Essential roles of lytic transglycosylases in l,d-transpeptidase-mediated peptidoglycan cross-linking in Escherichia coli

Abstract

ABSTRACT Bacteria resist the turgor pressure of the cytoplasm thanks to peptidoglycan, a mesh-like macromolecule that completely surrounds the cytoplasmic membrane and consists of glycan chains cross-linked to each other by short peptides. Protecting the cell from swelling and lysing during the entire cell cycle, while enabling cell expansion and division, requires the coordinated action of enzymes that form (glycosyltransferases and transpeptidases) and cleave (lytic transglycosylases, endopeptidases, and amidases) peptidoglycan chemical bonds. In Escherichia coli, cleavage of glycosidic bonds is potentially performed by eight lytic transglycosylases with partially redundant functions. Here, we explore the roles of these enzymes in bacteria that overproduce the penicillin-insensitive L,D-transpeptidase YcbB (LdtD), leading to the bypass of conventional D,D-transpeptidases belonging to the penicillin-binding protein family and to resistance to β-lactam antibiotics. Construction of lytic transglycosylase-deficient mutants and cross-complementation with other members of the autolysin family identified combinations of enzymes that are essential either for the expression of YcbB-mediated β-lactam resistance or for the prevention of toxic effects resulting from YcbB overproduction in the absence of β-lactams. We show that, among the eight lytic transglycosylases, MltG and MltD have both essential and non-redundant roles in these conditions and that endopeptidase MepK can compensate for the absence of MltG. We propose that MltG and MltD specifically alleviate defects in peptidoglycan cross-linking by eliminating toxic peptidoglycan intermediates that potentially accumulate in the inner and outer side of the peptidoglycan layer of bacterial cells overproducing YcbB, respectively. IMPORTANCE The role of lytic transglycosylases has been extensively studied in bacteria that rely on conventional D,D-transpeptidases (PBPs) for PG cross-linking. In contrast, little is known about the contribution of these enzymes in the PG metabolism that results after the bypass of PBPs by L,D-transpeptidase YcbB (aka LdtD). Here, we show that lytic transglycosylases MltG and MltD have specific and non-redundant roles in YcbB-mediated cross-linking. We also show that the absence of these autolysins can be partially compensated by the overproduction of endopeptidase MepK. MltG fulfilled a dual function involving both the production of the substrate of YcbB and the elimination of aberrant toxic peptidoglycan intermediates. The non-redundant roles of MltG and MltD could be accounted for by the elimination of these toxic intermediates from the inner and outer side of the PG layer, respectively. These results expand the repertoire of the physiological roles of lytic transglycosylases in PG metabolism. The role of lytic transglycosylases has been extensively studied in bacteria that rely on conventional D,D-transpeptidases (PBPs) for PG cross-linking. In contrast, little is known about the contribution of these enzymes in the PG metabolism that results after the bypass of PBPs by L,D-transpeptidase YcbB (aka LdtD). Here, we show that lytic transglycosylases MltG and MltD have specific and non-redundant roles in YcbB-mediated cross-linking. We also show that the absence of these autolysins can be partially compensated by the overproduction of endopeptidase MepK. MltG fulfilled a dual function involving both the production of the substrate of YcbB and the elimination of aberrant toxic peptidoglycan intermediates. The non-redundant roles of MltG and MltD could be accounted for by the elimination of these toxic intermediates from the inner and outer side of the PG layer, respectively. These results expand the repertoire of the physiological roles of lytic transglycosylases in PG metabolism.