Evidence for a role of N-acetylmuramyl-L-alanine amidase in septum separation in Escherichia coli

Abstract

Septum formation and septum separation have been studied in a chain-forming mutant of Escherichia coli K-12 bearing the envA mutation and its parental strain. In comparison to the wild type, the mutant showed a sixfold reduction in the specific activity of the enzyme, N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), part of which was associated to the outer membrane. Genetic as well as physiological suppression of chain formation resulted in an increase in amidase activity. The addition of N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid to growing wild-type cells and to cells bearing the envA mutation caused an inhibition of cell separation and an increased frequency of visible septa. The kinetics of septum formation and separation was followed in chains by the use of ampicillin and nalidixic acid. The latter drug inhibited initiation of new septa but allowed preformed ones to go to cell separation at a rate corresponding to that of steady-state growing cells. Ampicillin treatment, on the other hand, resulted in a more rapid decrease in the frequency of septa. The disparate effects of ampicillin and nalidixic acid were not explained by a difference in amidase activity but could be due to an inhibitory effect of ampicillin on a septal peptidoglycan fusing activity.