Abstract: The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of PG synthases and hydrolases. In Escherichia coli, the activity of the two bifunctional PG synthases is driven by lipoproteins anchored in the outer membrane. However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form multimeric complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and cell division biosynthetic complexes based on its localization and genetic interactions. In line with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. In addition, we find that NlpI interacts with the amidases AmiA, Amic and the amidase regulator EnvC. Using an HPLC-based PG hydrolase assay we show that NlpI affects the activity of AmiA in the presence or absence of EnvC. Our results indicate that PG regulators and adaptors are part of PG biosynthetic multi-enzyme complexes. Though, our findings raise the possibility that regulators are promiscuous and their regulation of PG hydrolases is more complex than originally understood.