characterization of Wall Teichoic Acid Degradation by the Bacteriophage ?29 Appendage Protein GP12 Using Synthetic Substrate Analogs

The genetics and enzymology of the biosynthesis of wall teichoic acid have been the extensively studied, however, comparatively little is known regarding the enzymatic degradation of this biological polymer. The GP12 protein from the Bacillus subtilis bacteriophage ϕ29 has been implicated as a wall teichoic acid hydrolase. We have studied the wall teichoic acid hydrolase activity of pure, recombinant GP12 using chemically defined wall teichoic acid analogs. The GP12 protein had potent wall teichoic acid hydrolytic activity in vitro and demonstrated ∼13-fold kinetic preference for glycosylated poly(glycerol phosphate) teichoic acid compared with non-glycosylated. Product distribution patterns suggested that the degradation of glycosylated polymers proceeded from the hydroxyl terminus of the polymer, whereas hydrolysis occurred at random sites in the non-glycosylated polymer. In addition, we present evidence that the GP12 protein possesses both phosphodiesterase and phosphomonoesterase activities.     

Identification of the teichoic acid phosphorylcholine esterase in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen and many interactions of this bacterium with its host appear to be mediated, directly or indirectly, by components of the bacterial cell wall, specifically by the phosphorylcholine residues which serve as anchors for surface-located choline-binding proteins and are also recognized by components of the host response, such as the human C-reactive protein, a class of myeloma proteins and PAF receptors. In the present study, we describe the identification of the pneumococcal pce gene encoding for a teichoic acid phosphorylcholine esterase (Pce), an enzymatic activity capable of removing phosphorylcholine residues from the cell wall teichoic acid and lipoteichoic acid. Pce carries an N-terminal signal sequence, contains a C-terminal choline-binding domain with 10 homologous repeating units similar to those found in other pneumococcal surface proteins, and the catalytic (phosphorylcholine esterase) activity is localized on the N-terminal part of the protein. The mature protein was overexpressed in Escherichia coli and purified in a one-step procedure by choline-affinity chromatography and the enzymatic activity was followed using the chromophoric p-nitrophenyl-phosphorylcholine as a model substrate. The product of the enzymatic digestion of 3H-choline-labelled cell walls was shown to be phosphorylcholine. Inactivation of the pce gene in S. pneumoniae strains by insertion-duplication mutagenesis caused a unique change in colony morphology and a striking increase in virulence in the intraperitoneal mouse model. Pce may be a regulatory element involved with the interaction of S. pneumoniae with its human host.     

The essential tacF gene is responsible for the choline-dependent growth phenotype of Streptococcus pneumoniae

Streptococcus pneumoniae has an absolute nutritional requirement for choline, and the choline molecules are known to incorporate exclusively into the cell wall and membrane teichoic acids of the bacterium. We describe here the isolation of a mutant of strain R6 in which a single G-->T point mutation in the gene tacF (formerly designated spr1150) is responsible for generating a choline-independent phenotype. The choline-independent phenotype could be transferred to the laboratory strain R6 and to the encapsulated strain D39 by genetic transformation with a PCR product or with a plasmid carrying the mutated tacF gene. The tacF gene product belongs to the protein family of polysaccharide transmembrane transporters (flippases). A model is presented in which TacF is required for the transport of the teichoic acid subunits across the cytoplasmic membrane. According to this model, wild-type TacF has a strict specificity for choline-containing subunits, whereas the TacF present in the choline-independent mutant strain is able to transport both choline-containing and choline-free teichoic acid chains. The proposed transport specificity of parental-type TacF for choline-containing subunits would ensure the loading of the cell wall with teichoic acid chains decorated with choline residues, which appear to be essential for the virulence of this pathogen.     

A common linkage saccharide unit between teichoic acids and peptidoglycan in cell walls of Bacillus coagulans

Teichoic acid-glycopeptide complexes were isolated from lysozyme digests of the cell walls of Bacillus coagulans AHU 1631, AHU 1634, and AHU 1638, and the structure of the teichoic acid moieties and their linkage regions was studied. On treatment with hydrogen fluoride, each of the complexes gave a hexosamine-containing disaccharide, which was identified to be glucosyl(beta 1----4)N-acetylglucosamine, in addition to dephosphorylated repeating units of the teichoic acids, namely, galactosyl(alpha 1----2)glycerol and either galactosyl(alpha 1----2)[glucosyl(alpha 1----1/3)]glycerol (AHU 1638) or galactosyl(alpha 1----2)[glucosyl(beta 1----1/3)]glycerol (AHU 1631 and AHU 1634). From the results of Smith degradation, methylation analysis, and partial acid hydrolysis, the teichoic acids from these strains seem to have the same backbone chains composed of galactosyl(alpha 1----2)glycerol phosphate units joined by phosphodiester bonds at C-6 of the galactose residues. The presence of the disaccharide, glucosyl(beta 1----4)N-acetylglucosamine, in the linkage regions between teichoic acids and peptidoglycan was confirmed by the isolation of a disaccharide-linked glycopeptide fragment from each complex after treatment with mild alkali and of a teichoic acid-linked saccharide from each cell wall preparation after treatment with mild acid. Thus, it is concluded that despite structural differences in the glycosidic branches, the teichoic acids in the cell walls of the three strains are linked to peptidoglycan through a common linkage saccharide, glucosyl (beta 1----4) N-acetylglucosamine.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.     

Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus

Bacteriophage-resistant strains of Staphylococcus aureus H were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Cell walls isolated from about half of these resistant strains were incapable of inactivating phages and were shown to lack N-acetyl-d-glucosamine (GlcNAc) in their cell wall teichoic acid. Apart from the lack of GlcNAc, two of these mutant strains were deficient in cell wall phosphorus and ester-linked d-alanine. These two strains were also found to be resistant to both phage K and a host-range mutant isolated from the parent phage. These two phages could lyse the other phage-resistant mutants which lacked GlcNAc in their teichoic acid. Cell walls from the remaining phage-resistant mutant strains did inactivate phages and were found to have normal cell wall teichoic acid. Although GlcNAc in teichoic acid was required for phage inactivation, no difference in phage inactivation ability was detected with cell walls isolated from strains of S. aureus having exclusively alpha- or exclusively beta-linked GlcNAc in their cell wall teichoic acid.