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Summary: The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for the maintenance of cell shape and survival. During growth, bacteria remodel their PG, releasing fragments that are predominantly re-internalized and recycled. Here, we show that Vibrio cholerae recycles PG fragments modified with non-canonical d-amino acids (NCDAA), which lead to the accumulation of cytosolic PG tetrapeptides. We demonstrate that the accumulation of recycled tetrapeptides has two regulatory consequences for the cell wall: reduction of d,d-cross-linkage and reduction of PG synthesis. We further demonstrate that l,d-carboxypeptidases from five different species show a preferential activity for substrates containing canonical (d-alanine) versus non-canonical (d-methionine) d-amino acids, suggesting that the accumulation of intracellular tetrapeptides in NCDAA-rich environments is widespread. Collectively, this work reveals a regulatory role of NCDAA linking PG recycling and synthesis to promote optimal cell wall assembly and composition in the stationary phase. : A critical step in peptidoglycan (PG) recycling is the transformation of PG tetrapeptides into tripeptides. Hernández et al. demonstrate that Vibrio cholerae accumulates tetrapeptide PG precursors to downregulate PG synthesis in the stationary phase. Tetrapeptide accumulation relies on the substrate preference of l,d-carboxypeptidases for d-ala versus NCDAA-modified substrates. Keywords: cell wall, Vibrio cholerae, peptidoglycan recycling, l,d-carboxypeptidase, l,d-transpeptidase, NCDAA, d-amino acids, Tn-seq