Colicin M Exerts Its Bacteriolytic Effect via Enzymatic Degradation of Undecaprenyl Phosphate-linked Peptidoglycan Precursors

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-MurNAcpentapeptide or 1-pyrophospho-MurNAc-(pentapeptide)-GlcNAc 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.Colicins are plasmid-encoded toxins, synthesized and released in the growth medium by Escherichia coli, which kill susceptible E. coli strains and closely related bacterial species (1-3). Strains are protected against the colicin they produce by concomitant expression of a highly specific immunity protein.The lethal action of colicins can be divided into three steps:binding to a specific outer membrane receptor protein, translocation through the cell envelope, and finally interaction with the target and killing effect. To each of these steps corresponds a specific protein domain, the different colicins showing a similar three-domain structural organization. Depending on the import pathway they parasitize to enter the cells, the Tol system or the TonB system, colicins are classified into two groups A and B, respectively (3). The mode of action of colicins is variable: formation of voltage-gated pores in the cytoplasmic membrane (e.g. colicins A, B, E1, Ia, N), inhibition of protein synthesis (e.g. colicins D and E3), enzymatic degradation of cellular DNA or 16S rRNA (e.g. colicin E2) and inhibition of cell envelope biosynthesis (colicin M, see below).Various bacterial cell envelope polysaccharides (peptidoglycan, O-antigen, teichoic acid, capsular polysaccharide) in both Gram-negative and Gram-positive bacteria have a lipid-linked intermediary stage in their biosynthesis that is dependent on the essential carrier lipid undecaprenyl phosphate (C 55 -P) 4 (4 -15) (Scheme 1). In the peptidoglycan pathway, this lipid is needed for the synthesis and trans...

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…These take the form of a nuclease domain that specifically targets DNA, tRNA, or rRNA or a pore-forming domain that targets the cytoplasmic membrane (9,10). In addition, colicin M and bacteriocins with homologous catalytic domains kill susceptible cells through a highly specific phosphatase activity that targets lipid II (11). Cleavage of lipid II at the phosphoester bond between the undecaprenyl and pyrophosphate moieties prevents recycling of undecaprenyl phosphate, thus preventing the translocation of peptidoglycan precursors across the inner membrane (12).…”

Lipid Intermediates in the Biosynthesis of Bacterial Peptidoglycan

“…Their ready availability is thus of primary importance. Lipid I and analogues were synthesized mainly by chemical methods, but the successful synthesis of lipid I by use of purified MraY (25,60) could be extended to that of analogues. A possible drawback of this approach is the easy reversibility of the MraY-catalyzed reaction requiring the use of high lipid phosphate concentrations or a specific way of removing formed UMP.…”

“…They involved the coupling of protected phospho-N-acetylmuramoylpentapeptide to undecaprenyl monophosphate by application of the 1,1Ј-carbonyldiimidazole (197) or the phosphoroimidazolidate (183) method. Finally, the availability of purified MraY transferase has enabled the enzymatic synthesis of natural A 2 pm-containing lipid I from undecaprenyl phosphate and UDP-MurNAc-pentapeptide (25,60). The synthesis of lipid II and its analogues was recently reviewed in detail (191).…”

“…2). The structures of the lipid intermediates from various organisms and those of analogues were studied by mass spectrometry without prior modification (29,153), after conversion to reduced muropeptides (107), or after specific enzymatic degradation by colicin M into pyrophospho-muropeptides (60). It is noteworthy that the ␣-anomeric configuration of the N-acetylmuramic acid residue in UDP-MurNAc-pentapeptide is conserved in lipids I and II (Fig.…”

Deciphering the Metabolism of Undecaprenyl-Phosphate: The Bacterial Cell-Wall Unit Carrier at the Membrane Frontier

“…While E. coli cells do not usually present any detectable amount of C 55 -OH within their membranes, the accumulation of this compound was found to occur when these cells were subjected to a bacteriocin called colicin M (ColM) 24 ( Fig. 2).…”