The cell wall of Bacillus licheniformis N.C.T.C. 6346. Linkage between the teichuronic acid and mucopeptide components

1. After extraction of teichoic acid from cell walls of Bacillus licheniformis with dilute alkali, the insoluble residue contains the teichuronic acid and mucopeptide components and a small amount of residual phosphorus. 2. A complex of teichuronic acid and a part of the mucopeptide was isolated from the soluble fraction obtained by lysozyme treatment of alkali extracted walls. 3. Small-molecular-weight mucopeptide fragments, not containing teichuronic acid, are obtained from the soluble fraction in yields similar to those obtained after treatment of whole walls or acid-extracted walls with lysozyme. 4. The covalent linkages between teichuronic acid and mucopeptide are broken by treatment with dilute acid. The release of teichuronic acid chains is accompanied by the hydrolysis of N-acetylgalactosaminide linkages and the exposed N-acetylgalactosamine residues form chromogen under very mild conditions, indicating that they are substituted on C-3. 5. The initial rate of formation of reactive N-acetylgalactosamine residues during mild acid hydrolysis is parallel to the rate of extraction under the same conditions of teichuronic acid from alkali-treated insoluble walls, and to the rate of acid hydrolysis of glucose 1-phosphate. 6. The results suggest that the teichuronic acid chains are attached through reducing terminals of N-acetylgalactosamine residues to phosphate groups in the mucopeptide. 7. Muramic acid phosphate was isolated from the insoluble mucopeptide remaining after extraction of walls with dilute alkali followed by dilute acid.     

The isolation of structural components present in the cell wall of Bacillus licheniformis N.C.T.C. 6346

1. Four of the known components of wall preparations of vegative cells of Bacillus licheniformis N.C.T.C. 6346 have been isolated free of each other after successive treatments of the walls with trichloroacetic acid and lysozyme: (a) a mucopeptide consisting of glucosamine, muramic acid, alphain-diaminopimelic acid, glutamic acid and alanine in the molar proportions 1.0:0.8:1.0:1.2:1.7; (b) an insoluble protein; (c) teichoic acid containing phosphorus and glucose in equimolar amounts; (d) teichuronic acid containing equimolar amounts of N-acetylgalactosamine and glucuronic acid, as found by Janczura, Perkins & Rogers (1961). 2. Evidence has been obtained for the presence in the soluble fraction obtained by lysozyme treatment of whole walls of a stable covalent complex of the teichoic acid and the mucopeptide components. 3. The molar ratio of phosphorus to glucose in the teichoic acid present in intact walls or the soluble fractions obtained by extraction of the walls with lysozyme or trichloroacetic acid is 1.0:0.25, in contrast with values of about unity obtained for the purified teichoic acid. 4. Intact walls have been shown to contain polyribitol phosphate chains bearing different amounts of glucose substituents. 5. Trichloroacetic acid extracts of walls also contain polyribitol phosphate compounds of different chain lengths. Dialysis of trichloroacetic acid extracts removes the short chains of polyribitol phosphate that have been found to carry only very low amounts of glucose side chains. By contrast, the longer chains present in the non-diffusible fraction contain phosphorus and glucose in almost equimolar amounts.     

The interrelationship between mucopeptide and ribitol teichoic acid formation as shown by the effect of inhibitors

1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90–100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10μg./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0·1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are discussed.     

Cell-wall thickening in Bacillus subtilis. Comparison of thickened and normal walls

1. Incubation of Bacillus subtilis 168 trp in a glucose-amino acids-salts medium lacking tryptophan leads to an inhibition of cellular growth without affecting cell-wall synthesis. The cell walls increased approximately two- to three-fold in thickness and at the same time the amount of mucopeptide in the cells measured chemically increased to about the same extent. 2. Synthesis of mucopeptide and teichoic acid as measured by the extent of incorporation of radioactivity continued linearly for approximately 1h and then stopped. No reason was found for the strictly limited synthesis of the wall polymers. 3. The initial rates of incorporation of [(32)P]P(i) or [(3)H]alanine into teichoic acid and of (3)H-labelled amino acids into mucopeptide were not appreciably inhibited by the addition of chloramphenicol to the glucose-amino acids-salts medium. 4. There was no selective turnover of the mucopeptide synthesized by the cells in a medium lacking tryptophan on resumption of growth in a complete medium. 5. Wall synthesis taking place during the thickening process was similar to normal wall synthesis proceeding in growing cells. Walls of different thicknesses prepared from cells incubated for various times in incomplete medium did not differ qualitatively in composition. The products of autolysis of thickened walls were isolated and the analyses indicated a close similarity in the details of their mucopeptide structure compared with the mucopeptide of cells growing in the exponential phase.     

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.     

