In vitro synthesis of the unit that links teichoic acid to peptidoglycan.

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.

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.

The location of N-acetylgalactosamine in the walls of Bacillus subtilis 168.

The N-acetylgalactosamine in the walls of Bacillus subtilis 168 occurs in two polymers. One of these contains N-acetylgalactosamine, glucose and phosphorus and is attached to the peptidoglycan through an alkali-labile bond; preliminary studies indicate that a repeating unit of this polymer is glucosyl-N-acetylgalactosamine 1-phosphate. N-Acetylgalactosamine is also associated with the peptidoglycan in a component that is not converted into the free sugar or other soluble compounds on treatment of the walls with alkali. The two polymers containing N-acetylgalactosamine are released on autolysis of the walls and can be separated by ion-exchange chromatography. As glucose 6-phosphate is produced by gentle hydrolysis of the wall with acid a third phosphate polymer, poly(glucose 1-phosphate), may occur in this wall. However, as no polymer with this structure could be separated from that containing galactosamine, its existence has not been established unequivocally. The methods described permit the study of variations in N-acetylgalactosamine content with respect to growth conditions.

The phospholipids of Pneumococcus I-192R, A.T.C.C. 12213. Some structural rearrangements occurring under mild conditions.

1. The phospholipids from the non-capsulated strain of Pneumococcus I-192R, A.T.C.C. 12213, were separated into three fractions by chromatography on columns of silicic acid and DEAE-cellulose (acetate form). 2. The water-soluble phosphate esters produced by deacylation of each fraction were separated by chromatography on columns of DEAE-cellulose (HCO(3) (-) form). 3. Three deacylated products, diglycerol phosphate, glycerylphosphorylglycerol phosphate and bis(glycerylphosphoryl)glycerol, were identified by analysis, by chemical degradations and by comparison with synthetic materials. 4. From a study of freshly isolated lipids prepared and worked up under conditions where exposure to acid was minimal, it was concluded that the Pneumococcus contains phosphatidylglycerol and bisphosphatidylglycerol, in the molar proportion 1:2.5-3.0, and that the deacylation product glycerylphosphorylglycerol phosphate was probably an artifact of the isolation procedure. 5. Acid-catalysed isomerization (phosphodiester migration) of diglycerol phosphate and bis(glycerylphosphoryl)glycerol and transesterification (glycerol phosphate transfer) of diglycerol phosphate were observed. The structures of the products were established by degradation. 6. A novel mechanism for the biosynthesis of bisphosphatidylglycerol is presented.

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.

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.