Bacteria are surrounded by a protective exoskeleton, peptidoglycan (PG), a cross-linked mesh-like macromolecule consisting of glycan strands interlinked by short peptides. Because PG completely encases the cytoplasmic membrane, cleavage of peptide cross-links is a prerequisite to make space for incorporation of nascent glycan strands for its successful expansion during cell growth. In most bacteria, the peptides consist of l-alanine, d-glutamate, meso-diaminopimelic acid (mDAP) and d-alanine (d-Ala) with cross-links occurring either between d-Ala and mDAP or two mDAP residues. In Escherichia coli, the d-Ala−mDAP cross-links whose cleavage by specialized endopeptidases is crucial for expansion of PG predominate. However, a small proportion of mDAP−mDAP cross-links also exist, yet their role in the context of PG expansion or the hydrolase(s) capable of catalyzing their cleavage is not known. Here, we identified an ORF of unknown function, YcbK (renamed MepK), as an mDAP−mDAP cross-link cleaving endopeptidase working in conjunction with other elongation-specific endopeptidases to make space for efficient incorporation of nascent PG strands into the sacculus. E. coli mutants lacking mepK and another d-Ala−mDAP–specific endopeptidase (mepS) were synthetic sick, and the defects were abrogated by lack of l,d-transpeptidases, enzymes catalyzing the formation of mDAP cross-links. Purified MepK was able to cleave the mDAP cross-links of soluble muropeptides and of intact PG sacculi. Overall, this study describes a PG hydrolytic enzyme with a hitherto unknown substrate specificity that contributes to expansion of the PG sacculus, emphasizing the fundamental importance of cross-link cleavage in bacterial peptidoglycan synthesis.

Peptidoglycan is a defining feature of the bacterial cell wall. Initially identified as a target of the revolutionary beta-lactam antibiotics, peptidoglycan has become a subject of much interest for its biology, its potential for the discovery of novel antibiotic targets, and its role in infection. Peptidoglycan is a large polymer that forms a mesh-like scaffold around the bacterial cytoplasmic membrane. Peptidoglycan synthesis is vital at several stages of the bacterial cell cycle: for expansion of the scaffold during cell elongation and for formation of a septum during cell division. It is a complex multifactorial process that includes formation of monomeric precursors in the cytoplasm, their transport to the periplasm, and polymerization to form a functional peptidoglycan sacculus. These processes require spatio-temporal regulation for successful assembly of a robust sacculus to protect the cell from turgor and determine cell shape. A century of research has uncovered the fundamentals of peptidoglycan biology, and recent studies employing advanced technologies have shed new light on the molecular interactions that govern peptidoglycan synthesis. Here, we describe the peptidoglycan structure, synthesis, and regulation in rod-shaped bacteria, particularly Escherichia coli , with a few examples from Salmonella and other diverse organisms. We focus on the pathway of peptidoglycan sacculus elongation, with special emphasis on discoveries of the past decade that have shaped our understanding of peptidoglycan biology.

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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.