The <i>Staphylococcus aureus</i> esterase FmtA is essential for wall teichoic acid D-alanylation

<i>Staphylococcus aureus</i> 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 <i>fmtA</i> results in a more negative net surface charge, impacting host adhesion, biofilm formation, and cell aggregation. We found that LTA from <i>fmtA</i>-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 <i>Staphylococcus aureus</i> 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 <i>S. aureus</i> 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.