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.     

Structure of the linkage units between ribitol teichoic acids and peptidoglycan.

The structure of the linkage regions between ribitol teichoic acids and peptidoglycan in the cell walls of Staphylococcus aureus H and 209P and Bacillus subtilis W23 and AHU 1390 was studied. Teichoic acid-linked saccharide preparations obtained from the cell walls by heating at pH 2.5 contained mannosamine and glycerol in small amounts. On mild alkali treatment, each teichoic acid-linked saccharide preparation was split into a disaccharide identified as N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and the ribitol teichoic acid moiety that contained glycerol residues. The Smith degradation of reduced samples of the teichoic acid-linked saccharide preparations from S. aureus and B. subtilis gave fragments characterized as 1,2-ethylenediol phosphate-(glycerolphosphate)3-N-acetylmannosaminyl beta(1----4)N- -acetylxylosaminitol and 1,2-ethylenediolphosphate-(glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylxylosaminitol, respectively. The binding of the disaccharide unit to peptidoglycan was confirmed by the analysis of linkage-unit-bound glycopeptides obtained from NaIO4 oxidation of teichoic acid-glycopeptide complexes. Mild alkali treatment of the linkage-unit-bound glycopeptides yielded disaccharide-linked glycopeptides, which gave the disaccharide and phosphorylated glycopeptides on mild acid treatment. Thus, it is concluded that the ribitol teichoic acid chains in the cell walls of the strains of S. aureus and B. subtilis are linked to peptidoglycan through linkage units, (glycerol phosphate)3-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine and (glycerol phosphate)2-N-acetylmannosaminyl beta(1----4)N-acetylglucosamine, respectively.     

Defect in biosynthesis of the linkage unit between peptidoglycan and teichoic acid in a bacteriophage-resistant mutant of Staphylococcus aureus.

The biosynthesis of the linkage region between peptidoglycan and the ribitol teichoic acid was investigated in the bacteriophage-resistant, teichoic acid-less mutant Staphylococcus aureus 52A5 (Chatterjee et al., J. Bacteriol. 100:846--853, 1969). Membrane preparations of this strain were found to be incapable of forming the first intermediate of the biosynthetic pathway, namely, the transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to the acceptor molecule, which presumbably is undecaprenol phosphate (R. Bracha and L. Glaser, Biochem. Biophys. Res. Commun. 72:1091--1098, 1976). The addition of heat-inactivated membrane preparations of S. aureus 52A2 (which normally has ribitol teichoic acid) that had been preincubated with UDP-GlcNAc to membranes of strain 52A5 enabled the synthesis of teichoic acid. These data suggest that the mutational defect in the teichoic acid-less organism is in the synthesis of the first compound of the linkage unit, and this is apparently the reason for its absence in the cell walls.     

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).     

Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyglycerol phosphate teichoic acids in Bacillus subtilis strains 168 and W23

A 60 kbp region of the Bacillus subtilis chromosome encompassing the genes concerned with teichoic acid biosynthesis has been subjected to physical analysis. No homology was detected by Southern hybridization between DNA segments encoding the tag genes of strain 168, concerned with polyglycerol phosphate (poly(groP)) biosynthesis, and the tar genes of strain W23, concerned with polyribitol phosphate (poly-(rboP)) biosynthesis. Analysis of 168/W23 interstrain hybrids that incorporate poly(rboP) instead of poly-(groP) into their cell walls revealed that, in every case, integral substitution of the W23 tar genes for the 168 tag genes had occurred. Interstrain hybrids of the 'W23-like' type have inherited larger segments of W23 DNA than interstrain hybrids of the 'mixed' type. The tag and tar genes are located at equivalent positions on the chromosomes of strains 168 and W23, behaving, in genetic crosses, like an allelic pair. They provide the first example of a pseudo-allelic relationship between non-homologous genes in B. subtilis.     

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.     

Structure and biosynthesis of teichoic acids in the cell walls of Staphylococcus xylosus DSM 20266

The simultaneous occurrence of a N-acetylglucosaminyl poly(ribitolphosphate) (-GlcNAc) and a N-acetylglucosaminyl poly(glycerolphosphate) (-GlcNAc) in the cell walls of Staphylococcus xylosus DSM 20266 was demonstrated by different experimental lines:(1) Fractionation of extracted cell wall teichoic acid on DEAE-cellulose, (2) investigation of the composition of cell walls in the growth cycle, (3) in vitro biosynthesis using crude membranes as the source of enzyme.The polymerization of these polymers starts from CDP-ribitol and CDP-glycerol, respectively. In the presence of UDP-N-acetylglucosamine both polymers are substituted with N-acetylglucosamine at a level and with the identical anomeric configuration found in the native cell wall teichoic acids. The in vitro biosynthesis of poly(glycerolphosphate) was unique in that it was highly stimulated by UDP-N-acetylglucosamine and to a lower extent by other UDP-activated sugars. Kinetic studies have provided evidence that this stimulation is due to an increase of V _max while K _m is unchanged. Competition experiments have indicated that poly(ribitolphosphate) and poly(glycerolphosphate) were synthesized in the in vitro system in a close spatial relationship.Abbreviations ADP adenosine 5-diphospho - CDP cytidine 5-diphospho - GDP guanosine 5-diphospho - GalNAc N-acetyl-galactosamine - Glc glucose, glucosyl - GlcNAc N-acetyl-glucosamine - N acetylglucosaminyl - GlcUA glucuronic acid - Gro glycerol - Man mannose, mannosyl - Rit ribitol - SDS sodium dodecyl sulfate - UDP uridine 5-diphospho     

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.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Studies on the linkage between teichoic acid and peptidoglycan in a bacteriophage-resistant mutant of Staphylococcus aureus H.

