The Action of the Bacterial Toxin Microcin B17

Abstract

Microcins are a family of toxins produced by Enterobacteriaceae to inhibit phylogenetically related species (1). They differ from colicins by their smaller molecular mass and production at the stationary phase of growth (2). Microcin B17 (MccB17) 1 is a 3.1-kDa post-translationally modified peptide encoded on a plasmid in a single operon consisting of seven genes, mcbA to mcbG, responsible for synthesis, export, and immunity (3). Post-translational modifications involve both amino acid side chains and peptide backbones to form oxazole and thiazole rings (4 -6) that give MccB17 its characteristic structure (Fig. 1A).The action of microcin on sensitive Escherichia coli cells leads to an arrest of DNA replication and, consequently, to the induction of the SOS response (7). Moreover, MccB17 induces, in vitro and in vivo, double-stranded cleavage of DNA mediated by DNA gyrase (8). A mutation in the B subunit of gyrase (W751R) was found to confer resistance to MccB17, confirming that DNA gyrase is a cellular target of the toxin (8). MccB17 blocks gyrase by trapping the enzyme-DNA cleavage complex, a mode of action reminiscent of that of quinolones (8, 9). Of the two pairs of tandem, fused 4,2-bisheterocycles in MccB17, the integrity of the second one (B-site tandem; Fig. 1A) was found to be crucial for the potency of MccB17 (10, 11).DNA gyrase is a prokaryotic type II topoisomerase that negatively supercoils closed circular DNA with the requirement of ATP hydrolysis for energy (12, 13). The active enzyme is an A 2 B 2 heterotetramer with the A subunit (GyrA, 97 kDa) largely responsible for DNA wrapping and the breakage and reunion of DNA and the B subunit (GyrB, 90 kDa) responsible for ATP hydrolysis and the interaction with GyrA and DNA. The enzyme introduces supercoils into DNA by wrapping a doublestranded segment around itself, cleaving this DNA (the gate segment) in both strands, passing the wrapped DNA (transported segment) through the break, and then resealing the DNA (12-15). Binding of ATP to GyrB causes the closure of the clamp formed by dimerization of GyrB, which captures the transported segment and directs it through the doublestranded break in the gate segment. ATP is then hydrolyzed, allowing the enzyme to return to its starting conformation (16 -20).In addition to MccB17, other inhibitors of DNA gyrase include the antibacterial agents coumarins and quinolones and the bacterial toxin CcdB (21-23). Coumarins competitively inhibit ATP hydrolysis, whereas quinolones, such as ciprofloxacin (CFX) and oxolinic acid (OXO), stabilize the covalent gyrase-DNA cleavage complex. CcdB also stabilizes a cleavage complex but only in the presence of ATP (21). The trapping of the gyrase-DNA cleavage complex by MccB17 is also reported to be ATP-dependent (9). DNA cleavage complexes can also be observed in the absence of drugs or toxins, if Mg 2ϩ is substituted by Ca 2ϩ , with both gyrase (24) and eukaryotic topo II (25).The elucidation of the mode of action of MccB17 on DNA gyrase is important in at least two respects. F...

Fragments of the Bacterial Toxin Microcin B17 as Gyrase Poisons

“…Second, is the question of the role of this motif in the toxin's mode of action: what is the relevance of the dimeric nature of MccB17 in the context of the complex with the gyrase-DNA intermediate? The stoichiometry of MccB17 binding to gyrase is unknown, and its action has directly been linked with strand passage [17], [22]: the conformational changes occurring during this catalytic event may create the MccB17 binding site. Considering this motif repeat in the context of the open C-gate might be of significance for future modelling studies.…”

“…Previous work has shown that these heterocycles are critical, and modifications of these residues proved to be detrimental to the antibacterial activity of MccB17 [16]. When MccB17 was first identified, it was reported to be active on a variety of organisms ( Escherichia , Enterobacter , Pseudomonas and Shigella ) [12], however its activity in vitro has only been characterised with E. coli gyrase [11], [17]. In spite of its attractive antibacterial properties, the poor physico-chemical properties of MccB17 prevent it from being a good drug candidate.…”

Microcins, gene-encoded antibacterial peptides from enterobacteria

“…200 MccB17 was actually shown to slow down but not to completely inhibit the supercoiling and relaxation reactions of DNA gyrase and to stabilize the cleavage complex. 201 Later, by establishing the proteolytic signature of the gyrase in the presence of MccB17, the same authors showed that the binding site of MccB17 was likely to be the C-terminal domain of GyrB. 202 They also demonstrated that DNA strand passage was involved in the MccB17 mechanism of action.…”

The manifold roles of microbial ribosomal peptide–based natural products in physiology and ecology

“…Pop5, respectively (77)). It has been firmly established that microcin B17 blocks DNA gyrase (74,78) and that phazolicin and klebsazolicin inhibit the ribosome (77), but the modes of action of other TOMM peptides remain largely unknown.…”