Remodelling of the peptidoglycan sacculus in Escherichia coli during envelope stress

Abstract

The cell envelope of diderm (Gram-negative) bacteria consists of a cytoplasmic membrane (CM), a periplasmic compartment accommodating the peptidoglycan (PG) sacculus and an outer membrane (OM) with lipopolysaccharide (LPS) in its outer leaflet. The PG layer maintains the shape of the cell and protects it from bursting due to the turgor. The pathway and enzymes involved in PG biosynthesis remain a major target for the development of new antimicrobials. The PG layer has a mesh-like structure consisting of glycan chains connected by short, cross-linked peptides. In Escherichia coli, most of the peptide cross-links are of the DD (or 4-3) type connecting D-Ala at position 4 of one peptide and meso-diaminopimelic acid (meso-Dpm) at position 3 of another, and these cross-links are formed by Penicillin-binding proteins (PBPs). LD (or 3-3) cross-links between two meso-Dpm residues occur at the lower abundance and are formed by certain LD-transpeptidases (LDTs) belonging to the YkuD-family. Other LDTs tether the OM to PG by attaching the OM-anchored lipoprotein (Braun's lipoprotein, Lpp) to meso-Dpm residues in the PG. Both types of LDTs contribute to the maintenance of the cell envelope integrity under environmental and OM stress conditions, for example when the export of lipopolysaccharide (LPS) to the OM is compromised. This study focused on three YkuD domain-containing proteins LdtD, LdtE and DpaA involved in the remodelling of PG in Escherichia coli and essential under OM assembly stress conditions. LdtD was identified as the major stress-induced LDT required to rescue the viability of cells upon OM assembly stress by introducing LD cross-links into PG, presumably to repair defects in the PG layer that arise under stress conditions. Furthermore, it is shown that LdtD is part of a multi-protein complex with the PG synthase PBP1B, its cognate activator LpoB and the DD-carboxypeptidase PBP6a. Direct interactions were confirmed between PBP1B and LpoB, PBP6a and LdtD. DpaA (previously called LdtF) was identified as the first protein that is capable of detaching a covalently attached protein from PG. DpaA is an amidase that hydrolyses the covalent bond between meso-Dpm in PG and the C-terminal lysine residue of Lpp. IV A dpaA mutant showed increased sensitivity to mecillinam, an inhibitor of the essential PG synthase PBP2 required for cell elongation, causing cells to adopt a spherical cell shape. This suggests that the detachment of Lpp from PG by DpaA helps the cell to cope with changes in cell shape. In addition, a dpaA mutant also lysed upon OM assembly defects but lysis was prevented upon the additional deletion of the actS (former ygeR) gene, which was shown to encode a novel amidase activator ActS. Purified ActS was demonstrated to activate all three major amidases, AmiA, AmiB and AmiC. While ActS-activated AmiC showed the highest amidase activity, AmiB activation increased under acidic conditions. A direct interaction between ActS and AmiC was observed. It is therefore established that OM assembly stress activates PG amidases via ActS, and this activation of the amidases is controlled by the Lpp-PG attachment/detachment dynamics involving DpaA. Overall, the data emphasize the different ways by which Gram-negative cells respond to a weakened cell envelope caused by OM assembly stress or toxic compounds in the environment. The study illustrates for the first time the amidase activity of DpaA and an amidase activator, ActS.