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Synthesis of peptidoglycan lipid-linked intermediates and mode of action of ColM. The inner (IM) and outer (OM) membranes are depicted by the gray boxes. The MraY enzyme catalyzes the transfer of the phospho-MurNAc-pentapeptide from the nucleotide precursor onto the carrier lipid undecaprenyl phosphate (C 55-P), yielding lipid I; subsequently, the MurG enzyme adds the GlcNAc moiety, yielding lipid II, which is translocated from the inner side of the membrane to the outer side (38, 39). Thereafter, the disaccharide-pentapeptide motif is polymerized and incorporated into the peptidoglycan through the action of the penicillin-binding proteins (PBPs) releasing the lipid carrier in a pyrophosphate form that will be recycled (40, 41). Incoming ColM was shown to cleave the lipid-linked intermediates between the C 55 and pyrophosphoryl-disaccharide-peptide motifs (6). In the present report, an independent ColM toxicity domain (green) was identified (residues 124 –271), which was active in vivo under conditions where the FhuA receptor was by-passed. In contrast to the full-length ColM, the independent toxicity domain did not require the periplasmic FkpA protein to be toxic. These data suggest that ColM exists in two active (A) and inactive (I) conformations, FkpA being involved in the activation process. The reception and translocation domains of ColM are represented in red and yellow, respectively.
Source publication
Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini...
Citations
... Inside each of the two groups, colicins and colicin-like bacteriocins may be differentiated according to their bactericidal mechanisms [21]. Colicins kill target cells through three different mechanisms: (a) by making voltage-dependent channels in the inner membrane of the target bacteria, (b) by a nuclease action in the cytoplasm (DNase, 16S rRNase, and tRNase activities), or (c) by inhibiting peptidoglycan synthesis [7,12,17,37]. In further detail, colicins undergo a series of conformational changes during the voyage from the extracellular space to the periplasmic membrane of the target cell. ...
Enterobacteriaceae are widely present in many environments related to humans, including the human body and the food that they consume, from both plant or animal origin. Hence, they are considered relevant members of the gastrointestinal tract microbiota. On the other hand, these bacteria are also recognized as putative pathogens, able to impair human health and, in food, they are considered indicators for the microbiological quality and hygiene status of a production process. Nevertheless, beneficial properties have also been associated with Enterobacteriaceae, such as the ability to synthesize peptides and proteins, which can have a role in the structure of microbial communities. Among these antimicrobial molecules, those with higher molecular mass are called colicins, while those with lower molecular mass are named microcins. In recent years, some studies show an emphasis on molecules that can help control the development of pathogens. However, not enough data are available on this subject, especially related to microcins. Hence, this review gathers and summarizes current knowledge on colicins and microcins, potential usage in the treatment of pathogen-associated diseases and cancer, as well as putative applications in food biotechnology.
... However, owing to their complex structures and mechanisms of action, pore-forming lysis systems are less competitive for building synthetic biological tools. Enzymatic lysis systems, such as colicin M (CoIM) 22 , can directly interfere with cell-wall integrity through enzymatic degradation, resulting in cell lysis. The action mode and structural organization of CoIM for lysing Escherichia coli are simple and efficient; thus, CoIM has been used in food preservation and for treating infections 23 . ...
... The CoIM-based lysis unit exhibited superior universality and controllability for lysing E. coli. On the one hand, recent studies on the structure and mechanism of action of CoIM revealed that wild-type CoIM consists of three functional domains: (i) the N-terminal translocation domain and (ii) the central FhuAbinding domain can bind to the receptor FhuA and transport CoIM across the outer membrane 33,34 ; and (iii) the C-terminal toxicity domain is required for bactericidal activity 22 . Thus, mutations of FhuA and TonB (translocation pathway) can prevent wild-type CoIM from exerting its effects, decreasing the susceptibility of E. coli to CoIM. ...
Microbial populations are a promising model for achieving microbial cooperation to produce valuable chemicals. However, regulating the phenotypic structure of microbial populations remains challenging. In this study, a programmed lysis system (PLS) is developed to reprogram microbial cooperation to enhance chemical production. First, a colicin M -based lysis unit is constructed to lyse Escherichia coli . Then, a programmed switch, based on proteases, is designed to regulate the effective lysis unit time. Next, a PLS is constructed for chemical production by combining the lysis unit with a programmed switch. As a result, poly (lactate- co -3-hydroxybutyrate) production is switched from PLH synthesis to PLH release, and the content of free PLH is increased by 283%. Furthermore, butyrate production with E. coli consortia is switched from E. coli BUT003 to E. coli BUT004, thereby increasing butyrate production to 41.61 g/L. These results indicate the applicability of engineered microbial populations for improving the metabolic division of labor to increase the efficiency of microbial cell factories.
