Selective action of novobiocin on Act. levoris, the producer of levorin and levoristatin]

The effect of sodium novobiocin on Act. levoris, strain LIA 0868 producing levorin and levoristatin was studied. High lethal effect of the antibiotic on Act. levoris was found. The effect increased with a rise in the antibiotic concentration in the agar from 2 to 6 lambda/ml. The inhibitory effect of novobiocin on Act, levoris was evident from a marked increase in the number of morphologically changed colonies with a low level of levorin production. The selective effect of novobiocin on the organism producing levorin and levoristatin was evident from selection of variants with high levels of levoristatin production on media containing novobiocin.     

Fibroblast-like transformation and c-myc gene alteration of human hepatocytes induced by novobiocin and butyrate

Topoisomerase II inhibitor novobiocin and deacetylase inhibitor sodium butyrate synergistically transformed Chang liver cells into fibroblast-like cells. In these fibroblast-like cells, the production of type III procollagen was increased and the DNase I hypersensitivity of c-myc gene was reduced. In addition, these changes were associated with an increased acetylation of nuclear proteins, especially those of DNase I sensitive nucleosomes. Therefore, it is suggested that chemical modulation of nuclear proteins by novobiocin and butyrate may be responsible, at least partly, for the alteration of the chromatin structure of c-myc gene and the fibroblast-like transformation of Chang liver cells.     

Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following gamma-irradiation

The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post-treated with novobiocin.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 micrograms of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid.

The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 micrograms of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec+ cells approximately one in 10(4) cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec- cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, we concluded that the Rec+ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome in which the chromosome acquired the novobiocin marker from the plasmid. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.     

Comparative study of different methods for detection and enumeration of Salmonella spp. in natural waters

Seven enrichment media (two proposed by the authors) for detecting salmonellas from polluted freshwater were compared. The Most Probable Number technique for enumeration of salmonellas in water samples was used, directly adding filtered water to buffered peptone water as the pre-enrichment medium. The results indicate that Rappaport-Vassiliadis/43 and Rappaport-Vassiliadis/43 supplemented with 10 μg of sodium novobiocin per ml are the best media for the recovery and enumeration of salmonellas from water samples.     

Nucleotide sequence of the structural gene for the penicillin-binding protein 2 of Staphylococcus aureus and the presence of a homologous gene in other staphylococci

Abstract During growth of Streptomyces niveus wild-type in the novobiocin production medium CDM the resistance of mycelia to novobiocin rises from about 25 μg/ml to over 200 μg/ml. ( S. lividans , a novobiocin-sensitive strain, is resistant to approx. 10 μg/ml novobiocin.) The initial period of low level resistance extends from the time of inoculation of the culture until approx. 70 h when the culture is still in the growth phase. High level resistance is initiated before the start of novobiocin production and rises rapidly to a maximum level beyond the end of the growth phase. The rise in pH of the unbuffered CDM medium which occurs during S. niveus fermentation was shown not to be the cause of the change in novobiocin resistance. However, mycelia-free CDM from S. niveus cultures expressing high level novobiocin resistance was shown to contain a factor which induced high level novobiocin resistance in germinating S. niveus spores. Kinetic studies revealed that the inducer first appears in the culture medium before the switch to high level resistance begins and reaches its highest concentration before resistance reaches its maximum level.     

Laboratory study of several enrichment broths for the detection of Salmonella spp. particularly in relation to water samples

The selectivity and efficiency of several enrichment broths used for the detection of salmonellas were comparatively evaluated under laboratory and environmental conditions. Media with selenite were less efficient in their inhibition of the growth of Gram-positive micro-organisms. Salmonellas grew slowly in tetrathionate broth and in media containing brilliant green. These media inhibited the growth of Salmonella typhi, which grew only in media containing selenite. The results obtained in the experiments with stressed salmonellas indicate that the media selenite F, selenite F with novobiocin, selenite cystine and Rappaport-Vassiliadis (RV/43), in conjunction with the double agar layer technique, showed an optimal efficiency for the detection of stressed salmonellas. When natural samples (freshwater and seawater) were used to evaluate the media, however, those containing malachite green, whether or not supplemented with sodium novobiocin, enhanced the recovery of salmonellas.     

Novobiocin-Induced Ultrastructural Changes and Antagonism of DNA Synthesis in Trypanosoma cruzi Amastigotes Growing in Cell-Free Medium

