d-lactate drives lysine d-lactylation to regulate metabolism in Escherichia coli

l-lactate-derived lysine l-lactylation (K L-la ) has emerged as a key regulator in diverse cellular processes and disease pathogenesis. While lactate predominantly exists as the l-isomer in eukaryotes, both l- and d-lactate are present in some bacteria. However, it remains unclear whether d-lactate can drive post-translational modification to exert biological functions. Here, we reported that d-lactate-derived lysine d-lactylation (K D-la ) serves as a post-translational modification in Escherichia coli . We demonstrated that acetate CoA-transferase (YdiF) catalyzes the formation of d-lactyl-CoA, the key d-lactyl donor, connecting d-lactate to K D-la . Notably, we identified 86 K D-la sites on 71 proteins in E. coli . In addition, our data demonstrated that anerobic conditions enhance glycolysis, increasing d-lactate production via l-lactate dehydrogenase A (LdhA) and further elevating K D-la levels. We also found that CobB functions as an endogenous de-d-lactylase. Our experiment further showed that K257 of GapA can regulate bacterial growth, whereas CobB can remove K D-la at this site, suggesting a potential CobB-mediated K D-la role. Briefly, this study shows that K D-la directly driven by d-lactate exists as a regulatory mechanism and provides insights into the functional roles of d-lactate in prokaryotes.