... The catalytic domain is organized in a half, open β-barrel, which presents structural homology with the β-domain of the Hia autotransporter of Haemophilus influenzae [25]. However, the delineation of the catalytic domain according to the 3D structure is not consistent with the minimal length of the catalytic domain as determined through truncation experiments, being composed of residues 122-271 [26]. [26]: blue for the translocation domain, green for the reception domain, and red for the catalytic domain. ...
... However, the delineation of the catalytic domain according to the 3D structure is not consistent with the minimal length of the catalytic domain as determined through truncation experiments, being composed of residues 122-271 [26]. [26]: blue for the translocation domain, green for the reception domain, and red for the catalytic domain. Secondary structure elements are numbered according to their order of appearance within the primary structure of the protein. ...
... In contrast, the residual activity of the P176A ColM variant remained FkpA-dependent when it penetrated the cell via FhuA or when it was addressed to the periplasm via the Sec system (by fusing the OmpA signal sequence to the N-terminus of ColM), both modes of transport implying ColM unfolding [41]. The latter observation suggested that the isomerisation of the Phe 175 -Pro 176 bond was not the only action of FkpA towards ColM [26,36]. ...
The misuse of antibiotics during the last decades led to the emergence of multidrug resistant pathogenic bacteria. This phenomenon constitutes a major public health issue. Consequently, the discovery of new antibacterials in the short term is crucial. Colicins, due to their antibacterial properties, thus constitute good candidates. These toxin proteins, produced by E. coli to kill enteric relative competitors, exhibit cytotoxicity through ionophoric activity or essential macromolecule degradation. Among the 25 colicin types known to date, colicin M (ColM) is the only one colicin interfering with peptidoglycan biosynthesis. Accordingly, ColM develops its lethal activity in E. coli periplasm by hydrolyzing the last peptidoglycan precursor, lipid II, into two dead-end products, thereby leading to cell lysis. Since the discovery of its unusual mode of action, several ColM orthologs have also been identified based on sequence alignments; all of the characterized ColM-like proteins display the same enzymatic activity of lipid II degradation and narrow antibacterial spectra. This publication aims at being an exhaustive review of the current knowledge on this new family of antibacterial enzymes as well as on their potential use as food preservatives or therapeutic agents.
... The catalytic domain is organized in a half, open β-barrel, which presents structural homology with the β-domain of the Hia autotransporter of Haemophilus influenzae [25]. However, the delineation of the catalytic domain according to the 3D structure is not consistent with the minimal length of the catalytic domain as determined through truncation experiments, being composed of residues 122-271 [26]. In order to bind its target cell, ColM parasitizes the FhuA protein from the outer membrane. ...
... In contrast, the residual activity of the P176A ColM variant remained FkpAdependent when it penetrated the cell via FhuA or when it was addressed to the periplasm via the Sec system (by fusing the OmpA signal sequence to the N-terminus of ColM), both modes of transport implying ColM unfolding [41]. The latter observation suggested that the isomerisation of the Phe 175 -Pro 176 bond was not the only action of FkpA towards ColM [26,36]. ...
... Whatever the manner by which the wild-type ColM reaches the periplasm (FhuA/Ton, osmotic shock or Sec system), its activity is FkpA-dependent [26,36,39]. However, two studies have shown that a truncated form of ColM, corresponding to its isolated catalytic domain, exerted a cytotoxic activity in an FkpA-independent fashion as long as the FhuA/Ton system was bypassed [26,39]. ...
The misuse of antibiotics during the last decades led to the emergence of multidrug resistant pathogenic bacteria. This phenomenon constitutes a major public health issue. Consequently, the discovery of new antibacterials in the short term is crucial. Colicins, due to their antibacterial properties, thus constitute good candidates. These toxin proteins, produced by E. coli to kill enteric relative competitors, exhibit cytotoxicity through ionophoric activity or essential macromolecule degradation. Among the 25-colicin types known to date, colicin M (ColM) is the only one colicin interfering with peptidoglycan biosynthesis. Accordingly, ColM develops its lethal activity in E. coli periplasm by hydrolyzing the last peptidoglycan precursor, lipid II, into two dead-end products, thereby leading to cell lysis. Since the discovery of its unusual mode of action, several ColM orthologs have also been identified based on sequence alignments; all of the characterized ColM-like proteins display the same enzymatic activity of lipid II degradation and narrow antibacterial spectra. This publication aims at being an exhaustive review about what is currently known on this new family of antibacterial enzymes as well as on their potential use of food preservatives or therapeutic agents.
