Matos, R. C.; Nikolopoulos, N.; Perrier, Q.; Robert, X.; Gueguen-Chaignon, V.; Hirayama, H.; Leulier, F.; Grangeasse, C.; Guerardel, Y.; Ravaud, S.

D-alanylation of teichoic acids is a widespread modification of Gram-positive bacterial cell envelopes that modulates resistance to environmental stresses and host interactions. Although the cytosolic steps of this pathway are well characterized, the extracellular reactions responsible for transferring D-alanine onto teichoic acids remain poorly understood. Here we investigate the role of DltD in the commensal bacterium Lactiplantibacillus plantarum. We determined the 2.3 A crystal structure of the extracellular catalytic domain of DltD, which adopts an SGNH-hydrolase fold with a conserved Ser-His-Asp catalytic triad. Docking analyses with lipoteichoic acids (LTA) fragments suggest that the glycerol-phosphate backbone of LTA is accommodated along a surface groove leading to the catalytic serine, with conserved residues contributing to substrate positioning. Biochemical measurements further reveal direct interactions between DltD, the acyl-carrier protein DltX, and the LTA esterase DltE. The conserved C-terminal motif of DltX binds DltD and is required for efficient D-alanylation and for L. plantarum-mediated promotion of Drosophila juvenile growth. Together, these findings support a DltX-dependent acyl-transfer mechanism and reveal an interaction network that coordinates LTA D-alanylation in a symbiotic bacterium.