The "core" structure of the cell wall of Mycobacterium and related genera is unique among prokaryotes, consisting of a covalently linked complex of mycolic acids, D-arabinan and D-galactan (mycolylarabinogalactan, mAG), which, in turn, is linked to peptidoglycan via a special linkage unit, -alpha-L-Rhap(1-->3)-D-GlcNAc-P-. Little is known of the biosynthesis of this complex, although it is the site of action of several common anti-tuberculosis drugs. Isolated cell membranes of Mycobacterium smegmatis catalyzed the incorporation of [14C]GlcNAc from UDP-[14C]GlcNAc into two glycolipids (1 and 2) and of [14C]Rha from TDP-[14C]Rha into glycolipid 2. These products were characterized as polyprenol-P-P-GlcNAc (glycolipid 1) and polyprenol-P-P-GlcNAc-Rha (glycolipid 2) based on sensitivity of synthesis to tunicamycin, chromatographic characterization of the products of mild acid hydrolysis, and mass spectral analysis of the glycosyl and polyprenyl units. Glycolipids 1 and 2 were shown to be precursors of the linkage unit in polymerized cell wall. The inclusion in the assays of UDP-[14C]Galp and a preparation of cell walls allowed the incorporation of [14C]Gal into two further glycolipids (3 and 4). Preliminary evidence indicates a precursor-product relationship among glycolipids 1, 2, 3, and 4. Thus, the first steps in the biosynthesis of the mycobacterial cell wall involve synthesis of the linkage disaccharide on a polyprenyl-P-P carrier followed by growth of the galactan unit. Assays are thus defined for the screening of new anti-tuberculosis drugs active against cell wall synthesis.

1. The effects of teichoic acids on the Mg(2+)-requirement of some membrane-bound enzymes in cell preparations from Bacillus licheniformis A.T.C.C. 9945 were examined. 2. The biosynthesis of the wall polymers poly(glycerol phosphate glucose) and poly(glycerol phosphate) by membrane-bound enzymes is strongly dependent on Mg(2+), showing maximum activity at 10-15mm-Mg(2+). 3. When the membrane is in close contact with the cell wall and membrane teichoic acid, the enzyme systems are insensitive to added Mg(2+). The membrane appears to interact preferentially with the constant concentration of Mg(2+) that is bound to the phosphate groups of teichoic acid in the wall and on the membrane. When the wall is removed by the action of lysozyme the enzymes again become dependent on an external supply of Mg(2+). 4. A membrane preparation that retained its membrane teichoic acid was still dependent on Mg(2+) in solution, but the dependence was damped so that the enzymes exhibited near-maximal activity over a much greater range of concentrations of added Mg(2+); this preparation contained Mg(2+) bound to the membrane teichoic acid. The behaviour of this preparation could be reproduced by binding membrane teichoic acid to membranes in the presence of Mg(2+). Addition of membrane teichoic acid to reaction mixtures also had a damping effect on the Mg(2+) requirement of the enzymes, since the added polymer interacted rapidly with the membrane. 5. Other phosphate polymers behaved in a qualitatively similar way to membrane teichoic acid on addition to reaction mixtures. 6. It is concluded that in whole cells the ordered array of anionic wall and membrane teichoic acids provides a constant reservoir of bound bivalent cations with which the membrane preferentially interacts. The membrane teichoic acid is the component of the system which mediates the interaction of bound cations with the membrane. The anionic polymers in the wall scavenge cations from the medium and maintain a constant environment for the membrane teichoic acid. Thus a function of wall and membrane teichoic acids is to maintain the correct ionic environment for cation-dependent membrane systems.

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.

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The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.