Bacterial cell walls and membranes. Discovery of the teichoic acids.

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.

Biosynthesis of wall teichoic acids in Staphylococcus aureus H, Micrococcus varians and Bacillus subtilis W23. Involvement of lipid intermediates containing the disaccharide N-acetylmannosaminyl N-acetylglucosamine.

The precursors for linkage unit (LU) synthesis in Staphylococcus aureus H were UDP-GlcNAc, UDP-N-acetylmannosamine (ManNAc) and CDP-glycerol and synthesis was stimulated by ATP. Moraprenol-PP-GlcNAc-ManNAc-(glycerol phosphate)1-3 was formed from chemically synthesised moraprenol-PP-GlcNAc, UDP-ManNAc and CDP-glycerol in the presence of Triton X-100. LU intermediates formed under both conditions served as acceptors for ribitol phosphate residues, from CDP-ribitol, which comprise the main chain. The initial transfer of GlcNAc-1-phosphate from UDP-GlcNAc was very sensitive to tunicamycin whereas the subsequent transfer of ManNAc from UDP-ManNAc was not. Poly(GlcNAc-1-phosphate) and LU synthesis in Micrococcus varians, with endogenous lipid acceptor, UDP-GlcNAc and CDP-glycerol, was stimulated by UDP-ManNAc. Synthesis of LU on exogenous moraprenol-PP-GlcNAc, with Triton X-100, was dependent on UDP-ManNAc and CDP-glycerol and the intermediates formed served as substrates for polymer synthesis. Membranes from Bacillus subtilis W23 had much lower levels of LU synthesis, but UDP-ManNAc was again required for optimal synthesis in the presence of UDP-GlcNAc and CDP-glycerol. Conditions for LU synthesis on exogenous moraprenol-PP-GlcNAc were not found in this organism. LU synthesis on endogenous acceptor in the absence of UDP-ManNAc was explained by contamination of membranes with UDP-GlcNAc 2-epimerase. Under appropriate conditions, low levels of this enzyme were sufficient to convert UDP-GlcNAc into a mixture of UDP-Glc-NAc and UDP-ManNAc and account for LU synthesis. The results indicate the formation of prenol-PP-GlcNAc-ManNAc-(glycerol phosphate)1-3 which is involved in the synthesis of wall teichoic acids in S. aureus H, M. varians and B. subtilis W23 and their attachment to peptidoglycan.

Synthesis of peptidoglycan and teichoic acid in Bacillus subtilis: role of the electrochemical proton gradient.

The effects of several ionophores and uncouplers on glycerol and N-acetylglucosamine incorporation by Bacillus subtilis 61360, a glycerol auxotroph, were tested at different pH values. In particular, the effect of valinomycin on the synthesis of teichoic acid and peptidoglycan was examined in more detail in both growing cells and in vitro biosynthetic systems. Valinomycin inhibited synthesis of wall teichoic acid and peptidoglycan in whole cells but not in the comparable in vitro systems. It did not inhibit formation of free lipid or lipoteichoic acid. The results were consistent with a role for the electrochemical proton gradient in maintaining full activity of cell wall synthetic enzymes in intact cells. Such an energy source would be required for a model in which rotation or reorientation of synthetic enzyme complexes is envisaged for the translocation of wall precursor molecules across the cytoplasmic membrane (Harrington and Baddiley, J. Bacteriol. 155:776-792, 1983).

Peptidoglycan synthesis by partly autolyzed cells of Bacillus subtilis W23.

Partly autolyzed, osmotically stabilized cells of Bacillus subtilis W23 synthesized peptidoglycan from the exogenously supplied nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl pentapeptide. Freshly harvested cells did not synthesize peptidoglycan. The peptidoglycan formed was entirely hydrolyzed by N-acetylmuramoylhydrolase, and its synthesis was inhibited by the antibiotics bacitracin, vancomycin, and tunicamycin. Peptidoglycan formation was optimal at 37 degrees C and pH 8.5, and the specific activity of 7.0 nmol of N-acetylglucosamine incorporated per mg of membrane protein per h at pH 7.5 was probably decreased by the action of endogenous wall autolysins. No cross-linked peptidoglycan was formed. In addition, a lysozyme-resistant polymer was also formed from UDP-N-acetylglucosamine alone. Peptidoglycan synthesis was inhibited by trypsin and p-chloromercuribenzenesulfonic acid, and we conclude that it occurred at the outer surface of the membrane. Although phospho-N-acetylmuramyl pentapeptide translocase activity was detected on the outside surface of the membrane, no transphosphorylation mechanism was observed for the translocation of UDP-N-acetylglucosamine. Peptidoglycan was similarly formed with partly autolyzed preparations of B. subtilis NCIB 3610, B. subtilis 168, B. megaterium KM, and B. licheniformis ATCC 9945. Intact protoplasts of B. subtilis W23 did not synthesize peptidoglycan from externally supplied nucleotides although the lipid intermediate was formed which was inhibited by tunicamycin and bacitracin. It was therefore considered that the lipid cycle had been completed, and the absence of peptidoglycan synthesis was believed to be due to the presence of lysozyme adhering to the protoplast membrane. The significance of these results and similar observations for teichoic acid synthesis (Bertram et al., J. Bacteriol. 148:406-412, 1981) is discussed in relation to the translocation of bacterial cell wall polymers.