... (-pentapeptide)-GlcNAc as lipid II degradation products, and the resulting depletion of the pool of lipid II rapidly led to the arrest of peptidoglycan synthesis and, ultimately, to cell lysis (3). The crystal structure of ColM was determined (4), and active site amino acid residues were identified using site-directed mutagenesis experiments (5). Several ColM homologues that are produced by a restricted number of strains from Pseudomonas, Burkholderia, and Pectobacterium species were subsequently identified and characterized both biochemically and structurally (6)(7)(8)(9). ...
... N-Dodecyl--D-maltopyranoside (DDM) was purchased from Thermo Fisher Scientific, isopropyl--D-thiogalactopyranoside (IPTG) was purchased from Eurogentec, and Ni 2ϩ -NTA-agarose was purchased from Qiagen. Colicin M was purified as described previously (5). Mutanolysin, antibiotics, and reagents were from Sigma. ...
Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C 55 -carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C 55 -carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.
... The lipid IIhydrolyzing activity of the PaeM2 variant was confirmed in vitro, and its specific activity, estimated at 12 Ϯ 0.6 nmol/min/mg of protein, was quite similar to that of PaeM1 (13 Ϯ 0.4 nmol/min/mg) in testing performed under the same assay conditions. As shown in Fig. 1, the amino acid sequences of PaeM1 and PaeM2 mainly differed (23 changes) in their N-terminal and central regions (residues 1 to 130), which are known to be involved in the translocation and reception steps for these bacteriocins, respectively, as established previously for ColM and other colicins (1,15,16). Only six differences (in most cases, conservative) were found in the C-terminal region corresponding to the activity domain, consistent with the similar specific enzyme activities of the two PaeM variants. ...
Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.
... ns indicates no significant difference. (Schaller et al., 1982;Harkness and Braun, 1989a, b;Harkness and Braun, 1989b;Barreteau et al., 2010), which may lead to a detrimental increase in membrane permeability to Ca 2+ and result in disruption of bacterial cell physiology. This hypothesis is based on the findings that bacterial cytosolic Ca 2+ must be tightly controlled at low levels akin to those found in eukaryotic cells, and the disturbance of calcium ion homeostasis can affect bacterial survival (Dominguez, 2004;Miyamoto et al., 2005). ...
Bacteriocins are regarded as important factors mediating microbial interactions, but their exact role in community ecology largely remains to be elucidated. Here, we report the characterization of a mutant strain, derived from Pseudomonas syringae pv. tomato DC3000 (Pst), that was incapable of growing in plant extracts and causing disease. Results showed that deficiency in a previously unannotated gene saxE led to the sensitivity of the mutant to Ca²⁺ in leaf extracts. Transposon insertions in the bacteriocin gene syrM, adjacent to saxE, fully rescued the bacterial virulence and growth of the ΔsaxE mutant in plant extracts, indicating that syrM‐saxE encode a pair of bacteriocin immunity proteins in Pst. To investigate whether the syrM‐saxE system conferred any advantage to Pst in competition with other SyrM‐sensitive pathovars, we compared the growth of a SyrM‐sensitive strain co‐inoculated with Pst strains with or without the syrM gene and observed a significant syrM‐dependent growth reduction of the sensitive bacteria on plate and in lesion tissues upon desiccation–rehydration treatment. These findings reveal an important biological role of SyrM‐like bacteriocins and help to understand the complex strategies used by P. syringae in adaptation to the phyllosphere niche in the context of plant disease.
... A subset of these modular bacteriocins in pseudomonads are those equipped with a ColM domain (24), a toxin module that was first identified in colicin M of E. coli (25,26) but actually occurs in a wide variety of proteobacterial genera (27), including other gammaproteobacteria (Pectobacterium) (28), as well as betaproteobacteria (Burkholderia) (29). The ColM domain acts in the periplasm and provokes cellular killing through degradation of lipid II (30,31). Based on phylogeny, 2 large subgroups of Pseudomonas ColM bacteriocins were previously discerned (27). ...
... aeruginosa BL03) share 29% AA sequence identity, whereas their amino-terminal receptor-binding regions cannot be meaningfully aligned. The catalytic motif in the ColM domain of PaeM4 can be recognized as DxYD(x 5 )QR, slightly deviating from the equivalent motif from colicin M and PaeM, DxYD(x 5 )HR (27,31,34). However, it was found previously that limited variation in this sequence motif does not necessarily affect the bacteriocin function of ColM-type toxins (29). ...
