Cleavage of Braun’s lipoprotein Lpp from the bacterial peptidoglycan by a paralog ofl,d-transpeptidases, LdtF

Abstract

The gram‐negative bacterial cell envelope is made up of an outer membrane (OM), an inner membrane (IM) that surrounds the cytoplasm, and a periplasmic space between the two membranes containing peptidoglycan (PG or murein). PG is an elastic polymer that forms a mesh-like sacculus around the IM, protecting cells from turgor and environmental stress conditions. In several bacteria, including Escherichia coli, the OM is tethered to PG by an abundant OM lipoprotein, Lpp (or Braun’s lipoprotein), that functions to maintain the structural and functional integrity of the cell envelope. Since its discovery, Lpp has been studied extensively, and although l,d-transpeptidases, the enzymes that catalyze the formation of PG−Lpp linkages, have been earlier identified, it is not known how these linkages are modulated. Here, using genetic and biochemical approaches, we show that LdtF (formerly yafK), a newly identified paralog of l,d-transpeptidases in E. coli, is a murein hydrolytic enzyme that catalyzes cleavage of Lpp from the PG sacculus. LdtF also exhibits glycine-specific carboxypeptidase activity on muropeptides containing a terminal glycine residue. LdtF was earlier presumed to be an l,d-transpeptidase; however, our results show that it is indeed an l,d-endopeptidase that hydrolyzes the products generated by the l,d-transpeptidases. To summarize, this study describes the discovery of a murein endopeptidase with a hitherto unknown catalytic specificity that removes the PG−Lpp cross-links, suggesting a role for LdtF in the regulation of PG–OM linkages to maintain the structural integrity of the bacterial cell envelope.

A Dynamic Network of Proteins Facilitate Cell Envelope Biogenesis in Gram-Negative Bacteria

“…After Initial Synthesis, Peptidoglycan Is Modified The modification of peptidoglycan extends far beyond the model in Figure 1 , and continues after de novo peptidoglycan insertion, by a series of proteins and protein complexes referred to as PBPs. During synthesis a lytic transglycosylase ( Figure A1 ) separates the existing strands of peptidoglycan to make space for new synthesis by cleaving the links between sugars [ 79 , 82 ], the glycan strand does not continue indefinitely and are typically between 7 and 32 sugars in length [ 83 ]. New peptidoglycan must also be attached to the outer membrane by L, D transpeptidase action through linking peptidoglycan to outer membrane proteins like Lpp ( Figure A2 ), roughly once every 100 Å to maintain cell envelope stability [ 69 , 80 ].…”

“…The attachment of protein partners to the peptidoglycan layer and the peptidoglycan recycling process additionally requires peptidoglycan cleavage. The cleavage and modifications of peptidoglycan varies across species and can be broadly split into two classes of enzymatic action: hydrolase and transferase [ 12 , 82 ] ( Figure A1 and Figure A2 ). Hydrolases carry out a range of lytic modifications to peptidoglycan, including cleavage of the peptide stem at the glycosidic bond between glycan molecules, and the removal of acetyl groups (a lysozyme resistance factor in pathogenic strains) [ 85 ] ( Figure A1 ).…”

“…The attachment of protein partners to the peptidoglycan layer and the peptidoglycan recycling process additionally requires peptidoglycan cleavage. The cleavage and modifications of peptidoglycan varies across species and can be broadly split into two classes of enzymatic action: hydrolase and transferase [ 12 , 82 ] ( Figure A1 and Figure A2 ).…”

ARED: Antibiotic Response determined by Euclidean Distance with highly sensitive Escherichia coli biosensors

“…The frameshift mutation in ldtF in C19 leads to a truncated version of the protein LdtF, a recently identi ed L,Dendopeptidase. LdtF has been shown to cleave the cross-links between peptidoglycan and Braun's lipoprotein, thereby disturbing the tethering of the OM to peptidoglycan (Bahadur et al 2021). The bamA missense mutation in C14 could lead to diminished assembly of β-barrel proteins, including TolC, directly (Werner and Misra 2005), or indirectly (Bennion et al 2010).…”

Divergent LD-transpeptidase-independent effects of peptidoglycan carboxypeptidases on intrinsic β-lactam and vancomycin resistance

“…E. coli has seven PG endopeptidases, which seem to play distinct roles in cross-link cleavage for PG synthesis (6), and seven PG carboxypeptidases (3). After the removal of the fifth D-alanine by PG carboxypeptidases, the third meso-DAP of the PG tetrapeptide can be covalently cross-linked to the third meso-DAP of adjacent peptide chains by some LD-transpeptidases (LdtD and LdtE) or to the lysine residue of an abundant outer membrane (OM) lipoprotein Lpp (Braun's lipoprotein) by other LD-transpeptidases (LdtA, LdtB, and LdtC) (7)(8)(9). The covalent cross-links between the peptide chain of PG and Lpp provide a tight connection between PG and OM, which increases the envelope integrity (10).…”