AhfA is required for mycolic acid homeostasis and cell envelope integrity in Corynebacteriales.

Mycolic acids are critical components of the Corynebacteriales cell envelope, contributing to cellular morphology, permeability and antibiotic resistance. While the core enzymes responsible for mycolic acid synthesis are relatively well described, little is known about the accessory factors that coordinate their biosynthesis and integration into the cell envelope. In this study, we investigated the role of AhfA in Corynebacterium glutamicum using genetic, biochemical and physiological approaches. Loss of AhfA resulted in broad effects on cell growth, morphology, lipid composition and cell envelope integrity, which were restored upon complementation. Our analysis suggests that AhfA acts as an early stage of mycolic acid synthesis supporting the formation of the lipid-rich outer cell envelope. Notably, a similar reduction in mycolic acid-containing glycolipids was observed in mycobacterial species, suggesting that the role of AhfA in mycolic acid metabolism is conserved across Corynebacteriales. This conservation suggests that accessory proteins involved in mycolic acid biosynthesis and homeostasis may represent potential targets for therapeutic intervention. These findings identify AhfA as a previously uncharacterized factor in cell envelope assembly and highlight the complexity of lipid integration in Corynebacteriales.