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Cell Reports Sep 2015Toxin-antitoxin (TA) modules are ubiquitous molecular switches controlling bacterial growth via the release of toxins that inhibit cell proliferation. Most of these...
Toxin-antitoxin (TA) modules are ubiquitous molecular switches controlling bacterial growth via the release of toxins that inhibit cell proliferation. Most of these toxins interfere with protein translation, but a growing variety of other mechanisms hints at a diversity that is not yet fully appreciated. Here, we characterize a group of FIC domain proteins as toxins of the conserved and abundant FicTA family of TA modules, and we reveal that they act by suspending control of cellular DNA topology. We show that FicTs are enzymes that adenylylate DNA gyrase and topoisomerase IV, the essential bacterial type IIA topoisomerases, at their ATP-binding site. This modification inactivates both targets by blocking their ATPase activity, and, consequently, causes reversible growth arrest due to the knotting, catenation, and relaxation of cellular DNA. Our results give insight into the regulation of DNA topology and highlight the remarkable plasticity of FIC domain proteins.
Topics: Bacterial Toxins; DNA Gyrase; DNA Topoisomerase IV; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Nucleic Acid Conformation; Pseudomonas aeruginosa
PubMed: 26299961
DOI: 10.1016/j.celrep.2015.07.056 -
Structure (London, England : 1993) Mar 2007DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a...
DNA gyrase, the only topoisomerase able to introduce negative supercoils into DNA, is essential for bacterial transcription and replication; absent from humans, it is a successful target for antibacterials. From biophysical experiments in solution, we report a structural model at approximately 12-15 A resolution of the full-length B subunit (GyrB). Analytical ultracentrifugation shows that GyrB is mainly a nonglobular monomer. Ab initio modeling of small-angle X-ray scattering data for GyrB consistently yields a "tadpole"-like envelope. It allows us to propose an organization of GyrB into three domains-ATPase, Toprim, and Tail-based on their crystallographic and modeled structures. Our study reveals the modular organization of GyrB and points out its potential flexibility, needed during the gyrase catalytic cycle. It provides important insights into the supercoiling mechanism by gyrase and suggests new lines of research.
Topics: Amino Acid Sequence; DNA Gyrase; Escherichia coli; Molecular Sequence Data; Protein Subunits; Scattering, Small Angle; Solutions; X-Ray Diffraction
PubMed: 17355868
DOI: 10.1016/j.str.2007.01.013 -
Microbial Drug Resistance (Larchmont,... Dec 2021Leprosy is a chronic infectious disease caused by and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of have been...
Leprosy is a chronic infectious disease caused by and the treatment of choice is ofloxacin (OFX). Specific amino acid substitutions in DNA gyrase of have been reported leading to resistance against the drug. In our previous study, WQ-3810, a fluoroquinolone with a new R1 group (6-amino-3,5-difluoropyridin-2-yl) was shown to have a strong inhibitory activity on OFX-resistant DNA gyrases of , and the structural characteristics of its R1 group was predicted to enhance the inhibitory activity. To further understand the contribution of the R1 group, WQ-3334 with the same R1 group as WQ-3810, WQ-4064, and WQ-4065, but with slightly modified R1 group, were assessed on their activities against recombinant DNA gyrase of . An study was conducted to understand the molecular interactions between DNA gyrase and WQ compounds. WQ-3334 and WQ-3810 were shown to have greater inhibitory activity against DNA gyrase than others. Furthermore, analysis using quinolone-resistant DNA gyrases showed that WQ-3334 had greater inhibitory activity than WQ-3810. The R8 group was shown to be a factor for the linkage of the R1 groups with GyrB by an study. The inhibitory effect of WQ compounds that have a new R1 group against DNA gyrase can be enhanced by improving the binding affinity with different R8 group molecules. The information obtained by this work could be applied to design new fluoroquinolones effective for quinolone-resistant and other bacterial pathogens.
Topics: Anti-Bacterial Agents; Azetidines; DNA Gyrase; Drug Resistance, Multiple, Bacterial; Fluoroquinolones; Genes, Bacterial; Microbial Sensitivity Tests; Mycobacterium leprae; Quinolones
PubMed: 34077282
DOI: 10.1089/mdr.2020.0408 -
The Journal of Biological Chemistry Feb 2006DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including...
DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including topoisomerase IV, to DNA takes place at the catalytic domain across the DNA gate, whereas DNA gyrase binds to DNA not only at the amino-terminal catalytic domain but also at the carboxyl-terminal domain (CTD) of the GyrA subunit. The binding of the GyrA CTD to DNA allows gyrase to wrap DNA around itself and catalyze the supercoiling reaction. Recent structural studies, however, have revealed striking similarities between the GyrA CTD and the ParC CTD, as well as the ability of the ParC CTD to bind and bend DNA. Thus, the molecular basis of gyrase-mediated wrapping of DNA needs to be reexamined. Here, we have conducted a mutational analysis to determine the role of the "GyrA-box," a 7-amino acid-long motif unique to the GyrA CTD, in determining the DNA binding mode of gyrase. Either a deletion of the entire GyrA-box or substitution of the GyrA-box with 7 Ala residues abolishes the ability of gyrase to wrap DNA around itself and catalyze the supercoiling reaction. However, these mutations do not affect the relaxation and decatenation activities of gyrase. Thus, the presence of a GyrA-box allows gyrase to wrap DNA and catalyze the supercoiling reaction. The consequence of the loss of the GyrA-box during evolution of bacterial type II topoisomerases is discussed.
Topics: Adenosine Triphosphate; Alanine; Amino Acid Motifs; Catalysis; Catalytic Domain; DNA; DNA Gyrase; DNA Topoisomerase IV; DNA, Bacterial; DNA, Superhelical; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Mutation; Nucleic Acid Conformation; Phylogeny; Protein Binding; Trypsin
PubMed: 16332690
DOI: 10.1074/jbc.M511160200 -
The Plant Cell Oct 2017
Topics: AAA Domain; Arabidopsis; Chloroplasts; DNA Gyrase; Genomic Instability; Humans; Ribonuclease H
PubMed: 28970337
DOI: 10.1105/tpc.17.00774 -
Antimicrobial Agents and Chemotherapy Mar 2015The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this...
The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.
Topics: Barbiturates; DNA Gyrase; Drug Resistance, Bacterial; Isoxazoles; Microbial Sensitivity Tests; Morpholines; Mutation; Neisseria gonorrhoeae; Oxazolidinones; Spiro Compounds; Topoisomerase II Inhibitors
PubMed: 25534723
DOI: 10.1128/AAC.04456-14 -
Nucleic Acids Research Oct 2008Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL...
Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.
Topics: Adenosine Triphosphate; Anti-Bacterial Agents; Base Sequence; DNA Damage; DNA Gyrase; DNA, Superhelical; Diterpenes; Enzyme Inhibitors; Molecular Sequence Data; Mutagenesis; Sequence Analysis, DNA; Streptococcus pneumoniae; Topoisomerase II Inhibitors
PubMed: 18723572
DOI: 10.1093/nar/gkn539 -
Journal of Molecular Biology Jul 2015Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II...
Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.
Topics: 2-Aminopurine; Adenosine Triphosphate; Antigens, Neoplasm; DNA Gyrase; DNA Topoisomerases, Type II; DNA, Superhelical; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Escherichia coli Proteins; Nucleic Acid Conformation; Protein Conformation; Salmonella
PubMed: 25902201
DOI: 10.1016/j.jmb.2015.04.006 -
Antimicrobial Agents and Chemotherapy Jan 2015AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus...
In vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione DNA gyrase/topoisomerase inhibitor with potent activity against Gram-positive, fastidious Gram-Negative, and atypical bacteria.
AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development.
Topics: Anti-Bacterial Agents; Atypical Bacterial Forms; Barbiturates; DNA Gyrase; Drug Resistance, Bacterial; Fluoroquinolones; Gram-Negative Bacteria; Gram-Positive Bacteria; Isoxazoles; Microbial Sensitivity Tests; Morpholines; Nucleic Acid Synthesis Inhibitors; Oxazolidinones; Spiro Compounds; Topoisomerase II Inhibitors
PubMed: 25385112
DOI: 10.1128/AAC.04124-14 -
Nucleic Acids Research Jan 2019Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription....
Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
Topics: Catalysis; DNA Gyrase; DNA, Superhelical; DNA-Binding Proteins; Escherichia coli; Protein Binding; Single Molecule Imaging
PubMed: 30445553
DOI: 10.1093/nar/gky1143