The

Staphylococcus aureus teichoic acids are anionic glycopolymers covalently attached to peptidoglycan (wall teichoic acids, WTAs) or anchored to the phospholipid membrane (lipoteichoic acids, LTAs). The post-synthetic addition of D-alanine (D-Ala) residues to these polymers modulates surface charge and contributes to pathogen survival in the host environment. Despite this importance, the underlying mechanisms controlling WTA D-alanylation remain a significant area for further investigation. Here, we demonstrate that the teichoic acid D-Ala esterase, FmtA, is essential for WTA D-alanylation. The inactivation of fmtA results in a more negative net surface charge, impacting host adhesion, biofilm formation, and cell aggregation. We found that LTA from fmtA-deficient strains retains normal D-alanylation levels, while WTA is almost devoid of D-Ala. These data support the notion that LTA provides the D-Ala for WTA modification, a process dependent on FmtA.IMPORTANCEThe D-alanine (D-Ala) modification of Staphylococcus aureus teichoic acids influences bacterial interactions and survival under stress. While this modification is important for host survival, the mechanisms underlying wall teichoic acid (WTA) D-alanylation remain unclear. A deeper understanding of this process could lead to the development of targeted therapies to combat S. aureus infections. We have identified FmtA as essential for this process, supporting the idea that lipoteichoic acid (LTA) provides the D-Ala used to modify WTAs. Our findings highlight a critical gap in understanding this mechanism: an acyltransferase must incorporate the D-Ala released from LTAs by FmtA into WTAs.