Fragments of the Bacterial Toxin Microcin B17 as Gyrase Poisons

Abstract

Fluoroquinolones are very important drugs in the clinical antibacterial arsenal; their success is principally due to their mode of action: the stabilisation of a gyrase-DNA intermediate (the cleavage complex), which triggers a chain of events leading to cell death. Microcin B17 (MccB17) is a modified peptide bacterial toxin that acts by a similar mode of action, but is unfortunately unsuitable as a therapeutic drug. However, its structure and mechanism could inspire the design of new antibacterial compounds that are needed to circumvent the rise in bacterial resistance to current antibiotics. Here we describe the investigation of the structural features responsible for MccB17 activity and the identification of fragments of the toxin that retain the ability to stabilise the cleavage complex.

The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells

“…Yet both mutants were able to cause some accumulation of linear DNA, indicating that they retained some residual ability to inhibit the gyrase. A similar result was recently reported by Collin et al (23).…”

“…Removing the terminal isoleucine residue at position 69 does not affect the number of heterocycles but decreases antibiotic activity 50-fold. A similar drop of activity was noticed by Collin et al (23), though in that work the composition of mutant McB with respect to heterocycle content was not determined. Both the transport of mutant antibiotic through an inner-membrane transporter, SbmA, and the ability to inhibit the intracellular target, the DNA gyrase, are affected.…”

“…Second, the importance of fused heterocycles for McB activity is supported by the observation that the ⌬ϩ1 form of McB is more active than the standard ⌬0 form (20). Third, mutations that prevent formation of site B fused heterocycle render McB inactive, while mutations interfering with site A fused heterocycle formation abolish McB production (20 and thus containing the site B fused heterocycle inhibited DNA gyrase in vitro, while a shorter fragment lacking the fused cycle was inactive (23). On the other hand, in the latter paper, data were presented on McB mutants lacking the ultimate and penultimate amino acids that showed that the ability to affect the gyrase is strongly affected by the deletions.…”

Structure of Microcin B-Like Compounds Produced by Pseudomonas syringae and Species Specificity of Their Antibacterial Action

“…Thus, although the N-terminal polyglycine sequence appears to be important for Ec-McB maturation in vitro (29), it is dispensable for activity, as was also recently suggested by Collin et al (30). Furthermore, since removal of the N-terminal polyglycine sequence does not allow the Ec-McB-based molecule to inhibit Pseudomonas, it follows that it is not involved in the species specificity of McB action.…”

Bacteriocins and their position in the next wave of conventional antibiotics

“…In addition, bacteriocins >10 kDa are parsed into a third class [12]. Bacteriocins from Gram-negative bacteria are divided [16,17] into small peptides, such as microcins, and large peptides, such as colicins [18][19][20]. Further subdivisions exist within these broader categories, including instances of homology in motifs [21].…”