Peptidoglycan hydrolase of an unusual cross-link cleavage specificity contributes to bacterial cell wall synthesis Pavan Kumar Chodisettia and Manjula Reddya,1 aCentre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India 500007 Edited by Carol A. Gross, University of California, San Francisco, CA, and approved March 13, 2019 (received for review October 2, 2018) Bacteria are surrounded by a protective exoskeleton, peptidoglycan Synthesis of PG is a complex process that occurs in two distinct (PG), a cross-linked mesh-like macromolecule consisting of glycan cellular compartments: the cytosol and the periplasm (8). The strands interlinked by short peptides. Because PG completely precursor UDP-MurNAc-pentapeptide is synthesized in the cytosol encases the cytoplasmic membrane, cleavage of peptide cross- by sequential enzymatic reactions catalyzed by MurA, -B, -C, -D, -E, links is a prerequisite to make space for incorporation of nascent and -F before being transferred to a membrane-bound lipid carrier glycan strands for its successful expansion during cell growth. In (C55-bactoprenol phosphate) to generate lipid I. Subsequently, a most bacteria, the peptides consist of L-alanine, D-glutamate, meso- molecule of UDP-GlcNAc is added to lipid I to yield lipid II, which diaminopimelic acid (mDAP) and D-alanine (D-Ala) with cross-links is then flipped across the inner membrane into the periplasm. Here, occurring either between D-Ala and mDAP or two mDAP residues. the membrane-bound D,D-transpeptidases catalyze the formation of Escherichia coli − 4 5 In ,theD-Ala mDAP cross-links whose cleavage by 4−3 cross-links by cleaving the D-Ala −D-Ala peptide bond of the 4 specialized endopeptidases is crucial for expansion of PG predomi- incoming disaccharide pentapeptide (donor) to link the D-Ala to − 3 nate. However, a small proportion of mDAP mDAP cross-links also the D-center of the mDAP residue of an adjacent peptide chain exist, yet their role in the context of PG expansion or the hydrolase(s) (acceptor) (6). On the contrary, L,D-transpeptidases (LdtD and capable of catalyzing their cleavage is not known. Here, we identi- LdtE) catalyze the formation of 3−3 cross-links by cleaving the fied an ORF of unknown function, YcbK (renamed MepK), as an 3 4 BIOCHEMISTRY mDAP −D-Ala peptide bond of an existing tetrapeptide of the PG mDAP−mDAP cross-link cleaving endopeptidase working in conjunc- 3 3 sacculus (donor) to link the mDAP to the D-center of the mDAP tion with other elongation-specific endopeptidases to make space residue of an adjacent peptide (acceptor) (9). for efficient incorporation of nascent PG strands into the sacculus. Because the PG sacculus is a continuous molecular network E. coli mutants lacking mepK and another D-Ala−mDAP–specific en- that completely encircles the cytoplasmic membrane, the growth dopeptidase (mepS) were synthetic sick, and the defects were abro- of a cell is tightly coupled to expansion of PG, requiring the co- gatedbylackofL,D-transpeptidases, enzymes catalyzing the ordinated activity of hydrolases that cleave the cross-links and formation of mDAP cross-links. Purified MepK was able to cleave synthases that form the cross-links (1). Given that the PG is the mDAP cross-links of soluble muropeptides and of intact PG sac- – – culi. Overall, this study describes a PG hydrolytic enzyme with a interconnected by two types of cross-bridges (4 3 and 3 3), it is hitherto unknown substrate specificity that contributes to expansion expected that the cleavage of both of these cross-links is a pre- of the PG sacculus, emphasizing the fundamental importance of requisite to make space for the incorporation of incoming murein cross-link cleavage in bacterial peptidoglycan synthesis. strands for its successful expansion. We previously showed cleav- age of 4−3 cross-links is crucial for PG enlargement as an E. coli bacteria | peptidoglycan | mDAP cross-link | MepK | YcbK mutant lacking three D,D-endopeptidases, MepS, MepM, and MepH ell envelopes of bacteria have a mesh-like exoskeleton called Significance Cpeptidoglycan (PG; also called murein) to protect them against turgor and environmental stress conditions. It also con- Bacteria contain peptidoglycan (PG) in their cell envelope to pro- fers mechanical strength and shape to bacterial cells. PG is a tect them against intracellular osmotic pressure and environmental single, large, covalently cross-linked macromolecule made up of stress. PG is a large elastic polymermadeupofglycanstrands interlinked by short peptide chains that form a mesh-like sacculus. multiple linear glycan strands that are interconnected by short In many bacteria, the peptide cross-links are of two types: the peptide chains (Fig. 1) (1–4). The glycan strands are polymers of predominant D-alanine−meso-diaminopimelic acid (mDAP) and the β alternating -1,4-linked N-acetylglucosamine (GlcNAc) and N- rare mDAP−mDAP cross-links. Here, we report the importance of acetylmuramic acid (MurNAc) disaccharide units in which the D- mDAP cross-links in PG synthesis during cell growth by identifying lactoyl moiety of each MurNAc residue is covalently attached to a previously unknown hydrolytic enzyme that cleaves such cross- the first amino acid of the peptide chain. Typically, the peptide links in the PG sacculi of Escherichia coli.Insummary,thisstudy chains consist of two to five amino acids, and in Escherichia coli, clarifies the role of PG hydrolysis in bacterial cell wall synthesis, 1 a pentapeptide consists of L-alanine (L-Ala )−D-glutamic acid (D- thereby rendering it an alternative drug target for development of 2 3 4 5 Glu )−meso-diaminopimelic acid (mDAP )−D-Ala −D-Ala . Nearly new antimicrobial agents. 40% of the neighboring peptide chains are cross bridged to each 4 3 − Author contributions: P.K.C. and M.R. designed research; P.K.C. performed research; other, either between the D-Ala and mDAP (D-Ala mDAP, or P.K.C. and M.R. analyzed data; and P.K.C. and M.R. wrote the paper. − 3 3 − − 4 3) or between mDAP and mDAP (mDAP mDAP, or 3 3) The authors declare no conflict of interest. − residues (2, 5). Of these, the 4 3 cross-links are more predominant This article is a PNAS Direct Submission. ∼ ( 93%) and are catalyzed by D,D-transpeptidase activity of high- Published under the PNAS license. molecular-weight penicillin-binding proteins (PBPs) that include 1To whom correspondence should be addressed. Email: [email protected]. − PBP1A, PBP1B, PBP2, and PBP3 (6). On the contrary, 3 3 cross- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. links are much less abundant (about 7%) and result from the ac- 1073/pnas.1816893116/-/DCSupplemental. tivity of L,D-transpeptidases LdtD and LdtE (7). www.pnas.org/cgi/doi/10.1073/pnas.1816893116 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 and SI Appendix,Fig.S1A). However, the ΔmepK single mutant did not exhibit any discernible phenotype except a moderate increase in sensitivity to certain β-lactam antibiotics such as ampicillin and cephalexin (ref. 13, SI Appendix,Fig.S1B). MepK belongs to the M15 family of peptidases that also comprises DdpX, a peptidase that cleaves D-Ala−D-Ala di- peptide in the cytosol of E. coli (14, 15). In addition, a mepK homolog of Klebsiella pneumoniae is predicted to be a PG hy- drolase (16). Hence, we tested whether increased expression of mepK is able to compensate the loss of MepS and/or MepM, the elongation-specific PG hydrolases (10). The absence of MepS results in sensitivity on NA at 42 °C (17), and this growth defect was significantly suppressed when mepK (Ptrc::mepK) was introduced in multiple copies (Fig. 2C). In addition, mepK overexpression suppressed the vancomycin sensitivity of a ΔmepM deletion mutant, indicating that MepK compensates for the loss of either mepS or mepM (Fig. 2D). A double Fig. 1. Schematic representation of the PG sacculus of E. coli. The structure mutant lacking mepS and mepM does not grow on rich media and composition of glycan strands and peptide chains are shown. Listed are the known PG hydrolases of E. coli (3, 4, 10). Cleavage sites of the hydrolases such as LB but grows well on minimal media (10). Additional are indicated by the scissors symbol. MepK (indicated in red) is identified in copies of mepK could also support, albeit partially, the growth this study. of a ΔmepS ΔmepM double mutant on LB (SI Appendix,Fig. S2A). Moreover, deletion of mepK conferred significant ad- ditive sickness to the ΔmepS ΔmepM strain growing on min- (specific to D-Ala−mDAP cross bridges), is unable to incorporate imal media, with the triple-deletion strain growing very poorly new murein and undergoes rapid lysis (10). However, it is not clear with extensive lysis and cell death (SI Appendix,Fig.S2B and how the 3−3 cross-linkages affect PG enlargement because they are C). These observations collectively indicated contribution of also expected to hinder opening of the mesh for the incorporation of MepK to the functions of elongation-specific D,D-endopepti- new PG material. dases, MepS and MepM. In this study, we show that 3−3 cross-link cleavage contributes to PG enlargement by identifying an enzyme of previously unknown MepK Modulates mDAP−mDAP Cross-Links of Peptidoglycan. As the specificity, YcbK (renamed murein endopeptidase K, MepK), as a above observations suggest that MepK is a PG peptidase, we murein hydrolase that cleaves 3−3 cross-links in E. coli. Extensive examined the composition of PG of a mutant lacking MepK. The genetic and molecular analyses indicate that mepK functions in PG sacculi from WT and the ΔmepK mutant were prepared and conjunction with other elongation-specific D,D-endopeptidases to digested with a muramidase (mutanolysin), and the resulting contribute to synthesis of PG.