The antimicrobial agent novobiocin, an inhibitor of the bacterial enzyme topoisomerase II (DNA gyrase), is known to antagonize Trypanosoma cruzi amastigotes growing in cell-free medium. To determine sites of antagonism of novobiocin, the effects of drug on parasite ultrastructure and incorporation of radiolabeled precursors of DNA, RNA and protein into macromolecules were determined. The predominant ultrastructural abnormality seen after exposure to 0.40 mM novobiocin for 24 h was the presence of electron-dense clumps in the mitochondrion-kinetoplast organelle in 95 of 257 (37%) of cells, in comparison to no clumps seen in 110 drug-free cells. In addition, in the nucleus, the karyosome was less distinct than in control cells and appeared to merge with the chromatin. In the radiolabcling studies, incorporation of thymidine was inhibited in a dose-dependent fashion by novobiocin (0.16–0.80 mM) in a range of drug concentrations that also inhibited parasite growth. For 0.16 and 0.24 mM novobiocin, incorporation of thymidine was inhibited up to 65% relative to drug-free control cells while uptake of uridine and leucine was unaltered. We interpret these ultrastructure and precursor-incorporation studies as suggesting that (i) the mitochondrion-kinetoplast and possibly the nucleus are sites of novobiocin antagonism of T. cruzi amastigotes and (ii) that novobiocin appears to antagonize DNA synthesis within these organisms. Whether the drug target is topoisomerase II, however, is as yet unknown.     

Novobiocin-induced ultrastructural changes and antagonism of DNA synthesis in Trypanosoma cruzi amastigotes growing in cell-free medium

The antimicrobial agent novobiocin, an inhibitor of the bacterial enzyme topoisomerase II (DNA gyrase), is known to antagonize Trypanosoma cruzi amastigotes growing in cell-free medium. To determine sites of antagonism of novobiocin, the effects of drug on parasite ultrastructure and incorporation of radiolabeled precursors of DNA, RNA and protein into macromolecules were determined. The predominant ultrastructural abnormality seen after exposure to 0.40 mM novobiocin for 24 h was the presence of electron-dense clumps in the mitochondrion-kinetoplast organelle in 95 of 257 (37%) of cells, in comparison to no clumps seen in 110 drug-free cells. In addition, in the nucleus, the karyosome was less distinct than in control cells and appeared to merge with the chromatin. In the radiolabeling studies, incorporation of thymidine was inhibited in a dose-dependent fashion by novobiocin (0.16-0.80 mM) in a range of drug concentrations that also inhibited parasite growth. For 0.16 and 0.24 mM novobiocin, incorporation of thymidine was inhibited up to 65% relative to drug-free control cells while uptake of uridine and leucine was unaltered. We interpret these ultrastructure and precursor-incorporation studies as suggesting that (i) the mitochondrion-kinetoplast and possibly the nucleus are sites of novobiocin antagonism of T. cruzi amastigotes and (ii) that novobiocin appears to antagonize DNA synthesis within these organisms. Whether the drug target is topoisomerase II, however, is as yet unknown.     

Effect of novobiocin on gamma-radiation-induced DNA repair in human lymphocyte subpopulations

1. DNA repair was measured in 3 Gy gamma-irradiated human peripheral lymphocyte subpopulations by means of nucleoid sedimentation. 2. The influence of the antibiotic, novobiocin (an inhibitor of inter alia topoisomerase II) on the repair process was investigated. 3. Repair of 33 37% of the DNA lesions induced by gamma-irradiation in enriched B lymphocyte fractions, was retarded by novobiocin. 4. Repair in enriched T lymphocyte fractions was unaffected by novobiocin.     

Increase in the frequency of gamma-ray induced chromosomal aberrations in mammalian cells by post-treatment with novobiocin

The effect of novobiocin (an inhibitor of DNA topoisomerase and polymerase) on the frequency of chromosomal aberrations was examined in Chinese hamster V79 cells irradiated with gamma-rays in the plateau phase of growth and subcultured in the presence of novobiocin until the first mitosis after irradiation. Novobiocin alone affected cell survival, DNA synthesis and the mitotic frequency of unirradiated cells in a dose-dependent manner, without causing any significant increase in the frequency of chromosome- or chromatid-type aberrations. The frequency of chromosome-type aberrations induced by gamma-radiation was not influenced by novobiocin at 200 microM, but the frequency of chromosome deletions (but not rings and dicentrics) showed a two-fold increase when 300 microM novobiocin was present. Irradiation produced a low level of chromatid-type aberrations and post-treatment with novobiocin at concentrations greater than 100 microM significantly increased the frequency of chromatid gaps and breaks. The results support the idea that different radiation-induced lesions lead to chromosome- as opposed to chromatid-type aberrations.     

Morphological changes associated with novobiocin resistance in Bacillus licheniformis.

Spontaneously occurring novobiocin-resistant (Nov) mutants of Bacillus licheniformis ATCC 9945, resistant to low levels of novobiocin (15 mug/ml), were isolated with a frequency of 3 in 106 organisms. Such isolates grew well, but nearly all exhibited consistent plleiotropic alterations in colonial and cell morphologies. One mutant, nov-12, grew as chains of unseparated but clearly distinct daughter cells in the absence of novobiocin in liquid culture. When novobiocin was present, nov-12 grew as very long "filaments" which were, however, septate. Septa formed in the presence of the antibiotic were normal, except that no annular clevage of the septal wall was observed. Septa were also irregularly positioned along the filament. These observations were compared with previous findings on the effects of novobiocin and novobiocin resistance described for other organisms. It was concluded that the primary action of novobiocin might differ in gram-positive and gram-negative organisms. However, when the low-level novobiocin sensitivity, normally associated with gram-positive organisms, was genetically abolished in Nov strains of B. licheniformis they became susceptible to an action of novobiocin more analogous to that found for gram-negative organisms. The morphological alterations associated with the Nov phenotype in this organism, together with observations in other organisms, indicate that novobiocin resistance might be generally useful in the search for mutants of gram-positive organisms with altered cell walls.     

