Maintenance of D-alanine ester substitution of lipoteichoic acid by reesterification in Staphylococcus aureus

Abstract

Toluene-treated Staphylococcus aureus cells did not synthesize teichoic acid and lipoteichoic acid under the conditions used. The organism displayed, however, a high capacity of incorporating D-[14C]alanine into previously formed polymers. The reaction was dependent on ATP and enhanced by magnesium ions. The incorporation rate into lipoteichoic acid correlated with the rate of loss of alanine ester which occurred through transfer to teichoic acid and base-catalyzed hydrolysis. At pH 6.5 the loss (20% within 4 h) was completely compensated for by reesterification. At pH 7.5 the loss was 60%, but by accelerated incorporation it was reduced to 10%. Incorporation was also enhanced when the original substitution of lipoteichoic acid was lowered by previous growth of S. aureus at high salt concentration. The newly added alanine was randomly distributed along the poly(glycerophosphate) chain. The decreased alanine substitution of lipoteichoic acid after growth at high salt concentration was shown to result from a direct inhibition of alanine incorporation. Lipoteichoic acid and teichoic acid are widespread membrane and cell wall components of gram-positive bacteria. In many cases both polymers are replaced with D-alanine ester (1, 3, 6). A concomitant decrease of the alanine content of teichoic acid (15) and lipoteichoic acid (10) in Staphylococcus aureus at increasing salt concentrations in the growth medium led us to hypothesize that the incorporation of D-alanine into both polymers may be under the same control. In Lactobacillus casei the incorporation into lipoteichoic acid was shown to occur by a two-step reaction (5, 19): Enzyme + D-alanine + ATP = enzyme alanylAMP + PPa Enzyme alanylAMP + lipoteichoic acid alanyl lipoteichoic acid + AMP + enzyme Recent pulse-chase experiments of growing S. aureus with D-[14C]alanine revealed a rapid turnover of the alanine ester

A partial reconstitution implicates DltD in catalyzing lipoteichoic acid d-alanylation

“…obstacles, we utilized the full DLT pathway using D-[ 14 C]alanylation of LTA in vivo and in vitro to detect activity of individual components. This work focused on LTA rather than WTA D-alanylation because it is clear from previous studies that D-alanine is added to LTA before WTA (35,36). Genetic and wholecell labeling experiments with null mutants of each of the four individual dlt genes demonstrated that each was essential for LTA D-alanylation in vivo.…”

“…Lipoteichoic and wall teichoic acids become D-alanylated through a poorly understood process that depends on the dlt operon. LTA D-alanylation occurs first, and D-alanines are subsequently transferred to WTA (35,36). The dlt operon encodes a set of proteins that activate D-alanine as a thioester in the cytoplasm before moving the D-alanine across the plasma membrane and onto teichoic acids (4, 36 -39).…”

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

“…Cell wall teichoic acids are thought to be uniformly distributed over the entire peptidoglycan exoskeleton (806). Some teichoic acid decorations, for example the esterified D-Ala, are unstable, and S. aureus continuously reesterifies D-Ala residues (431,433).…”

The absence of D-alanine from lipoteichoic acid and wall teichoic acid alters surface charge, enhances autolysis and increases susceptibility to methicillin in Bacillus subtilis

“…The gene dltC encodes the d -alanine carrier protein (Dcp). The proposed functions of the proteins encoded by the other genes are briefly: dltA , ligation of d -alanine to Dcp via d -alanyl AMP; dltB , transport of activated d -alanine in a not yet defined form through the cytoplasmic membrane; dltD , transfer from the transport form to LTA and possibly WTA, unless WTA is alanylated by d -alanyl transfer from d - Ala-LTA as shown earlier with Staphylococcus aureus (Haas et al ., 1984; Koch et al ., 1985). The fifth gene of the dlt operon of B. subtilis, dltE , has no function in the d -alanylation process (Perego et al ., 1995).…”