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At least 19 recordsLinked to original sources
PubMed1995

Expression of novobiocin resistance genes in the novobiocin-producing organism Streptomyces niveus.

RNA isolated at intervals during fermentation from the novobiocin-producing wild-type strain of Streptomyces niveus and from a series of novobiocin-non-producing (Nov-) mutants was hybridized to DNA probes containing sequences which specify novobiocin resistance. The probes were made from inserts contained in the clones pGL101 and pGL103 which increase the level of novobiocin resistance of S. lividans transformants from 10 micrograms ml-1 to 50 micrograms ml-1 and 150 micrograms ml-1, respectively. No hybridization was detected with the pGL101 probe. The pGL103 probe hybridized to RNA extracted during the later stages of growth--a pattern corresponding to the transition from low to high level novobiocin resistance during growth of S. niveus wild-type cultures. Neither probe hybridized to RNA extracted from four Nov- mutants. These mutants showed variable levels of novobiocin resistance but none expressed the high wild-type levels. The authors conclude that expression of the DNA sequence in pGL103 is associated with high level novobiocin resistance.

DNA, Bacterial↗
PubMed1990

Novobiocin-resistance sequences from the novobiocin-producing strain Streptomyces niveus.

Two distinct DNA sequences expressing novobiocin resistance in Streptomyces lividans were cloned from the novobiocin-producing species Streptomyces niveus. Clone pGL101 (5kb) conferred resistance to 50 micrograms ml-1 novobiocin, whereas clones pGL102 and pGL103, which carry the same 6.5kb insert but in opposite orientations, expressed resistance to 150 micrograms ml-1. The cloned inserts from pGL101 and pGL103 failed to hybridize with each other or with the cloned novobiocin-resistant gyrB sequence from Streptomyces sphaeroides. Both probes hybridized strongly with DNA from the novobiocin-producing species S. niveus and S. sphaeroides but no hybridization (pGL103) or very weak hybridization (pGL101) was detected with DNA from the non-producing species S. lividans, Streptomyces griseus and Streptomyces antibioticus. S. niveus contains at least three novobiocin-resistance determinants with the pGL101 and pGL103 cloned sequences specific for novobiocin-producing strains of Streptomyces.

Blotting, Southern↗
PubMed1967

Development of resistance to novobiocin, tetracycline, and a novobiocin-tetracycline combination in Staphylococcus aureus populations.

The antibiotic sensitivity of the individual organisms of a bacterial population was determined to study the comparative rates of development of resistance of Staphylococcus aureus to novobiocin, tetracycline, and to a combination of these antibiotics. Serial subculture of S. aureus with the combination of novobiocin-tetracycline (N-T 2.5:1; the ratio in serum of patients dosed with Panalba) showed a significant retardation of resistance outgrowth compared with subculture in the presence of the antibiotics individually. Increase in organisms resistant to novobiocin seen after one N-T subculture was related to the "concentration gap" between novobiocin and tetracycline. Two additional subcultures with N-T caused little or no increase in organisms resistant to novobiocin, tetracycline, or to the combination. The data suggest that the retardation of further development of resistance was the result of tetracycline inhibition of novobiocin-resistant strains and vice versa.

Drug Resistance, Microbial↗
PubMed1986

Chromatid aberrations in a novobiocin-resistant subline of Chinese hamster V79 cells after exposure to novobiocin.

We examined effects of novobiocin alone or in combination with gamma-irradiation, on the frequencies of chromatid-type aberrations in a novobiocin-resistant subline of Chinese hamster V79 cells (NOVOr-1). NOVOr-1 cells were significantly resistant to novobiocin, as compared to wild-type V79 (WT) cells, with respect to cell survival and DNA synthesis. Survival responses of WT and NOVOr-1 cells to gamma-rays in the range 2-10 Gy differed only slightly and the number of chromatid aberrations produced by irradiation at 1 Gy was fairly comparable in the two cell types. Post-irradiation treatment of cells with novobiocin at concentrations exceeding 200 micrograms/ml significantly increased the number of chromatid gaps plus breaks in WT cells as compared with NOVOr-1 cells. With 200 micrograms/ml the increase was 1.2-fold (t-test, P less than 0.05) and with 400 micrograms/ml, 2.3-fold (P less than 0.01) the number produced in NOVOr-1 cells.

Animals↗
PubMed1988

Inhibition of RNA polymerase I-directed transcription by novobiocin. Potential use of novobiocin as a general inhibitor of eukaryotic transcription initiation.

