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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.

SUBMITTER: Collin F

PROVIDER: S-EPMC3622597 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Fragments of the bacterial toxin microcin B17 as gyrase poisons.

Collin Frédéric F Thompson Robert E RE Jolliffe Katrina A KA Payne Richard J RJ Maxwell Anthony A

PloS one 20130410 4

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 antibacteri ...[more]

PMID: 23593482

Similar Datasets

The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents.

Project description:Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase-DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial-bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40?years about this still enigmatic molecule and place it in the wider context of antibacterials.

| S-EPMC6722960 | biostudies-literature

A major portion of DNA gyrase inhibitor microcin B17 undergoes an N,O-peptidyl shift during synthesis.

Project description:Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the McbBCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N ? O shift of the Ser(52) residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser(52) is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.

| S-EPMC3143593 | biostudies-literature

Architecture of Microcin B17 Synthetase: An Octameric Protein Complex Converting a Ribosomally Synthesized Peptide into a DNA Gyrase Poison.

Project description:The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.

| S-EPMC6395948 | biostudies-literature

Complete nucleotide sequence and comparative genomic analysis of microcin B17 plasmid pMccB17.

Project description:We present a comprehensive sequence and bioinformatic analysis of the prototypical microcin plasmid, pMccb17, which includes a definitive sequence for the microcin operon, mcb. Microcin B17 (MccB17) is a ribosomally synthesized and posttranslationally modified peptide produced by Escherichia coli. It inhibits bacterial DNA gyrase similarly to quinolone antibiotics. The mcb operon, which consists of seven genes encoding biosynthetic and immunity/export functions, was originally located on the low copy number IncFII plasmid pMccB17 in the Escherichia coli strain LP17. It was later transferred to E. coli K-12 through conjugation. In this study, the plasmid was extracted from the E. coli K-12 strain RYC1000 [pMccB17] and sequenced twice using an Illumina short-read method. The first sequencing was conducted with the host bacterial chromosome, and the plasmid DNA was then purified and sequenced separately. After assembly into a single contig, polymerase chain reaction primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete circular DNA sequence is 69,190 bp long and includes 81 predicted genes. These genes were initially identified by Prokka and subsequently manually reannotated using BLAST. The plasmid was assigned to the F2:A-:B- replicon type with a MOBF12 group conjugation system. A comparison with other IncFII plasmids revealed a large proportion of shared genes, particularly in the conjugative plasmid backbone. However, unlike many contemporary IncFII plasmids, pMccB17 lacks transposable elements and antibiotic resistance genes. In addition to the mcb operon, this plasmid carries 25 genes of unknown function.

| S-EPMC10912980 | biostudies-literature

Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42.

Project description:Here we report on a novel thiazole/oxazole-modified microcin (TOMM) from Bacillus amyloliquefaciens FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from B. amyloliquefaciens FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ∼10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.

| S-EPMC3019963 | biostudies-literature

Vibrio cholerae ParE2 poisons DNA gyrase via a mechanism distinct from other gyrase inhibitors.

Project description:DNA gyrase is an essential bacterial enzyme required for the maintenance of chromosomal DNA topology. This enzyme is the target of several protein toxins encoded in toxin-antitoxin (TA) loci as well as of man-made antibiotics such as quinolones. The genome of Vibrio cholerae, the cause of cholera, contains three putative TA loci that exhibit modest similarity to the RK2 plasmid-borne parDE TA locus, which is thought to target gyrase although its mechanism of action is uncharacterized. Here we investigated the V. cholerae parDE2 locus. We found that this locus encodes a functional proteic TA pair that is active in Escherichia coli as well as V. cholerae. ParD2 co-purified with ParE2 and interacted with it directly. Unlike many other antitoxins, ParD2 could prevent but not reverse ParE2 toxicity. ParE2, like the unrelated F-encoded toxin CcdB and quinolones, targeted the GyrA subunit and stalled the DNA-gyrase cleavage complex. However, in contrast to other gyrase poisons, ParE2 toxicity required ATP, and it interfered with gyrase-dependent DNA supercoiling but not DNA relaxation. ParE2 did not bind GyrA fragments bound by CcdB and quinolones, and a set of strains resistant to a variety of known gyrase inhibitors all exhibited sensitivity to ParE2. Together, our findings suggest that ParE2 and presumably its many plasmid- and chromosome-encoded homologues inhibit gyrase in a different manner than previously described agents.

| S-EPMC3001019 | biostudies-literature

Mechanism of Action of Mycobacterium tuberculosis Gyrase Inhibitors: A Novel Class of Gyrase Poisons.

