Genetic Evidence for Distinct Functions of Peptidoglycan Endopeptidases in Escherichia coli.

Si Hyoung Park,Han Byeol Lee,Chang-Ro Lee,Yung Jae Kim,Yeong-Jae Seok

doi:10.3389/fmicb.2020.565767

Si Hyoung Park, Han Byeol Lee + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2020.565767

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Abstract

Peptidoglycan (PG) is an essential component of the bacterial exoskeleton that plays a pivotal role in the maintenance of cell shape and resistance to cell lysis under high turgor pressures. The synthesis and degradation of PG must be tightly regulated during bacterial cell elongation and division. Unlike enzymes involved in PG synthesis, PG hydrolases show high redundancy in many bacteria including Escherichia coli. In this study, we showed that PG endopeptidases have distinct roles in cell growth and division. Phenotypic analysis of mutants lacking one of seven PG endopeptidases identified a MepM-specific phenotype, salt sensitivity, and a MepS-specific phenotype, EDTA sensitivity. Complementation test in each phenotype showed that the phenotype of the mepM mutant was restored only by MepM, whereas the phenotype of the mepS mutant was restored by MepS or by overexpression of MepH, PbpG, or MepM. These distinct phenotypes depend on both the specific localizations and specific domains of MepM and MepS. Finally, using the identified phenotypes, we revealed that MepM and MepH were genetically associated with both penicillin-binding protein 1a (PBP1a) and PBP1b, whereas MepS and PbpG were genetically associated with only PBP1b. Notably, a defect in PBP1a or PBP1b phenocopied the mepM mutant, suggesting the importance of MepM on PG synthesis. Therefore, our results indicate that each PG endopeptidase plays a distinct role in cell growth and division, depending on its distinct domains and cellular localizations.

Highlights

Peptidoglycan (PG) is a macromolecule that forms a rigid mesh-like exoskeleton, which is required for shape maintenance and protection of bacteria from harsh environmental stresses, such as turgor pressure (Vollmer and Bertsche, 2008)
To analyze the physiological roles of redundant PG endopeptidases, we constructed deletion mutants of all proteins with PG endopeptidase activity and examined the bacterial growth under various stress conditions, including envelope, oxidative, ethanol, EDTA, and carbon/nitrogen starvation stresses (Supplementary Figure S1). These experiments revealed that the growth of the mepM mutant was completely inhibited under salt stress, whereas the growth of the mepS mutant was completely inhibited under EDTA stress (Figure 1)
Western blot analysis using an anti–Flag-tag antibody showed that all chimeric proteins were sufficiently expressed in the cells (Supplementary Figure S11). These results strongly suggest that the distinct roles of MepM and MepS are inimitable by targeting other PG endopeptidases to the inner membrane (IM) and outer membrane (OM), respectively

Summary

Introduction

Peptidoglycan (PG) is a macromolecule that forms a rigid mesh-like exoskeleton, which is required for shape maintenance and protection of bacteria from harsh environmental stresses, such as turgor pressure (Vollmer and Bertsche, 2008). The glycan strands are made of alternating β-1,4-linked sugars, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), and a short-peptide chain composed of 2–5-amino-acid residues is covalently attached to the D-lactoyl moiety of each MurNAc. In Escherichia coli, the peptide chain is composed of L-alanine, D-glutamic acid, meso-diaminopimelic acid (meso-DAP), D-alanine, and D-alanine, and the cross-links between the Distinct Functions of Peptidoglycan Endopeptidases neighboring peptide chains take place predominantly between the fourth D-alanine and the third meso-DAP or minorly between the third meso-DAP and the third meso-DAP (Glauner et al, 1988; Vollmer and Bertsche, 2008). Lytic transglycosylases cleave the β-1,4-glycosidic bond between MurNAc and GlcNAc and catalyze the formation of an anhydro linkage between the C1 and C6 residues of MurNAc, which results in the formation of 1,6-anhydromuramic acid products (GlcNAc-anhydroMurNAc-peptide) (Holtje et al, 1975; Dhar et al, 2018). Based on the two isomeric forms of the cleaved amino acids, peptidases can be divided into DD, DL, and LD peptidases (Vollmer et al, 2008)

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