Control of synthesis of wall teichoic acid during balanced growth of Bacillus subtilis W23.

Enzymes involved in the synthesis of teichoic acid and its linkage to the wall in Bacillus subtilis W23 were measured in chemostat cultures growing at equilibrium at a dilution rate of 0.2 h-1 in different concentrations of inorganic phosphate. All the enzymes, except teichoic acid glucosyl transferase, which was insensitive to changes in phosphate concentration, were almost undetectable at 0.5 mM-phosphate. At higher phosphate concentrations the changes in activity of the enzymes of linkage unit synthesis were sufficient to account for the changes in the rate of incorporation of teichoic acid into the wall in vivo. Between 3.5 and 4.5 mM-phosphate the amount of teichoic acid synthesized in vivo increased, but no increase in the ability of toluenized bacteria to synthesize teichoic acid could be detected. Allosteric regulation might therefore be important at high phosphate concentrations. Bacteria maintained a constant ATP content and a constant adenylate energy charge during chemostat growth at all phosphate concentrations.

Synthesis of teichoic acid by Bacillus subtilis protoplasts.

Protoplasts of Bacillus subtilis W23 readily synthesized ribitol teichoic acid from nucleotide precursors in the surrounding medium. With cytidine diphosphate-ribitol they made poly(ribitol phosphate), presumably attached to lipoteichoic acid carrier; when cytidine diphosphate-glycerol and uridine diphosphate-N-acetylglucosamine were also present a 10-fold increase in the rate of polymer synthesis occurred, and the product contained both the main chain and the linkage unit. Synthesis was inhibited by trypsin or p-chloromercuribenzenesulfonate in the medium, and we concluded that it occurred at the outer surface of the membrane. During synthesis, which was also achieved readily by whole cells after a brief period of wall lysis, the cytidine phosphate portion of the nucleotide precursors did not pass through the membrane. No evidence could be obtained for a transphosphorylation mechanism for the translocation process. It is suggested that reaction with exogenous substrates was due to temporary exposure of a protein component of the enzyme complex at the outer surface of the membrane during the normal biosynthetic cycle.

Control of synthesis of wall teichoic acid in phosphate-starved cultures of Bacillus subtilis W23.

CDP-glycerol pyrophosphorylase, CDP-ribitol pyrophosphorylase and poly(ribitol phosphate) synthetase activities have been measured in cultures of Bacillus subtilis W23 as they became phosphate-starved either in batch culture or during changeover from potassium limitation to phosphate limitation in a chemostat. The results indicated that repression of synthesis of all three enzymes occurred at the onset of phosphate starvation and that this was accompanied by inhibition of inactivation of CDP-glycerol pyrophosphorylase and poly(ribitol phosphate) synthetase. These results show that the initial response to phosphate starvation involves more than inhibition of one enzyme as proposed by Glaser and Loewy [Glaser L. and Loewy, A. (1979) J. Biol. Chem. 254, 2184-2186]. Synthesis of both linkage unit and poly(ribitol phosphate) are inhibited independently.

Lipid intermediate in the synthesis of the linkage unit that joins teichoic acid to peptidoglycan in Bacillus subtilis.

Membranes from Bacillus subtilis W23 synthesized a lipid precursor of the linkage unit that attaches teichoic acid to the cell wall. It contained glycerophosphoryl-N-acetylglucosamine, linked through an acid-labile bond to a lipid.

Fractionation studies of the enzyme complex involved in teichoic acid synthesis.

