Abstract
Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.
Highlights
A key component of the bacterial cell envelope is the cell wall matrix, an essential structure that fortifies the cytoplasmic membrane against osmotic rupture and confers cell shape[1]
Mutants lacking the cell wall synthase PBP1b are viable, they lyse at an elevated frequency 26 and are hypersensitive to beta-lactam antibiotics 27, indicating they have an attenuated capacity for PG synthesis
We reasoned that a screen for genes rendering PBP1b essential when they are overexpressed from a multicopy plasmid might identify previously uncharacterized inhibitors of PBP1a/LpoA or novel PG-cleaving enzymes that damage the PG matrix to an extent that is not tolerated when cell wall synthesis is compromised by PBP1b inactivation
Summary
A key component of the bacterial cell envelope is the cell wall matrix, an essential structure that fortifies the cytoplasmic membrane against osmotic rupture and confers cell shape[1]. Any perturbations to the process of PG biogenesis can lead to a catastrophic breach in the cell wall and cause cell lysis. This sequence of events is exploited by many antibiotics used clinically to treat infections, including penicillin and vancomycin. An attractive avenue towards the development of such therapies is the identification of additional weak points in the cell wall biogenesis pathway amenable to antibiotic targeting. These novel vulnerabilities are most likely to be discovered through a greater understanding of the fundamental mechanisms underlying the process
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