A gene, mpl, encoding UDP-N-acetylmuramate:L-alanyl-␥-D-glutamyl-meso-diaminopimelate ligase was recognized by its amino acid sequence homology with murC as the open reading frame yjfG present at 96 min on the Escherichia coli map. The existence of such an enzymatic activity was predicted from studies indicating that reutilization of the intact tripeptide L-alanyl-␥-D-glutamyl-meso-diaminopimelate occurred and accounted for well over 30% of new cell wall synthesis. Murein tripeptide ligase activity could be demonstrated in crude extracts, and greatly increased activity was produced when the gene was cloned and expressed under control of the trc promoter. A null mutant totally lacked activity but was viable, showing that the enzyme is not essential for growth.The biosynthesis of bacterial cell wall peptidoglycan (murein) is a complex process involving many different cytoplasmic and membrane steps (for review, see references 11, 24, and 30). The main cytoplasmic precursors of Escherichia coli are a series of seven uridine nucleotides, from UDP-N-acetylglucosamine to UDP-N-acetylmuramyl-L-Ala-␥-D-Glu-meso-A 2 pm-D-Ala-D-Ala (where A 2 pm represents diaminopimelic acid). Their sequential formation is catalyzed by a set of highly specific enzymes, designated as Mur activities (MurA to MurF [ Fig. 1]). Subsequent steps located in the membrane result in the synthesis of the disaccharide pentapeptide MurNAc(-pentapeptide)-GlcNAc, which is anchored in the membrane via the lipid carrier undecaprenyl-phosphate. The disaccharide pentapeptide units are then used in polymerization reactions (transglycosylation and transpeptidation) catalyzed by the wellknown bifunctional penicillin-binding proteins. In order to enlarge the murein sacculus during normal cell growth and division, new peptidoglycan subunits have to be inserted into the preexisting network, a process which probably begins with a specific cleavage of some covalent bonds by peptidoglycan hydrolases (9,29).A large portion of the murein sacculus of E. coli is degraded each generation, resulting in the formation of various degradation products which can be detected in the culture medium (8). One of them, the tripeptide L-alanyl-␥-D-glutamyl-meso-A 2 pm, is of particular interest, since it was shown to be efficiently reutilized by the cell to form new cell wall murein via a reaction sequence termed "the recycling pathway" (6-8, 25, 26). This pathway involves five different known hydrolases and a permease. However, reutilization of the tripeptide was ultimately dependent on a hypothetical ligase which could link tripeptide to UDP-N-acetylmuramic acid (Fig. 1). This paper demonstrates the existence of such an enzyme and presents the initial characterization of its properties as well as identification and mapping of its gene, mpl (which codes for murein peptide ligase). MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The E. coli strains used in this study are listed in Table 1. Plasmid pFD12, a pBR322 derivative carrying an 18.5-kb chromosomal fr...