Peptidoglycan hydrolase of an unusual cross-link cleavage specificity contributes to bacterial cell wall synthesis

Abstract

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.

Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi‐enzyme complexes in Escherichia coli

“…Escherichia coli contains a repertoire of more than 20 periplasmic hydrolases providing specificity to almost every bond present in PG (Vollmer et al, 2008b;van Heijenoort, 2011;Singh et al, 2012;Yunck et al, 2016;Chodisetti & Reddy, 2019). However, with the exception of amidases, it is unclear how these hydrolases are regulated to prevent autolysis (Uehara et al, 2009).…”

An inducible AraC that responds to blue light instead of arabinose

“…Since light is particularly well-suited for medium to highthroughput studies due to its low cost, scalability, and effortless application, we used BLADE to characterize some of these genes in terms of their intracellular localization and effect on cell growth and morphology. We randomly selected 34 completely uncharacterized genes and included 5 additional genes, for which some information was available: ydaT, which was shown to lead to cell elongation and reduced survival when overexpressed in E. coli 37 ; ydiY, which was shown to be induced by acid and was predicted to be an outer membrane protein 38 ; ycbK (renamed MepK), which was shown to be a murein hydrolase involved in cell wall synthesis 39 ; yehS, whose downregulation has been shown to improve the growth of E. coli in n-butanol and n-hexane ; and yebE, which was shown to be induced by copper in a CpxA/CpxR-dependent manner 41 , and was predicted to be localized to the inner membrane 42 . Importantly, fluorescence microscopy-based localization studies have not been so far carried out for any of these 39 genes.…”

Building peptidoglycan inside eukaryotic cells: A view from symbiotic and pathogenic bacteria

“…A vast number of biosynthetic and hydrolytic extracytosolic enzymes contribute to this PG structural plasticity. On an average, it is estimated that a minimum of 40 extracytosolic enzymes act on different bonds of the PG structure (Chodisetti & Reddy, 2019;Otten, Brilli, Vollmer, Viollier, & Salje, 2018;Sanders & Pavelka, 2013;Scheurwater, Reid, & Clarke, 2008;Vermassen et al, 2019). The bases for so many extracytosolic enzymes that exceed in number to what is needed to synthetize or cleave the bonds existing in the PG (Figure 1b), remain poorly understood.…”

“…In Gramnegative bacteria, l,d-transpeptidation also play a housekeeping role in the covalent anchoring of the outer membrane Braun lipoprotein to the PG (Magnet et al, 2007). l,d (3-3)-bridges are also recognized by dedicated endopeptidases (Chodisetti & Reddy, 2019;Vollmer & Bertsche, 2008) and the lack of some of these hydrolases result in growth defects that are abrogated in the absence of l,d-transpeptidases (Chodisetti & Reddy, 2019). Such loss of cleavage in the l,d (3-3)-bridges could compromise the transit of facultative intracellular pathogens between host and non-host environments, a tempting hypothesis to explore in future studies.…”