Inhibition of the D-alanine:D-alanyl carrier protein ligase from Bacillus subtilis increases the bacterium's susceptibility to antibiotics that target the cell wall.

The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram-positive cell wall is controlled by the D-alanylation of the lipoteichoic acid. The incorporation of D-Ala into lipoteichoic acid requires the D-alanine:D-alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC. We found that apo-DltC is recognized by both endogenous 4'-phosphopantetheinyl transferases AcpS and Sfp. After the biochemical characterization of DltA and DltC, we designed an inhibitor (D-alanylacyl-sulfamoyl-adenosine), which is able to block the D-Ala adenylation by DltA at a K(i) value of 232 nM vitro. We also performed in vivo studies and determined a significant inhibition of growth for different Bacillus subtilis strains when the inhibitor is used in combination with vancomycin.