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Antimicrobial Agents and Chemotherapy May 2016Condensins play a key role in the global organization of bacterial chromosomes. In Escherichia coli, the inactivation of its sole condensin MukBEF induces severe growth...
Condensins play a key role in the global organization of bacterial chromosomes. In Escherichia coli, the inactivation of its sole condensin MukBEF induces severe growth defects and renders cells hypersusceptible to novobiocin. We report here that this hypersusceptibility can be observed in TolC-deficient cells and is therefore unrelated to multidrug efflux. We further show that mutations in MukE that impair its focal subcellular localization potentiate novobiocin and that the extent of the potentiation correlates with the residual activity of MukE. Finally, both DNA gyrase and topoisomerase IV might partially complement novobiocin susceptibility in a temperature-dependent manner. These data indicate that the observed antibiotic susceptibility resides in both type II DNA topoisomerases and is efflux independent. Furthermore, novobiocin susceptibility is associated with the activity of MukBEF and can be induced by its partial inactivation, which makes the protein a plausible target for inhibition.
Topics: Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; DNA Topoisomerases; Escherichia coli; Escherichia coli Proteins; Novobiocin
PubMed: 26926630
DOI: 10.1128/AAC.03102-15 -
Journal of Microbiological Methods Aug 2022The detection of Salmonella in food is based on the use of a selective enrichment broth such as Muller-Kauffman Tetrathionate-Novobiocin (MKTTn), in which tetrathionate...
Improvement of Mueller-Kauffman Tetrathionate-Novobiocin (MKTTn) enrichment medium for the detection of Salmonella enterica by the addition of ex situ-generated tetrathionate.
The detection of Salmonella in food is based on the use of a selective enrichment broth such as Muller-Kauffman Tetrathionate-Novobiocin (MKTTn), in which tetrathionate plays a key role by providing Salmonella with a growth advantage. As sodium tetrathionate is unstable, it is generated in situ by the addition of iodine (Lugol's solution) before seeding. This step is cumbersome as the solution is easily spilled, compromising the performance of the medium and hindering the work of technicians. The aim of this study was to optimize MKTTn broth by generating tetrathionate ex situ through an external reaction between iodine and thiosulphate followed by lyophilization. Quality control procedures were performed to compare the modified and original media, testing pure productivity (enrichment with 50-120 CFU of Salmonella Thyphimurium ATCC 14028 and Salmonella Enteritidis ATCC 13076 and plating on Xylose Lysine Deoxycholate agar, XLD), mixed productivity (50-120 CFU of Salmonella strains and Pseudomonas aeruginosa and Escherichia coli at ≥10 CFU and XLD plating) and selectivity (≥10 CFU of P. aeruginosa and Enterococcus faecalis and plating on Tryptone Casein Soy agar, TSA). The modified MKTTn medium (S/L) performed comparably with the original medium in terms of growth of both Salmonella strains (>300 colonies in XLD), alone or with P. aeruginosa and E. coli. Quantitative assays showed no statistically significant differences in the number of colonies grown on XLD after 10 dilution (p = 0.7015 with S. Thyphimurium ATCC 14028 and p = 0.2387 with S. Enteritidis ATCC 13076; ANOVA test). MKTTn medium (S/L) was also selective against E. coli (≤100 colonies) and E. faecalis (<10 colonies). These results suggest that adding tetrathionate as a lyophilisate (S/L) is a feasible alternative to the use of Lugol's solution for the preparation of MKTTn enrichment broth and does not affect the properties of the medium.
Topics: Agar; Culture Media; Escherichia coli; Iodine; Novobiocin; Salmonella enterica; Salmonella enteritidis
PubMed: 35732231
DOI: 10.1016/j.mimet.2022.106524 -
DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in .Genes Nov 2022produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments...
produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in and may be relevant to microbial genetic exchange in natural ecosystems.
Topics: Rhodobacter capsulatus; Topoisomerase II Inhibitors; Gene Expression Regulation, Bacterial; Bacteriophages; Novobiocin; Ecosystem; Bacterial Proteins; Anti-Bacterial Agents
PubMed: 36360308
DOI: 10.3390/genes13112071 -
Journal of Bacteriology Mar 1980Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and...
Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and Micrococcus luteus in its enzymatic requirements, substrate specificity, and sensitivity to several antibiotics. The enzyme was purified from the wild type and nalidixic acid-resistant and novobiocin-resistant mutants of B. subtilis and was functionally characterized in vitro. The genetic loci nalA and novA but not novB were shown to code for portions of the functional gyrase. Enzyme from the antibiotic-resistant mutants was resistant to the drug in vitro. The most striking observation was the remarkable similarity between the B. subtilis enzyme and other DNA gyrases, especially with respect to the oxolinic acid-induced DNA cleavage in the presence of sodium dodecyl sulfate. All of the enzymes appeared to possess the same specificity of cutting sites regardless of the source or type of DNA used. This result implies that gyrase binding to DNA is highly specific.
Topics: Adenosine Triphosphate; Bacillus subtilis; Chromosome Mapping; DNA Topoisomerases, Type II; DNA, Bacterial; Genes; Magnesium; Novobiocin; Oxolinic Acid; Substrate Specificity
PubMed: 6245067
DOI: 10.1128/jb.141.3.1331-1339.1980 -
Biochimica Et Biophysica Acta.... Apr 2018Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in...
Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.
Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Dipeptides; Doxorubicin; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Kinetics; Minocycline; Molecular Structure; Multidrug Resistance-Associated Proteins; Naphthalenes; Novobiocin; Piperazines; Protein Binding; Surface Plasmon Resonance
PubMed: 28890187
DOI: 10.1016/j.bbamem.2017.08.024 -
Journal of Applied Microbiology Dec 2012To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic...
AIM
To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic differences between the novobiocin-resistant strain and its virulent parent strain AH11P.
METHODS AND RESULTS
A novobiocin-resistant strain AH11NOVO was obtained from a virulent Aer. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus), whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challenged with AH11P at 14 days postvaccination, relative per cent of survival of vaccinated fish was 100%. The cell proliferation rate of AH11NOVO was found to be significantly (P < 0.05) less than that of AH11P. In vitro motility assay revealed that AH11NOVO was nonmotile, whereas AH11P was motile. AH11NOVO had significantly (P < 0.05) lower in vitro chemotactic response to catfish mucus than that of AH11P. Although the ability of AH11NOVO to attach catfish gill cells was similar to that of AH11P, the ability of AH11NOVO to invade catfish gill cells was significantly (P < 0.05) lower than that of AH11P.
CONCLUSIONS
The novobiocin-resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity.
SIGNIFICANCE AND IMPACT OF THE STUDY
The significantly lower chemotactic response and invasion ability of AH11NOVO compared with that of its virulent parent strain AH11P might shed light on the pathogenesis of Aer. hydrophila.
Topics: Aeromonas hydrophila; Animals; Anti-Bacterial Agents; Bacterial Vaccines; Cells, Cultured; Chemotaxis; Fish Diseases; Gills; Ictaluridae; Novobiocin; Vaccination; Vaccines, Attenuated; Virulence
PubMed: 22897434
DOI: 10.1111/j.1365-2672.2012.05430.x -
Pharmaceutical Biology Dec 2022A novobiocin derivative, XN4, has been shown to promote cell apoptosis in chronic myeloid leukaemia.
CONTEXT
A novobiocin derivative, XN4, has been shown to promote cell apoptosis in chronic myeloid leukaemia.
OBJECTIVE
This study explores the mechanism by which XN4 promotes ferroptosis of gastric cancer (GC) cells.
MATERIALS AND METHODS
Human GC SGC-7901 and BGC-823 cells were treated with different XN4 concentrations (0, 0.1, 0.5, 1.0, 5.0, and 10.0 μmol/L) to evaluate effects of XN4. Additionally, cells were pre-treated for 24 h with si-NOX4, for 1 h with the iron chelator deferoxamine mesylate (DFO) or for 1 h with the lipid peroxidation inhibitor liproxstatin-1 before being treated with XN4 to analyse the mechanism of XN4.
RESULTS
XN4 increased cell death (IC values of XN4 on SGC-7901 and BGC-823 cells: 1.592 ± 0.14 μmol/L and 2.022 ± 0.19 μmol/L) and Fe levels in SGC-7901 and BGC-823 cells. These effects of 2.0 μmol/L XN4 were abolished by 100 μmol/L DFO treatment. XN4 enhanced transferrin and transferrin receptor expression to induce Fe accumulation. XN4 decreased mitochondrial membrane potentials in GC cells, similar to erastin. Additionally, XN4 increased MDA, hydrogen peroxide, and ROS levels, but diminished total glutathione levels. Liproxstatin-1 (200 nmol/L) nullified the effects of XN4 (2.0 μmol/L) on MDA levels and cell death. Moreover, GPX4 levels decreased, but NOX4 and ferroptosis-related protein PTGS2 levels increased in GC cells following XN4 treatment, which was nullified by NOX4 knockdown.