The effect of novobiocin, a coumarin class antibiotic, on rat ribosomal gene (rDNA) transcription in a fractionated extract derived from adenocarcinoma ascites cells (fraction DE-B) was studied. This drug inhibited transcription of rDNA by blocking initiation of transcription, whereas it had no effect on the elongation of the rRNA transcript. Order of addition experiments indicated that the novobiocin effect was at a step(s) in preinitiation complex formation. Preincubation of fraction DE-B with ATP before exposure to this antibiotic prevented inhibition of rDNA transcription. Since novobiocin has been shown to inhibit RNA polymerases II- and III-directed transcription of linear DNAs by interfering with a step(s) in the initiation reaction, these data suggest that initiation of transcription of all classes of RNA is inhibited by novobiocin by a mechanism independent of its effect on DNA topoisomerase II.

Adenocarcinoma↗
PubMed1989

[Features of glycolysis and pentose phosphate pathway in novobiocin sensitive and novobiocin resistant staphylococci].

Intensity of glycolysis and the pentose phosphate cycle in staphylococci sensitive and resistant to novobiocin was studied. The resistant variants did not practically store lactate and the activity of glycolytic enzymes i.e. hexokinase and aldolase was lowered by 15-20 and 53-59 per cent, respectively. Monoiodoacetate, a glycolysis inhibitor suppressed the glucose oxidation rate by 53.3-66.9 per cent in the sensitive variants and by 16-21.8 per cent in the resistant variants. At the same time it was characteristic of the resistant variants to increase the activity of the pentose phosphate cycle enzymes; glucose-6-phosphate dehydrogenase by 25-38.1 per cent transketolase by 21.5-27.3 per cent and transaldolase by 30-57.1 per cent. No differences in the transhydrogenase reaction kinetics of both the novobiocin sensitive and the novobiocin resistant variants were observed.

Drug Resistance, Microbial↗
PubMed1978

[Separation of novobiocin, isnovobiocin and descarbamyl novobiocin by a thin-layer chromatographic method].

A thin-layer chromatographic method for separation of novobiocin, isonovobiocin and descarbamylnovobiocin using Silufol plates was developed. The method is simple and rapid. It provided clear separation of the components and using of diethyl ether, a simple individual separating solvent. The method allowed determination of novobiocin and the products of its isomerization in the fermentation broth, extracts and dry substances, as well as performance of operative control and regulation of the technological processes of fermentation and chemical purification of novobiocin.

Chromatography, Thin Layer↗
PubMed1989

Interplay of novobiocin-resistant and -sensitive DNA gyrase activities in self-protection of the novobiocin producer, Streptomyces sphaeroides.

The novobiocin (Nb)-producing organism, Streptomyces sphaeroides, possesses two gyrB genes: gyrBS and gyrBR (encoding the DNA gyrase B subunit-the normal target for Nb) whose products differ in their response to the drug. Novobiocin-sensitive gyrase is the predominant form of the enzyme in this strain and is produced constitutively but at variable levels, whereas Nb-resistant gyrase appears when growth takes place in the presence of the drug. The promoter isolated from the Nb-resistance determinant responds sharply to changes in DNA topology, being activated when the (negative) superhelical density is reduced and vice versa when the supercoiling of DNA is increased. Thus, resistance to Nb in S. sphaeroides is induced by a reduction in DNA supercoiling due to the action of autogenous drug on the sensitive gyrase.

Blotting, Southern↗
PubMed1984

Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae.

Plasmids pNov1 and pNov1s , coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1 , measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s , could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.

Anaerobiosis↗
PubMed1992

Potentiation by novobiocin of the cytotoxic activity of etoposide (VP-16) and teniposide (VM-26).

The coumermycin antibiotic novobiocin, which interacts with the nuclear enzyme topoisomerase II, produced supra-additive toxicity to WEHI-3B D+ leukemia cells at clinically achievable concentrations, when combined with teniposide (VM-26) or etoposide (VP-16). Simultaneous exposure of cells to both agents was required for maximum efficacy of the combination. Novobiocin also produced supra-additive toxicity to A549 human lung carcinoma cells when combined with VM-26 or VP-16. At concentrations above the peak plasma levels achievable in patients, novobiocin lost its potentiating activity. Exposure of WEHI-3B D+ cells to novobiocin did not modify the cytotoxicity produced by the topoisomerase II inhibitor m-AMSA, whereas, in contrast, novobiocin antagonized the cytotoxicity of m-AMSA in A549 cells. Although it has been suggested that inhibitors of the syntheses of DNA and RNA interfere with the cytotoxic activity of the epipodophyllotoxins, maximum potentiation of the cytotoxicities of VP-16 and VM-26 occurred at novobiocin concentrations that decreased the rates of synthesis of both DNA and RNA in WEHI-3B D+ cells by about 50%. The number of DNA-topoisomerase-II covalent complexes stabilized by VM-26 in WEHI-3B D+ cells was greatly increased when cells were exposed simultaneously to VM-26 and novobiocin for 1 hr, but not when cells were treated with m-AMSA and novobiocin for the same period of time. Novobiocin did not affect the amount of covalent complexes produced by VM-26 in isolated nuclei, suggesting that the potentiating activity of novobiocin was not due to its direct interaction with the nuclear topoisomerase II enzyme. Our findings suggest that therapeutic levels of novobiocin may be capable of enhancing the clinical activities of VP-16 and VM-26.

