N-acetylmuramyl-
L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic
amidase has been described until now, which is suggested to play a role in
murein recycling. Here, we report that three
amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the
murein septum during cell division. Moreover, the
amidases were shown to act as powerful autolytic
enzymes in the presence of
antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of
aztreonam and
bulgecin. Isolated
murein sacculi of these chain-forming mutants showed rings of thickened
murein at the site of blocked septation. In vitro, these
murein ring structures were digested more slowly by muramidases than the surrounding
murein. In contrast, when treated with the
amidase AmiC or the
endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the
murein sacculi. These results are taken as evidence that highly stressed
murein cross-bridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As
amidase deletion mutants accumulate trimeric and tetrameric cross-links in their
murein, it is suggested that these structures mark the division site before cleavage of the septum.