Involvement of N-acetylmuramyl-L-alanine amidases in cell separation and antibiotic-induced autolysis of Escherichia coli

N-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. H owever, in Escherichia coil, only one periplasmic amidase has been describe d until now, which is suggested to play a role in murein recycling, Here, w e report that three amidases, named AmiA, B and C, exist in E, coil and tha t 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, S and C were growing in long chains of unseparated cells and displayed a tolerant response to t he normally lytic combination of aztreonam and bulgecin, Isolated murein sa cculi of these chain-forming mutants showed rings of thickened murein at th e site of blocked septation. In vitro, these murein ring structures were di gested more slowly by muramidases than the surrounding murein, In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings di sappeared, and gaps developed at these sites in the murein sacculi, These r esults are taken as evidence that highly stressed murein crossbridges are c oncentrated at the site of blocked cell division, which, when cleaved, resu lt in cracking of the sacculus at this site. As amidase deletion mutants ac cumulate trimeric and tetrameric cross-links in their murein, it is suggest ed that these structures mark the division site before cleavage of the sept um.