ROLE OF PEPTIDOGLYCAN REMODELING IN OVERCOMING LPS BIOGENESIS DEFECTS IN ESCHERICHIA COLI

The three layered Gram-negative bacteria envelope consists of an inner membrane (IM), the periplasm‐containing peptidoglycan (PG), and an asymmetric outer membrane (OM) decorated with lipopolysaccharide (LPS) in the outer leaflet. Growth and assembly of cell envelope is orchestrated by action of dedicated protein machineries which span the entire envelope and whose coordinated activity guarantees proper envelope stiffness. Defects in biogenesis in any of these layers compromise the whole cell integrity and lead to cell death. In this thesis we show that Escherichia coli remodels the PG structure by increasing the level of 3-3 crosslinks produced by LD – Transpeptidases (LDTs), to avoid cell lysis when the LPS transport to the OM is disrupted. E. coli codes for six LDTs (LdtA-F): LdtA, LdtB, and LdtC covalently attach Lpp to PG while LdtD and LdtE introduce 3-3crosslinks. LdtF has no LD-Transpeptidase (LD-Tpase) activity but enhances the enzymatic activity of LdtD and LdtE. Our data outlines a major contribution of LdtD in PG remodelling and suggest that LdtD works in concert with the PG synthase PBP1B, its activator LpoB and the DD-CPase PBP6a to form a dedicated PG repair machine that runs a PG remodeling program to counteract damages to the OM. We also show that the lysis phenotype and morphological defects seen in mutants with an impaired LPS transport and lacking ldtF, are rescued and suppressed, respectively, by the loss of YgeR an uncharacterized lipoprotein predicted to be OM anchored. YgeR belongs to the family of LytM-domain factors which are hydrolases or hydrolase regulators implicated in PG remodeling/turnover. Important PG hydrolases are amidases which promote PG septal splitting and daughter cell separation. Our biochemical data reveal that YgeR is an amidase regulator able to activate AmiA, AmiB and AmiC the three amidases encoded by E. coli. We also show that YgeR binds purified PG and physically interacts with the amidase AmiC. Our biochemical analyses are complemented by in vivo data showing that YgeR preferentially activates AmiC and that it does it through its LytM domain. Altogether, our results point out an unexplored protective role of the 3-3 crosslinks in PG to overcome severe OM biogenesis defects and propose YgeR as a novel amidase activator whose action seems required upon envelope stress.