The membrane-bound enzymes participating in the syntheses of the teichoic acid main chain and linkage unit have been solubilized with Triton X-100 and fractionated by sucrose density gradient centrifugation. Two main fractions were obtained: a heavy fraction, containing enzymes effecting synthesis of the main chain attached to the linkage unit, which was associated with only a small amount of lipid, and a light fraction which was rich in prenyl phosphate and catalyzed only linkage-unit synthesis. The separation by density was not based entirely on polypeptide chain length, as some of the shortest chains appeared in the denser fractions and some relatively high-molecular-weight peptides occurred in the lightest fraction. High activity for linkage-unit synthesis was observed in a fraction containing only a few peptides. Addition of ficaprenyl phosphate to the enzyme preparations had no stimulatory effect. It is concluded that the enzymes for main-chain and linkage unit syntheses frm one or more fairly tightly associated complexes and that polyprenyl phosphate is an integral firmly bound component of the complex in which the linkage unit is synthesized.

Attachment of the main chain to the linkage unit in biosynthesis of teichoic acids.

The main chain of teichoic acids can be assembled in cell-free membrane preparations by the transfer of residues from the appropriate nucleotide precursors to an incompletely characterized amphiphilic molecule, lipoteichoic acid carrier (LTC). However, in the cell wall, the main chain is attached to peptidoglycan through a linkage unit which is synthesized independently. It is believed that, in these cell-free systems, lipid intermediates carrying linkage units are also able to accept residues directly from nucleotide precursors to build up the main chain. In this paper, we have shown that the main chain attached to LTC was transferred from LTC to lipids containing the linkage unit. Thus, in these systems, there appear to be two routes to the biosynthesis of teichoic acid-linkage unit complexes, one by direct assembly of the main chain on linkage unit lipids and the other by transfer of the preassembled main chain from LTC to the linkage unit. It was also shown that linkage unit lipids from different organisms were interchangeable and that these were used for polymer synthesis by Bacillus subtilis 3610, in which the teichoic acid is a poly(glycerol phosphate).

The teichuronic acid from the walls of Bacillus licheniformis A.T.C.C. 9945.

The teichuronic acid of Bacillus licheniformis A.T.C.C. 9945 grown under phosphate limitation was isolated from the cell walls and purified by ion-exchange and Sephadex chromatography. The detailed structure of the polysaccharide was established by methylation analysis, periodate oxidation and partial acid hydrolysis. The polymer is composed of tetrasaccharide repeating units with the structure [GlcA beta(1 leads to 4)GlcA beta(1 leads to 3)GalNAc beta(1 leads to 6)GalNAc alpha(1 leads to 4)n. 13C n.m.r. analysis has confirmed most of the structural features of the polysaccharide and, in particular, the anomeric configurations and linkage positions of substituents. The teichuronic acid from glucose-limited cells was identical with that from cells grown under phosphate limitation.

The identification of polypeptides synthesised during the acquisition of teichoic acid synthetic activity in Bacillus licheniformis.

An attempt has been made to identify proteins synthesised during induction of teichoic acid synthesis in Bacillus licheniformis ATCC 9945. The proteins are recognised as those produced on the change from teichuronic acid to teichoic acid synthesis that occurs after the transfer of the bacteria from phosphate-limited to phosphate-rich conditions. B. licheniformis was grown in phosphate-limiting conditions in the presence of threonine to stimulate threonine uptake. The bacteria were then transferred to phosphate-rich conditions and were pulse-labelled with [14C]threonine during the change to teichoic acid synthesis. All of the proteins were extracted from the cells with sodium dodecyl sulphate and were examined by sodium dodecyl sulphate-polyacrylamide gel elecstrophoresis. Radioactive polypeptides were identified by fluorography of the polyacrylamide gels. The radioactive polypeptides that were formed on change from teichuronic acid to teichoic acid synthesis were compared with the polypeptides present in a membrane sub-fraction that had high teichoic acid-synthesising activity. The labelling of nine polypeptides with [14C]threonine was dependent on new RNA synthesis. Of these nine polypeptides, five were also present in the membrane sub-fraction with the highest teichoic acid-synthesising activity.

The structure of C-polysaccharide from the walls of Streptococcus pneumoniae.

The well-known immologically active component of pneumococci, C-polysaccharide, is a teichoic acid that can be isolated from the cell walls and purified by Sephadex and ion-exchange chromatography. Further details of the structure of C-teichoic acid were established by chemical degradation, including hydrolysis in acid and alkali, treatment with HF, periodate oxidation and methylation. In addition, the use of 13C n.m.r. has confirmed some of these structural features and resulted in a proposal for the order of substituents, the location of positions of substitution and the configuration of anomeric centres in the repeating unit of the polymer.

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.

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.