Project description:Tuberculosis is one of the leading causes of morbidity worldwide, and the incidences of drug resistance and intolerance are prevalent. Thus, there is a desperate need for the development of new antitubercular drugs. Mycobacterium tuberculosis gyrase inhibitors (MGIs) are napthyridone/aminopiperidine-based drugs that display activity against M. tuberculosis cells and tuberculosis in mouse models [Blanco, D., et al. (2015) Antimicrob. Agents Chemother. 59, 1868-1875]. Genetic and mutagenesis studies suggest that gyrase, which is the target for fluoroquinolone antibacterials, is also the target for MGIs. However, little is known regarding the interaction of these drugs with the bacterial type II enzyme. Therefore, we examined the effects of two MGIs, GSK000 and GSK325, on M. tuberculosis gyrase. MGIs greatly enhanced DNA cleavage mediated by the bacterial enzyme. In contrast to fluoroquinolones (which induce primarily double-stranded breaks), MGIs induced only single-stranded DNA breaks under a variety of conditions. MGIs work by stabilizing covalent gyrase-cleaved DNA complexes and appear to suppress the ability of the enzyme to induce double-stranded breaks. The drugs displayed little activity against type II topoisomerases from several other bacterial species, suggesting that these drugs display specificity for M. tuberculosis gyrase. Furthermore, MGIs maintained activity against M. tuberuclosis gyrase enzymes that contained the three most common fluoroquinolone resistance mutations seen in the clinic and displayed no activity against human topoisomerase IIα. These findings suggest that MGIs have potential as antitubercular drugs, especially in the case of fluoroquinolone-resistant disease.

| S-EPMC6309371 | biostudies-literature

Targeting the gyrase of Plasmodium falciparum with topoisomerase poisons.

Project description:Drug-resistant malaria poses a major public health problem throughout the world and the need for new antimalarial drugs is growing. The apicoplast, a chloroplast-like organelle essential for malaria parasite survival and with no counterpart in humans, offers an attractive target for selectively toxic new therapies. The apicoplast genome (plDNA) is a 35 kb circular DNA that is served by gyrase, a prokaryotic type II topoisomerase. Gyrase is poisoned by fluoroquinolone antibacterials that stabilize a catalytically inert ternary complex of enzyme, its plDNA substrate, and inhibitor. We used fluoroquinolones to study the gyrase and plDNA of Plasmodium falciparum. New methods for isolating and separating plDNA reveal four topologically different forms and permit a quantitative exam of perturbations that result from gyrase poisoning. In keeping with its role in DNA replication, gyrase is most abundant in late stages of the parasite lifecycle, but several lines of evidence indicate that even in these cells the enzyme is present in relatively low abundance: about 1 enzyme for every two plDNAs or a ratio of 1 gyrase: 70 kb DNA. For a spectrum of quinolones, correlation was generally good between antimalarial activity and gyrase poisoning, the putative molecular mechanism of drug action. However, in P. falciparum there is evidence for off-target toxicity, particularly for ciprofloxacin. These studies highlight the utility of the new methods and of fluoroquinolones as a tool for studying the in situ workings of gyrase and its plDNA substrate.

| S-EPMC4449814 | biostudies-literature

Prion-like characteristics of the bacterial protein Microcin E492.

Project description:Microcin E492 (Mcc) is a pore-forming bacteriotoxin. Mcc activity is inhibited at the stationary phase by formation of amyloid-like aggregates in the culture. Here we report that, in a similar manner as prions, Mcc naturally exists as two conformers: a β-sheet-rich, protease-resistant, aggregated, inactive form (Mccia), and a soluble, protease-sensitive, active form (Mcca). The exogenous addition of culture medium containing Mccia or purified in vitro-generated Mccia into the culture induces the rapid and efficient conversion of Mcca into Mccia, which is maintained indefinitely after passaging, changing the bacterial phenotype. Mccia prion-like activity is conformation-dependent and could be reduced by immunodepleting Mccia. Interestingly, an internal region of Mcc shares sequence similarity with the central domain of the prion protein, which is key to the formation of mammalian prions. A synthetic peptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducing the conversion of Mcca into Mccia in vivo, suggesting that this region corresponds to the prion domain of Mcc. Our findings suggest that Mcc is the first prokaryotic protein with prion properties which harnesses prion-like transmission to regulate protein function, suggesting that propagation of biological information using a prion-based conformational switch is an evolutionary conserved mechanism.

| S-EPMC5374632 | biostudies-literature

Driving forces of gyrase recognition by the addiction toxin CcdB.

Project description:Gyrase, an essential bacterial topoisomerase, is the target of several antibiotics (e.g. quinolones) as well as of bacterial toxin CcdB. This toxin, encoded by Escherichia coli toxin-antitoxin module ccd, poisons gyrase by causing inhibition of both transcription and replication. Because the molecular driving forces of gyrase unfolding and CcdB-gyrase binding were unknown, the nature of the CcdB-gyrase recognition remained elusive. Therefore, we performed a detailed thermodynamic analysis of CcdB binding to several fragments of gyrase A subunit (GyrA) that contain the CcdB-binding site. Binding of CcdB to the shorter fragments was studied directly by isothermal titration calorimetry. Its binding to the longer GyrA59 fragment in solution is kinetically limited and was therefore investigated via urea induced unfolding of the GyrA59-CcdB complex and unbound GyrA59 and CcdB, monitored by circular dichroism spectroscopy. Model analysis of experimental data, in combination with the relevant structural information, indicates that CcdB binding to gyrase is an enthalpic process driven mainly by specific interactions between CcdB and the highly stable dimerization domain of the GyrA. The dissection of binding energetics indicates that CcdB-gyrase recognition is accompanied by opening of the tower and catalytic domain of GyrA. Such extensive structural rearrangements appear to be crucial driving forces for the functioning of the ccd toxin-antitoxin module.

| S-EPMC2740426 | biostudies-literature

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