DpaA and Lpp affect LD-cross-link formation in Escherichia coli

Peptidoglycan (PG) is an essential component of the cell envelope, required to mechanically sustain the turgor pressure of the cytoplasm. In Escherichia coli, the PG consists of glycan chains with attached stem peptides mainly cross-linked via DD-cross-links formed between Alanine (D-Ala4) and meso-diaminopimelic acid (DAP3) by DD transpeptidases (Vollmer et al., 2008). A small amount of LD-cross-links occurs between two DAP3 residues, formed by LD-transpeptidases members of the YkuD family of proteins (Magnet et al., 2008). Although not essential under standard laboratory conditions, LD-cross-links become crucial for β-lactam resistance, PG homeostasis and survival under stress conditions (Hugonnet et al., 2016, Morè et al., 2019, Alvarez et al., 2024). E. coli has two LD transpeptidases, LdtD and LdtE for LD-cross-link formation (Magnet et al., 2008). LdtD is the cell envelope-stress-induced LDT and LdtE is the housekeeping LDT active under non-stress conditions (Morè et al., 2019). Interestingly, the formation of LD-cross-links by LdtE requires DpaA, another YkuD protein family member, which detaches the outer membrane (OM)-anchored lipoprotein Lpp from the PG (Badour et al., 2021; Winkle et al., 2021). Lpp (Braun lipoprotein) is the most abundant OM lipoprotein which is attached to PG via its terminal Lys and a DAP3 residue in the PG. This is the only covalent linkage between the PG and OM and is needed to maintain a robust cell envelope. By analysing the PG composition of wild type and mutants lacking ldt genes, dpaA, and/or lpp, we found that DpaA and Lpp are both required for LD-cross-link formation by LdtE, highlighting the unexpected functional interaction between Lpp detachment dynamics and LD-cross-link formation.