Adenocarcinoma↗
PubMed1992

Novobiocin-induced accumulation of etoposide (VP-16) in WEHI-3B D+ leukemia cells.

A previous report from this laboratory demonstrated that novobiocin produced supra-additive cytotoxicity when combined with etoposide (VP-16) or teniposide (VM-26) in WEHI-3B D+ and A549 cells. The increase in cytotoxicity was accompanied by an increase in the formation of drug-stabilized protein-DNA covalent complexes. We now report that novobiocin increased the amount of VP-16-induced covalent complexes between the 170 kDa form of topoisomerase II and DNA in WEHI-3B D+ cells, as measured by the band-depletion immunoblotting assay, while it did not affect the extractable topoisomerase II activity, measured by the unknotting of P4 phage DNA and by a DNA cleavage assay. Novobiocin progressively increased the steady-state concentration of intracellular VP-16. Removal of novobiocin resulted in a rapid return of VP-16 to levels comparable to those seen with VP-16 alone. The increased accumulation of VP-16 was accounted for by an increase in the exchangeable fraction only. The novobiocin-mediated increase in the steady-state concentration of VP-16 occurred whether novobiocin was added simultaneously with VP-16 or was added after a steady-state level of VP-16 had been achieved. Novobiocin did not affect the initial rate of uptake of VP-16; however, it inhibited the efflux of the epipodophyllotoxin. In fact, when cells were loaded with the same level of VP-16 in the presence or absence of novobiocin, the efflux curves in the presence or absence of novobiocin were significantly different. We conclude that the inhibition of VP-16 efflux by novobiocin is responsible for the increase in VP-16 accumulation, leading to increased formation of VP-16-stabilized topoisomerase-II-DNA covalent complexes and increased cytotoxicity.

Animals↗
PubMed1981

Dependence of mammalian DNA synthesis on DNA supercoiling. III. Characterization of the inhibition of replicative and repair-type DNA synthesis by novobiocin and nalidixic acid.

Novobiocin and nalidixic acid, inhibitors of the bacterial enzyme DNA gyrase, inhibit DNA, RNA and protein synthesis in several human and rodent cell lines. The sensitivity of DNA synthesis (both replicative and repair) to inhibition by novobiocin and nalidixic acid is greater than that of protein synthesis. Novobiocin inhibits RNA synthesis about half as effectively as it does DNA synthesis, whereas nalidixic acid inhibits both equally well. Replicative DNA synthesis, as measured by incorporation of [3H]thymidine, is blocked by novobiocin in a number of cell strains; the inhibition is reversible with respect to both DNA synthesis and cell killing, and continues for as long as 20--30 h if the cells are kept in novobiocin-containing growth medium. Both novobiocin and nalidixic acid inhibit repair DNA synthesis (measured by BND-cellulose chromatography) induced by ultraviolet light or N-methyl-N'-nitro-N-nitrosoguanidine (but not that induced by methyl methanesulfonate) at lower concentration (as low as 5 micrograms/ml) than those required to inhibit replicative DNA synthesis (50 micrograms/ml or greater). Neither novobiocin nor nalidixic acid alone induces DNA repair synthesis. Incubation of ultraviolet-irradiated cells with 10--100 micrograms/ml novobiocin results in little, if any, further reduction of colony-forming ability (beyond that caused by the ultraviolet irradiation). Novobiocin at sufficiently low concentrations (200 micrograms/ml) apparently generates a quiescent state (in terms of cellular DNA metabolism) from which recovery is possible. Under more drastic conditions of time in contact with cells and concentration, however, novobiocin itself induces mammalian cell killing.

Cell Line↗
PubMed1985

Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases.

Novobiocin inhibits DNA topoisomerases. It also inhibits excision repair of DNA photodamage, blocking both repair synthesis and the earlier step of incision at u.v. damage sites (as measured by the accumulation of DNA strand breaks in u.v.-irradiated interphase cells treated with DNA synthesis inhibitors such as hydroxyurea or cytosine arabinoside). It has been supposed, therefore, that novobiocin affects repair by blocking a putative topoisomerase step prior to incision. But we find that novobiocin also has a marked dose- and time-dependent effect on mitochondria: in cells exposed to novobiocin, mitochondria swell and their cristae become disrupted, and the intracellular ATP:ADP ratio is lowered, though the membrane potential is maintained as judged by rhodamine 123 fluorescence. Mitotic cells are more resistant to mitochondrial disruption by novobiocin than are interphase cells. This correlates with a relative resistance of u.v.-irradiated mitotic cells to the inhibition of incision by novobiocin. The chromosomal decondensation that results from the accumulation of DNA breaks due to incision when u.v.-irradiated mitotic cells are treated with hydroxyurea and cytosine arabinoside is largely suppressed by novobiocin. Furthermore, the suppression of induced strand break accumulation is partly due to a suppression by novobiocin of the uptake and phosphorylation of cytosine arabinoside; breaks accumulated in u.v.-irradiated cells in the presence of aphidicolin, an inhibitor of DNA polymerase alpha that does not require phosphorylation, are less novobiocin-sensitive. We conclude that the effects of novobiocin on excision repair are more likely to be due to a non-specific effect on ATP metabolism than to a specific effect on a repair-related topoisomerase.

