ABSTRACTThe bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized by the cell, where they are recycled for synthesis of new PG. Although the PG recycling pathway is widely conserved, its components are not essential and its roles in cell wall homeostasis are not well-understood. Here, we identified LdcV, a Vibrio cholerae L,D-carboxypeptidase that cleaves the terminal D-Alanine from recycled murotetrapeptides. In the absence of ldcV, recycled tetrapeptides accumulated in the cytosol, leading to two toxic consequences for the cell wall. First, incorporation of tetrapeptide-containing PG precursors into the cell wall led to reduction in D,D-cross-linkage between stem peptides, diminishing PG integrity. Second, tetrapeptide accumulation led to a decrease in canonical UDP-pentapeptide precursors, reducing PG synthesis. Thus, LdcV and the recycling pathway promote optimal cell wall assembly and composition. Furthermore, Ldc substrate preference for murotetrapeptides containing canonical (D-Alanine) vs. non-canonical (D-Methionine) D-amino acids is conserved, suggesting that accumulation of tetrapeptide recycling intermediates may modulate PG homeostasis in environments enriched in non-canonical-muropeptides via substrate competition.