Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coliK12

Abstract

SummaryBacterial peptidoglycan (PG or murein) is a single, large, covalently cross-linked macromolecule and forms a mesh-like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre-existing crosslinks for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross-bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross-link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.

Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape

“…Sacculus growth requires at least three types of enzymes: transglycosylases to synthesize new glycan strands, transpeptidases to crosslink them together, and endopeptidases to cleave existing peptide crosslinks to open space for new material (9,42,43). E. coli has other hydrolases that can modify PG, but none of them have been shown to be essential for rod shape maintenance (2,44,45).…”

“…This remodeling process requires the presence of not only transglycosylases and transpeptidases, also known as penicillin-binding proteins (PBPs), to polymerize and crosslink new glycan strands into the existing network (2,8) but also, endopeptidases to cleave covalent bonds to open space for the new material (9). How these small enzymes work together to maintain the order and rod shape of a sacculus three orders of magnitude larger is not understood.…”

Dynamic protein complexes for cell growth

“…For example, although PBP1A is associated with cell elongation, a second bifunctional PG synthase, PBP1B, can cover for its absence. A similar redundancy exists for the hydrolases involved in cell elongation (8). Furthermore, in E. coli, the bifunctional PG synthases, PBP1A and PBP1B, have dedicated regulators, LpoA and LpoB, which localize independently to elongation and divisions sites, respectively (9,10).…”

Molecular Dissection of Phage Endolysin

“…The YebA of E. coli is a murein hydrolase that has metal-dependent endopeptidase activity (35) and contains a LysM domain that is known to bind peptidoglycan (47,48). The purified preparation of YebA showed binding to PG of E. coli, M. smegmatis, and M. tuberculosis when probed with anti-His 6 tag antibodies.…”

“…The green fluorescent protein (GFP) was expressed and purified as detailed earlier (34). The plasmid, pMN86 (kindly provided by Manjula Reddy, Centre for Cellular and Molecular Biology, Hyderabad) was used for expressing and purifying C-terminal histidine-tagged YebA 40 -440 as described (35). Zymography-Zymograms were prepared to test the activity of the purified proteins using 0.2% Micrococcus lysodeikticus cells (Sigma) as substrate in a 12% SDS-polyacrylamide gel.…”