Suppressive effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in the G1 phase of human lymphocytes

1-beta-D-Arabinofuranosylcytosine (ara C) induces chromosome-type aberrations in mammalian cells by inhibiting repair replication in the G1 phase. The effect of novobiocin, an inhibitor of prokaryotic gyrases, on G1 repair in human cells was studied cytogenetically using this characteristic of ara C. The experiment was based on the assumption that if novobiocin inhibits the relaxation of chromatin required prior to repair replication, it would reduce the frequency of chromosome-type aberrations in cells treated with a mutagen followed by posttreatment with ara C. It has also been shown that in lymphocytes ara C induces chromosome-type aberrations which were not caused by any induced DNA lesion, and that the frequency of these aberrations changes with the age of the blood donor. The effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in lymphocytes without mutagen pretreatment was also investigated for blood samples from donors of different ages. Human peripheral blood lymphocytes, which were either untreated of treated with 4-nitroquinoline-N-oxide (4NQO) or methyl methanesulfonate (MMS), were posttreated in their early G1 phase with ara C only or ara C and novobiocin. The resulting chromosome-type aberrations were observed in cells in their first mitoses, and a comparison was made between the frequency of aberrations occurring in the presence of novobiocin and in its absence. The results showed that novobiocin reduced the frequency of chromosome-type aberrations induced by ara C in both mutagen-pretreated and -non-pretreated cells, and that lymphocytes from younger donors were less sensitive to novobiocin. The present study demonstrated cytogenetically the existence of a novobiocin-sensitive process to induce chromosome recombination in G1 lymphocytes.     

Effect of permeabilizers on antibiotic sensitivity of Pseudomonas aeruginosa

Agents which had previously been shown to act as permeabilizers against Pseudomonas aeruginosa or other Gram-negative bacteria were tested to determine whether susceptibility to various antibiotics could be increased. In the absence of a permeabilizer, Ps. aeruginosa was resistant to several hydrophobic antibiotics and vancomycin, but not to gentamicin. Tartaric and gluconic acids had weak potentiating activity, whereas ethylenediamine tetraacetic acid and citric acid were more effective permeabilizers. However, sodium polyphosphate enhanced the activity of erythromycin, fucidin, novobiocin, rifampicin and methicillin; vancomycin was unaffected and the activity of gentamicin was reduced considerably.     

A note on novobiocin in XLD and HE agars: the optimum levels required in two commercial sources of media to improve isolation of salmonellas

Media containing novobiocin were tested for their selectivity for salmonelias. The optimum concentrations of novobiocin that gave the greatest percentage recovery without altering the colony morphology were 7 μg/ml in XLD Agar (Oxoid) and 40 μg/ml in HE Agar (Oxoid). At these concentrations some strains of Proteus mirabilis were suppressed and those that did survive did not produce hydrogen sulphide. The addition of novobiocin altered the colony morphology of Citrobacter freundii and Escherichia coli. Salmonella typhi was not used in this investigation and isolation of this pathogen on novobiocin supplemented media is questionable.     

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors,clorobiocin

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3′ position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5′-adenylyl-β,γ-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented. © 1997 Wiley-Liss Inc.     

Supplementation of enrichment broths by novobiocin for detecting Shiga toxin-producing Escherichia coli from food: a controversial use

AIMS: To investigate the assumption that usage of novobiocin (20 mg l(-1)) in Shiga toxin-producing Escherichia coli (STEC) enrichment broths could achieve false-negative results. METHODS AND RESULTS: First, the minimum inhibitory concentration (MIC) of 74 E. coli O157:H7 and 55 non-O157:H7 STEC strains to novobiocin was determined. Second, to visualize the potential impact of novobiocin on the STEC growth during the enrichment step, the growth experiments were carried out in trypticase soy broth (TSB) with and without 20 mg l(-1) of novobiocin. The MIC values varied from 32 to > 64 mg l(-1) for the 74 E. coli O157:H7 strains, and from 16 to > 64 mg l(-1) for the 55 non-O157:H7 STEC strains. The E. coli O157:H7 strains were significantly (P < 0.001) more resistant to novobiocin than the non-O157:H7 STEC strains. The present study shows that the addition of novobiocin into enrichment broths inhibits the growth of some non-O157:H7 STEC strains, and slows down the growth of some STEC strains. CONCLUSIONS: Enrichment broths supplemented by novobiocin could lead to false-negative results for detecting STEC from food. SIGNIFICANCE AND IMPACT OF THE STUDY: We strongly suggest that novobiocin should not be systematically added into enrichment broths for detecting STEC from food.     

Mutants suppressing novobiocin hypersensitivity of a mukB null mutation

The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.