The antimicrobial armamentarium of a bacterium is a major asset for colonizing competitive environments. Bacteriocins comprise a subset of these compounds. Pyocins are an example of such antibacterial proteins produced by Pseudomonas aeruginosa , killing other P. aeruginosa strains. A large group of these molecules show a modular protein architecture that includes a receptor-binding domain for initial target cell attachment and a killer domain. In this study, we have shown that a novel modular pyocin (PaeM4) that kills target bacteria via interference with peptidoglycan assembly takes advantage of the HxuC heme receptor. Cells can protect themselves from killing by the presence of a dedicated immunity partner, an integral inner membrane protein that adopts a transmembrane topology distinct from that of proteins currently known to provide immunity against such toxin activity. Understanding the receptors with which pyocins interact and how immunity to pyocins is achieved is a pivotal step toward the rational design of bacteriocin cocktails for the treatment of P. aeruginosa infections.
... Its carboxy-terminal catalytic domain (ColM) displays phosphatase activity and cleaves the peptidoglycan building block lipid II, accessible from the periplasm. This leads to accumulation of undecaprenol (C 55 -OH) and 1-pyrophospho-MurNAc (N-acetylmuramic acid)-GlcNAc (N-acetylglucosamine), with MurNAc carrying the stem pentapeptide, which cannot be reused for murein biosynthesis (13)(14)(15). This enzymatic activity distinguishes the "protein antibiotic" ColM from antibiotics of different chemical classes acting on lipid II (16), and pesticin, which cleaves the glycan chain of murein (17). ...
... The catalytic mechanism of ColM-mediated lipid II degradation remains elusive, although mutagenesis studies with E. coli and P. aeruginosa ColM bacteriocins allowed the identification of a DXYDX 5 HR motif required for the hydrolase function (14,28). Some degeneracy in this signature sequence appears to exist, e.g., in the ColM-type burkhocin from Burkholderia ambifaria MEX-5 as DXFKX 5 R (see Fig. S1 in the supple- mental material). ...
... Toxin activation by permissive function. Following passage through the outer membrane, full-length colicin M requires toxin refolding and activation by the peptidylprolyl-cis-trans-isomerase FkpA (14,61,62). This modulating process requires FkpA's enzymatic activity, although the proline(s) affected has not been identified unambiguously (Fig. S2A) (63). ...
Bacteria host an arsenal of antagonism-mediating molecules to combat for ecologic space. Bacteriocins represent a pivotal group of secreted antibacterial peptides and proteins assisting in this fight, mainly eliminating relatives. Colicin M, a model for peptidoglycan-interfering bacteriocins in Gram-negative bacteria, appears to be part of a set of polymorphic toxins equipped with such a catalytic domain (ColM) targeting lipid II. Diversifying recombination has enabled parasitism of different receptors and has also given rise to hybrid bacteriocins in which ColM is associated with another toxin module. Remarkably, ColM toxins have recruited a diverse array of immunity partners, comprising cytoplasmic membrane-associated proteins with different topologies. Together, these findings suggest that different immunity mechanisms have evolved for ColM, in contrast to bacteriocins with nuclease activities.
... Site-directed mutagenesis studies of colicin M identified residues Asp-226, Tyr-228, Asp-229, His-235, and Arg-236 as extremely important for both in vitro catalytic activity and in vivo potency [19]. All these residues are conserved also in PaeM; however, it was shown that for PaeM, mutation of His-235 affects pyocin activity only slightly [20]. ...
... The plant-produced pyocins are soluble, contains no tags, and can be easily purified by two-step protein chromatography. We believe that the Residues essential for activity of ColM [19] are marked by asterisks; residues essential for activity of PaeM [20] are marked by black asterisks. ...
The emergence, persistence and spread of antibiotic-resistant human pathogenic bacteria heralds a growing global health crisis. Drug-resistant strains of gram-negative bacteria, such as Pseudomonas aeruginosa, are especially dangerous and the medical and economic burden they impose underscore the critical need for finding new antimicrobials. Recent studies have demonstrated that plant-expressed bacteriocins of the colicins family can be efficient antibacterials against all major enteropathogenic strains of E. coli. We extended our studies of colicin-like bacteriocins to pyocins, which are produced by strains of P. aeruginosa for ecological advantage against other strains of the same species. Using a plant-based transient expression system, we expressed six different pyocins, namely S5, PaeM, L1, L2, L3 and one new pyocin, PaeM4, and purified them to homogeneity. Among these pyocins, PaeM4 demonstrated the broadest spectrum of activity by controlling 53 of 100 tested clinical isolates of P. aeruginosa. The activity of plant-made pyocins was confirmed in the agar drop, liquid culture susceptibility and biofilm assays, and in the Galleria mellonella animal infection model.