1. In addition to poly(ribitol phosphate) the walls of a bacteriophage-resistant mutant of Staphylococcus aureus H contain glycerol phosphate residues that are not removed on digestion with trypsin or extraction with phenol. 2. The glycerol phosphate is present in a chain, containing three or four glycerol phosphate residues, which is covalently attached to the peptidoglycan through a phosphodiester linkage to muramic acid; this linkage is readily hydrolysed by dilute alkali. 3. The degradative studies described suggest that the poly(ribitol phosphate) chains of the wall teichoic acid may be attached to the wall by linkage to this glycerol phosphate oligomer.     

Lipid intermediates in the biosynthesis of the wall teichoic acid in Staphylococcus lactis I3

1. Particulate enzyme systems have been prepared from Staphylococcus lactis I3 which effect the synthesis of wall teichoic acid (a polymer containing a repeating unit in which d-glycerol 1-phosphate is attached to the 4-position on N-acetylglucosamine 1-phosphate) from the nucleotide precursors CDP-glycerol and UDP-N-acetylglucosamine. By using nucleotides labelled with (32)P and (14)C it has been shown that the synthesis proceeds via lipid intermediates. 2. Two intermediates have been found. In one of these N-acetylglucosamine 1-phosphate is present, whereas in the other the repeating unit of the teichoic acid occurs. 3. The simultaneous formation of the teichoic acid, a poly-(N-acetylglucosamine 1-phosphate) and an unidentified lipid, together with the poor ability of most particulate systems to synthesize polymer and the instability of the lipid intermediates themselves, have interfered with pulse-labelling experiments. Nevertheless, the biosynthetic sequence has been elucidated. It is concluded that the intermediates are derivatives of undecaprenol phosphate.     

Antibodies to Staphylococcus aureus teichoic acids in the pathogenesis of chronic osteomyelitis

The titers of antibodies to S. aureus cell-wall teichoic acids have been determined in 97 orthopedic and traumatic patients with purulent diseases, differing by the activity of the process, by means of the enzyme immunoassay. These antibodies appeared in the patients' blood in active osteomyelitic process of staphylococcal etiology.     

Distinct and essential morphogenic functions for wall‐ and lipo‐teichoic acids in Bacillus subtilis

Teichoic acids (TAs) are anionic polymers that constitute a major component of the cell wall in most Gram‐positive bacteria. Despite decades of study, their function has remained unclear. TAs are covalently linked either to the cell wall peptidoglycan (wall TA (WTA)) or to the membrane (lipo‐TA (LTA)). We have characterized the key enzyme of LTA synthesis in Bacillus subtilis, LTA synthase (LtaS). We show that LTA is needed for divalent cation homoeostasis and that its absence has severe effects on cell morphogenesis and cell division. Inactivation of both LTA and WTA is lethal and comparison of the individual mutants suggests that they have differentiated roles in elongation (WTA) and division (LTA). B. subtilis has four ltaS paralogues and we show how their roles are partially differentiated. Two paralogues have a redundant role in LTA synthesis during sporulation and their absence gives a novel absolute block in sporulation. The crystal structure of the extracytoplasmic part of LtaS, solved at 2.4‐Å resolution, reveals a phosphorylated threonine residue, which provides clues about the catalytic mechanism and identifies the active site of the enzyme.     

Binding of magnesium ions to cell walls of Bacillus subtilis W23 containing teichoic acid or teichuronic acid.

When grown in a chemostat under various nutritional conditions, cells of Bacillus subtilis W23 produce walls containing teichoic acid or teichuronic acid. The binding of Mg2+ to these walls and to the isolated anionic polymers in solution was measured by equilibrium dialysis. In solution the ribitol teichoic acid bound Mg2+ in the molar ratio Mg2+/P=1:1 with an apparent association constant (Kassoc.) of 0.61 X 10(3)M-1, and the teichuronic acid bound Mg2+ in the ratio Mg2+/CO2-=1.1, Kassoc.=0.3 X 10(3)M-1. Cell walls containing teichuronic acid exhibited closely similar binding properties to those containing teichoic acid; in both cases Mg2+ was bound in the ratio Mg/P or Mg/CO2- of 0.5:1 and with a greater affinity than displayed by the isolated polymers in solution. It was concluded that Mg2+ ions are bound bivalently between anionic centres in the walls and that the incorporation of teichoic acid or teichuronic acid into the walls gives rise to similar ion-binding and charged properties. The results are discussed in relation to the possible functions of anionic polymers in cell walls.