Abstract
Colicin M was earlier demonstrated to provoke Escherichia coli cell lysis via inhibition of cell wall peptidoglycan (murein) biosynthesis. As the formation of the O-antigen moiety of lipopolysaccharides was concomitantly blocked, it was hypothesized that the metabolism of undecaprenyl phosphate, an essential carrier lipid shared by these two pathways, should be the target of this colicin. However, the exact target and mechanism of action of colicin M was unknown. Colicin M was now purified to near homogeneity, and its effects on cell wall peptidoglycan metabolism reinvestigated. It is demonstrated that colicin M exhibits both in vitro and in vivo enzymatic properties of degradation of lipid I and lipid II peptidoglycan intermediates. Free undecaprenol and either 1-pyrophospho-MurNAc-pentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-Glc-NAc were identified as the lipid I and lipid II degradation products, respectively, showing that the cleavage occurred between the lipid moiety and the pyrophosphoryl group. This is the first time such an activity is described. Neither undecaprenyl pyrophosphate nor the peptidoglycan nucleotide precursors were substrates of colicin M, indicating that both undecaprenyl and sugar moieties were essential for activity. The bacteriolytic effect of colicin M therefore appears to be the consequence of an arrest of peptidoglycan polymerization steps provoked by enzymatic degradation of the undecaprenyl phosphate-linked peptidoglycan precursors.
Highlights
Binding to a specific outer membrane receptor protein, translocation through the cell envelope, and interaction with the target and killing effect
To identify the step of peptidoglycan synthesis blocked by colicin M, these authors analyzed the distribution of radiolabeled diaminopimelic acid (A2pm) in pulse-labeled colicin-treated cultures of E. coli and they observed an accumulation of the soluble peptidoglycan precursors and the depletion of the pools of the two lipid intermediates I and II
Expression and Purification of Colicin M—The cma gene encoding colicin M was cloned into plasmid pTrc99A and derivative vectors allowing its expression under the control of the strong IPTG-inducible trc promoter in either wildtype, N-terminal, or C-terminal His6-tagged form
Summary
Colicin M exhibits a unique mode of action as it is the only colicin known to inhibit peptidoglycan synthesis and cause cell lysis [31]. As the C55-P carrier lipid is required in both processes, Harkness and Braun [32] earlier hypothesized that the synthesis or recycling of this compound could be the target of colicin M. To identify the step of peptidoglycan synthesis blocked by colicin M, these authors analyzed the distribution of radiolabeled diaminopimelic acid (A2pm) in pulse-labeled colicin-treated cultures of E. coli and they observed an accumulation of the soluble peptidoglycan precursors (mainly UDP-MurNAc-pentapeptide) and the depletion of the pools of the two lipid intermediates I and II. The depletion of the pools of these precursors results in an inhibition of peptidoglycan polymerization steps and cell lysis. To the best of our knowledge, this is the first time such an activity has been described to date
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