Cell Wall Composition of Hyphomicrobium Species

Chemical analysis of cell walls obtained from Hyphomicrobium B-522 and from a morphologically and nutritionally distinct organism, Hyphomicrobium neptunium (ATCC 15444), showed that the organisms have a similar cell wall composition, which is typical of gram-negative bacteria. The walls of both strains contained many amino acids, including the characteristic mucopeptide components diaminopimelic acid and muramic acid. Isolation of the mucopeptide by use of sodium dodecyl sulfate was successful only with cell walls of H. neptunium, thus revealing a difference between the walls of the two strains. The mucopeptide preparation contained glucosamine, muramic acid, alanine, glutamic acid, diaminopimelic acid, and glycine in molar ratios of 1.05:1.21:1.84:1.0:1.04:0.31, respectively. The concentration of glycine was sufficiently high to suggest that it is a mucopeptide component rather than an impurity.     

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

The phytohemagglutinin, concanavalin A (Con A), interacts specifically and reversibly with the polyglucosyl glycerol phosphate teichoic acid of Bacillus subtilis 168 cell walls. Advantage has been taken of this interaction to examine the organization of the surface teichoic acid at the ultrastructural level. Con A-treated whole cells and cell walls contain an irregular, fluffy layer 25 to 60 nm thick which is absent in untreated or alpha-methyl glucoside-treated preparations. This discontinuous layer is present only on the outer profile of Con-A-treated cell walls. The surface teichoic acid is proposed to be oriented perpendicular to the long axis of the cell. Fixation and embedment for electron microscopy result in condensation of this layer which then contributes to the stainable portion of the wall. Con A treatment binds adjacent teichoic acid molecules in their native configuration producing the irregular, fluffy layer visualized.     

Characterization of the N-acetylmuramic acid L-alanine amidase from Bacillus subtilis.

The N-acetylmuramic acid L-alanine amidase from Bacillus subtilis W-23 has been purified to apparent homogeneity. The enzyme is a monomer of molecular weight 51,000, which binds extremely tightly to homologous cell walls but not to heterologous cell walls, even of the closely related strain B. subtilis ATCC 6051. This difference in binding is only in part due to differences in teichoic acid between these two strains and to a large extent appears to represent differences in the arrangement of the peptidoglycan. A comparison of the amidase from B. subtilis W-23 and the enzyme previously purified from B. subtilis ATCC 6051 (Herbold and Glaser, 1975) shows that the two proteins, which cleave the same bond and are of the same size, do not cross-react immunologically and that the two enzymes are, therefore, not closely related in structure.     

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.     

The cell wall binding domain of Listeria bacteriophage endolysin PlyP35 recognizes terminal GlcNAc residues in cell wall teichoic acid

The cell wall binding domains (CBD) of bacteriophage endolysins target the enzymes to their substrate in the bacterial peptidoglycan with extraordinary specificity. Despite strong interest in these enzymes as novel antimicrobials, little is known regarding their interaction with the bacterial wall and their binding ligands. We investigated the interaction of Listeria phage endolysin PlyP35 with carbohydrate residues present in the teichoic acid polymers on the peptidoglycan. Biochemical and genetic analyses revealed that CBD of PlyP35 specifically recognizes the N-acetylglucosamine (GlcNAc) residue at position C4 of the polyribitol-phosphate subunits. Binding of CBDP35 could be prevented by removal of wall teichoic acid (WTA) polymers from cell walls, and inhibited by addition of purified WTAs or acetylated saccharides. We show that Listeria monocytogenes genes lmo2549 and lmo2550 are required for decoration of WTAs with GlcNAc. Inactivation of either gene resulted in a lack of GlcNAc glycosylation, and the mutants failed to bind CBDP35. We also report that the GlcNAc-deficient phenotype of L. monocytogenes strain WSLC 1442 is due to a small deletion in lmo2550, resulting in synthesis of a truncated gene product responsible for the glycosylation defect. Complementation with lmo2550 completely restored display of characteristic serovar 1/2 specific WTA and the wild-type phenotype.     

Effect of Phosphate Limitation on the Morphology and Wall Composition of Bacillus licheniformis and Its Phosphoglucomutase-Deficient Mutants

Two very poorly lytic mutants of Bacillus licheniformis 6346 that had no teichuronic acid or glucose in their walls were phosphoglucomutase deficient. The walls of the mutants were less autolytic, and the lesion in the phosphoglucomutase gene and the formation of lytic amidase seemed to be interrelated. When phosphoglucomutase was regained or the effects of the deficiency were circumvented by the presence of galactose in the medium, the lytic enzyme was partially regained. When subjected to growth limitation by the supply of inorganic phosphate, the mutants ceased to make teichoic acid, and their walls contained a greatly increased proportion of mucopeptide. Under these conditions they formed irregular spheres which changed back to rods when inorganic phosphate was supplied. Both wall and protein synthesis were necessary for the changes in morphology. An intermediate crescent-shaped cell was formed in the change from sphere to a rod. The possible relationship of this morphological change to the distribution of biosynthetic sites is discussed.     

An electron microscopic study of the location of teichoic acid and its contribution to staining reactions in walls of Streptococcus faecalis 8191.

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.     

