Role of endopeptidases in lateral cell wall expansion in Escherichia coli.

Abstract

Peptidoglycan, the major constituent of bacterial cell walls, is a giant macromolecule made of glycan strands cross-linked by short peptides, which provides resistance to the turgor pressure of the cytoplasm. We explored the mechanism of insertion of newly synthesized subunits into the existing peptidoglycan mesh by high-resolution mass spectrometry analysis of peptidoglycan labeled with heavy carbon and nitrogen isotopes in Escherichia coli mutants conditionally producing essential peptidoglycan biosynthesis enzymes. We show that side-wall expansion at a constant diameter is mediated by endopeptidases that cleave existing peptidoglycan cross-links in order to enable the one-at-a-time insertion of newly synthesized glycan strands into the stress-bearing peptidoglycan layer by transpeptidases. These results provide direct experimental evidence for the essential participation of endopeptidases in the biosynthesis of the lateral wall. They also suggest that transpeptidases and endopeptidases might act in a coordinated, rather than sequential, manner.