Ian C. Hancock

Occurrence and function of membrane teichoic acids

Membrane teichoic acids, sometimes described as lipoteichoic acids, are important but not major components of nearly all Gram-positive bacteria. They appear on the outer surface of the cytoplasmic membrane and possess antigenic properties. Several functions have been ascribed to these glycerol phosphate polymers, including the binding of divalent cations required for optimal activity of membrane-bound enzymes, and the control of certain lytic enzymes. A substance that is identical or closely similar to membrane teichoic acid, lipoteichoic acid carrier, plays an important part in the biosynthesis of wall teichoic acid; it accepts polyol phosphate residues from CDP-glycerol or CDP-ribitol to form a polyol phosphate chain which is then transferred after the incorporation of a tri(glycerol phosphate) linkage unit, to the growing glycan chain of peptidoglycan.

Cell Wall Assembly in Bacillus subtilis: Visualization of Old and New Wall Material by Electron Microscopic Examination of Samples Stained Selectively for Teichoic Acid and Teichuronic Acid

Uranyl acetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation.

Cell Wall Assembly in Bacillus subtilis: Partial Conservation of Polar Wall Material and the Effect of Growth Conditions on the Pattern of Incorporation of New Material at the Polar Caps

The use of phage SP50 as marker for cell wall containing teichoic acid in Bacillus subtilis showed clear differences in the rates at which new wall material becomes exposed at polar and cylindrical regions of the wall, though the poles were not completely conserved. Following transition from phosphate limitation to conditions that permitted synthesis of teichoic acid, old polar caps fairly rapidly incorporated enough teichoic acid to permit phage binding. Electron microscopy suggested that the new receptor material spread towards the tip of the pole from cylindrical wall so that phages bound to an increasing proportion of the pole area until only the tip lacked receptor. Eventually, receptor was present over the whole polar surface. Direct electron microscopic staining of bacteria collected during transitions between magnesium and phosphorus limitations showed that new material was incorporated at the inner surface of polar wall and later became exposed at the outer surface by removal of overlying older wall. The apparent partial conservation of the pole reflected a slower degradation of the overlying outer wall at the pole than at the cylindrical surface, the rate being graded towards the tip of the pole. The relative proportions of the new wall material incorporated into polar and cylindrical regions differed in bacteria undergoing transitions that were accompanied by upshift or downshift in growth rate. These differences can be explained on the basis that growth rate affected the rate of synthesis of cylindrical but not septal wall.

Synthesis of glycosyl phosphates from acetylated glycosyl nitrates

Abstract Acetylated α-glycosyl nitrates were efficiently converted under mild conditions into protected β-glycosyl phosphates by treatment with cesium dibenzyl phosphate or into thermodynamically more stable α-glycosyl phosphate derivatives upon interaction with cesium diphenyl phosphate. These reactions were found to be applicable both to 2-azido-2,6-dideoxy- and 2-azido-2-deoxygalactopyranosyl nitrates as well as to 6-deoxygalactopyranosyl and galactopyranosyl derivatives.

The Outer Membrane of Methylobacterium organophilum

SUMMARY: Inner and outer membranes were isolated from Methylobacterium organophilum by sucrose density centrifugation after disruption of bacteria by shaking with glass beads. The outer membrane (OM) contained all the pink oxocarotenoid pigment of the cell and unusually small amounts of phospholipid and lipopolysaccharide. An unidentified glucan was present in both membrane fractions. Several major OM proteins had molecular sizes in the range 49 kDa to 80 kDa and most of the OM proteins remained insoluble when OM or cell wall was treated with 2% (w/v) sodium dodecyl sulphate (SDS) at temperatures up to 50°C. Neither polysaccharide nor phospholipid was solubilized under the same conditions. Increasing the concentration of methanol in the growth medium led to an increase in the bacterial phospholipid content and to increased solubility of the OM in 2% SDS. It is suggested that the resistance of the OM to solubilization by the detergent is due in part to the presence of large amounts of three unidentified polar, phosphate-free lipids that might be related to hopane polyols. Phospholipids in isolated walls and OM were rapidly degraded by endogenous phospholipase when incubated in Tris buffer at pH 8 but the unidentified lipids were retained in the particulate fraction.

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.

The synthesis ofN-acetyl-β-l-fucosamine-1-phosphate and uridine 5′-diphospho-N-acetyl-β-l-fucosamine

Uridine 5′-(2-acetamido-2,6-dideoxy-β-l-galactopyranosyl) diphosphate (uridine 5′-diphospho-N-acetyl-β-l-fucosamine) was synthesized. The key intermediate, 3,4-di-O-acetyl-2-azido-2,6-dideoxy-β-l-galactopyranosyl dibenzyl phosphate, was prepared by a previously unknown reaction of cesium dibenzyl phosphate with the corresponding α-glycosyl nitrate and was then converted into theN-acetylated glycosyl phosphate and nucleoside diphosphate sugarsvia 3,4-di-O-acetyl-2-amino-2,6-dideoxy-β-l-galactopyranosyl phosphate using mildN-acetylation andO-deacetylation as the last synthetic steps.