Adenosine Triphosphate↗
PubMed1977

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.

Bacillus↗
PubMed1992

Development and characterization of a WEHI-3B D+ monomyelocytic leukemia cell line resistant to novobiocin and cross-resistant to other topoisomerase II-targeted drugs.

A novobiocin-resistant subline of WEHI-3B D+ murine monomyelocytic leukemia cells was developed by the continuous exposure of cells to this agent in vitro. Sensitive (WEHI-3B/S) and novobiocin-resistant (WEHI-3B/NOVO) sublines were cloned in vitro. WEHI-3B/NOVO cells were stable in the absence of novobiocin for more than 3 months, and the sensitive and resistant clones displayed the same growth rate, cell cycle distribution, cell size, DNA and protein content, and cloning efficiency. Novobiocin has been shown to compete with ATP for the ATP-binding site of topoisomerase II; therefore, intracellular ATP levels can influence the cellular sensitivity to novobiocin. High-performance liquid chromatographic analysis of total cell extracts demonstrated that no difference exists between WEHI-3B/S and WEHI-3B/NOVO cells in the content of ATP. Furthermore, exposure of both cell lines to novobiocin did not affect intracellular ATP levels. In addition to an approximately 2-fold level of resistance to novobiocin, the WEHI-3B/NOVO subline was also 7- and 11-fold cross-resistant to the topoisomerase II-targeted drugs, teniposide and etoposide (VP-16), respectively. A lower level of cross-resistance, comparable to that of novobiocin, was observed in WEHI-3B/NOVO cells for the intercalating topoisomerase II-reactive drugs, doxorubicin, 4'-(9-acridinylamino)methanesulfon-m-anisidide and aclacinomycin A, while the sensitivity to the cytotoxic action of the non-topoisomerase II-acting agents, camptothecin and vincristine, was not altered. After 3-6 h of exposure to 1 microM VP-16, WEHI-3B/S cells accumulated in the S and G2 + M phases of the cell cycle. Similar changes were detected in WEHI-3B/NOVO cells only after exposure to a 10-fold higher concentration of VP-16. Exposure to 150 microM novobiocin caused an accumulation of WEHI-3B/S cells in the G0-G1 phase of the cell cycle but did not affect the cell cycle distribution of WEHI-3B/NOVO cells, while camptothecin induced the same type and extent of changes in the cell cycle distribution of both cell lines. Although the WEHI-3B/NOVO subline appeared to be less responsive to the differentiation-inducing activity of novobiocin and teniposide, the capacity of WEHI-3B/NOVO cells to respond to the differentiation-inducing agent 13-cis-retinoic acid was not significantly different from that of WEHI-3B/S cells. A slight decrease in the accumulation of VP-16 occurred in the resistant cell line, which did not appear to be of sufficient magnitude to account for the 11-fold increase in the degree of resistance to this agent.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1988

Na transport stimulation by novobiocin: transepithelial parameters and evaluation of ENa.

The action of the antibiotic novobiocin on transepithelial Na transport was studied in isolated skins obtained from two different frog species. In Rana esculenta addition of novobiocin to the outer bath (1 mM) resulted in a sustained and reversible stimulation of the short-circuit current, transepithelial potential, and transepithelial conductance. Similar, though more variable and much less pronounced changes were observed in Rana temporaria. In the presence of amiloride (0.1 mM) novobiocin had no effect on any of the investigated transport parameters and all novobiocin induced changes were fully reversed when amiloride was given subsequently. At reduced external Na concentration or low pH the action of novobiocin was found to be greatly attenuated. In the presence of novobiocin an increased affinity to amiloride and a linearization of the transepithelial current-voltage relationship was observed. The results are consistent with the view that novobiocin increases the Na permeability of the outer membrane, possibly by an attenuation of an Na self-inhibition mechanism. In addition, the driving force of transepithelial Na transport was estimated by means of novobiocin. Several different methods were employed, providing varying results. As shown in an Appendix, for the most part the discrepancies can be explained by changes in the intracellular Na and K concentration. In some cases, novobiocin induced large secondary increases in the skin conductance which can be referred to an increased Cl permeability.

Amiloride↗