Wall turnover deficiency of Bacillus subtilis Ni15 is due to a decrease in teichoic acid

Bacillus subtilis Ni15 is deficient in cell wall turnover. The deficiency is removed if the medium contains 0.2 M NaCl, which does not affect growth. The levels of amidase and glucosaminidase, the most likely enzymes involved in turnover, were, in stationary phase Ni15 cells, similar to those in late-exponential phase cells of a standard strain. The Ni15 enzymes were not salt sensitive. However, the Ni15 walls contained 4.7-fold less phosphorus than the walls of the standard strain. Since the phosphorus content of B. subtilis walls reflects the level of teichoic acid, it is proposed that the turnover deficiency of this strain is due to a decrease in wall teichoic acid.     

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.     

Tetracycline resistance element of pBR322 mediates potassium transport

High concentrations of choline and phosphorylcholine blocked the adsorption of pneumococcal autolytic enzyme to homologous cell walls and inhibited enzymatic cell wall hydrolysis in a noncompetitive manner. Enzyme adsorption had an absolute requirement for the presence of choline residues in the wall teichoic acid. Other amino alcohols and derivatives such as ethanolamine, monomethylaminoethanolamine , and phosphorylethanolamine had no effect on enzyme adsorption or hydrolytic activity. It is proposed that enzymatic hydrolysis of cell walls requires prior adsorption of enzyme molecules to the insoluble wall substrate and that cholin residues of the wall teichoic acid have the role of adsorption ligands in this process.     

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.     

Autolysis of Bacillus cereus cell walls and isolation of structural components

Autolysis of Bacillus cereus N.R.R.L. 569 cell walls was accompanied by hydrolysis of the majority of the 4-O-beta-N-acetylglucosaminyl-N-acetylmuramic acid linkages in mucopeptide, presumably by an endo-beta-N-acetylglucosaminidase. Hydrolysis of the N-acetylmuramyl-l-alanine linkages by an amidase also occurred. Free d-alanine residues were detected in isolated cell walls and the proportion of these residues increased during autolysis, presumably due to d-alanine carboxypeptidase action. Fractionation and analysis of the products of autolysis confirmed these results. Among the products originating from mucopeptide were a disaccharide, N-acetylmuramyl-N-acetylglucosamine, and a tetrapeptide of sequence l-Ala-d-Glu-meso-Dap-d-Ala (Dap=diaminopimelate). A dimer fraction containing a d-Ala-meso-Dap cross-link was also isolated. Two polysaccharides were obtained from the products of autolysed cell walls and from walls made soluble by Chalaropsis B glycosidase. A neutral polysaccharide accounted for about 40% of the wall and contained N-acetylglucosamine, N-acetylgalactosamine and glucose. The neutral polysaccharide isolated from wall autolysates was attached to a part of the glycan moiety of mucopeptide. The molecular weight of the complex was approx. 28000. Stoicheiometric amounts of phosphorus were present, possibly in linkages between the polysaccharide and mucopeptide moieties. The second polysaccharide accounted for 12% of the wall and was very acidic. After acidic hydrolysis of the polysaccharide, glucosamine, galactosamine and unidentified acidic substances were detected. The acid polysaccharide isolated from wall autolysates contained only traces of mucopeptide constituents and no phosphorus.     

Teichoicase from Bacillus subtilis Marburg.

The properties of a teichoic acid degrading enzyme (teichoicase) isolated from Bacillus subtilis Marburg are described. The purified enzyme showed phosphodiesterase activity but not phosphomonoesterase activity, and it had an absolute substrate specificity for alpha-glucosylated glycerol teichoic acid, the endogenous cell wall teichoic acid of the enzyme-producing cell. The substrate was degraded by an exo-mechanism yielding the monomer alpha-D-glucose 1 leads to 2 (sn)glycero-3-phosphate. When B. subtilis Marburg was grown in a rich medium, enzyme activity was detected in extracts from sporulating cells. Teichoicase activity was present in a mutant blocked in stage II of the sporulation process but was absent in a mutant blocked in stage O. It was concluded that teichoicase is active on enzyme-producing cells since the reaction product could be detected in their culture supernatant. Attempts to demonstrate analogous enzyme activity in other Bacillus strains failed. The enzyme could be used for the rapid detection of alpha-glucosylated glycerol teichoic acid and for the controlled alteration of native bacterial cell surfaces exhibiting the appropriate structure.     

Control of teichoic acid synthesis in Bacillus licheniformis ATCC 9945

Analysis of cell walls of Bacillus licheniformis ATCC 9945 grown under phosphate limitation showed that teichoic acid could be replaced by teichuronic acid under these conditions. Teichuronic acid, however, was always present in the walls to some extent irrespective of the growth conditions. The enzymes involved in teichoic acid synthesis were investigated and the synthesis of these was shown to be repressed when the intracellular Pi level fell. CDP-glycerol pyrophosphorylase was studied in some detail and evidence is presented to show that the enzyme is inactivated under phosphate-limited conditions. The mechanism of inactivation is unknown but it has been shown that it does not require protein synthesis de novo.     

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.