DISCUSSION AND CONCLUSIONS
The pro-ferroptotic role of XN4 in GC might enable it to become a promising drug for GC treatment in the future despite the need for extensive research.
Topics: Apoptosis; Cell Death; Ferroptosis; Humans; Lipid Peroxidation; NADPH Oxidase 4; Novobiocin; Reactive Oxygen Species; Stomach Neoplasms
PubMed: 35938505
DOI: 10.1080/13880209.2022.2099431 -
Journal of Medicinal Chemistry Feb 2016Heat shock protein 90 (Hsp90) inhibition by modulation of its N- or C-terminal binding site has become an attractive strategy for the development of anticancer...
Heat shock protein 90 (Hsp90) inhibition by modulation of its N- or C-terminal binding site has become an attractive strategy for the development of anticancer chemotherapeutics. The first Hsp90 C-terminus inhibitor, novobiocin, manifested a relatively high IC50 value of ∼700 μM. Therefore, investigation of the novobiocin scaffold has led to analogues with improved antiproliferative activity (nanomolar concentrations) against several cancer cell lines. During these studies, novobiocin analogues that do not inhibit Hsp90 were identified; however, these analogues demonstrated potent antiproliferative activity. Compound 2, a novobiocin analogue, was identified as a MAPK pathway signaling disruptor that lacked Hsp90 inhibitory activity. In addition, structural modifications of compound 2 were identified that segregated Hsp90 inhibition from MAPK signaling disruption. These studies indicate that compound 2 represents a novel scaffold for disruption of MAPK pathway signaling and may serve as a useful structure for the generation of new anticancer agents.
Topics: Antineoplastic Agents; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; HSP90 Heat-Shock Proteins; Humans; Mitogen-Activated Protein Kinases; Models, Molecular; Molecular Structure; Novobiocin; Signal Transduction; Structure-Activity Relationship
PubMed: 26745854
DOI: 10.1021/acs.jmedchem.5b01354 -
PLoS Genetics Nov 2023Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red...
Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. We screened using three different antibiotics to identify core components of the biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC cefixime and novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon-controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions, and failed to increase further with the addition of sub-MIC cefixime. Using a tetrazolium dye and an in vivo NADH sensor, we showed spatial co-localization of increased metabolic activity with sub-lethal concentrations of the bactericidal antibiotics cefixime and novobiocin. Supporting a role for respiratory stress, the biofilm stimulation response to cefixime and novobiocin was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of a gene encoding part of the machinery for respiring nitrate abolished its ameliorating effects, and nitrate respiration increased during growth with sub-MIC cefixime. Finally, in probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors, unlike other antibiotic classes, was minimally affected by nitrate supplementation, suggesting that targeting the ribosome stimulates biofilm formation in distinct ways. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.
Topics: Anti-Bacterial Agents; Escherichia coli; Cefixime; Novobiocin; Nitrates; Biofilms; Microbial Sensitivity Tests
PubMed: 37917668
DOI: 10.1371/journal.pgen.1011013 -
Journal of Clinical Pathology Jul 1977Paper discs containing 5 microng of novobiocin were used as a presumptive test to differentiate peptococci and peptostreptococci. Zone diameters were measured and...
Paper discs containing 5 microng of novobiocin were used as a presumptive test to differentiate peptococci and peptostreptococci. Zone diameters were measured and minimum inhibitory concentrations (MIC) of the antibiotic for each group were performed to ascertain the activity of the antibiotic against these genera. All strains of peptococci showed no zone of inhibition in the disc test together with an MIC of 25 microng/ml or greater. All strains of peptostreptococci showed zones of inhibition of at least 15 mm diameter together with an MIC of 1-6 microng/ml or less.
Topics: Microbial Sensitivity Tests; Novobiocin; Peptococcus; Peptostreptococcus
PubMed: 886014
DOI: 10.1136/jcp.30.7.620