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PubMed1982

Effects of novobiocin, coumermycin A1, clorobiocin, and their analogs on Escherichia coli DNA gyrase and bacterial growth.

Novobiocin, coumermycin A1, and clorobiocin, structurally related compounds that antagonize the B subunit of the essential bacterial enzyme DNA gyrase, were compared with 18 of their analogs for the inhibition of Escherichia coli DNA gyrase supertwisting activity in vitro and of bacterial multiplication. This family of compounds has a 4-hydroxy-8-methylcoumarin core substituted in the 7 and 3 positions. Important for enzyme inhibition in vitro is a 7 ether linkage to a 3'-substituted noviose sugar. The 3'-ester-linked 5-methylpyrrole, found in the coumermycin series, conferred at least 10-fold more inhibitory activity than did the similarly linked amide, found in the novobiocin series; lack of the pyrrole and amide results in the loss of inhibitory activity. Of many aryl and alkyl substituents linked as an amide at the 3 position, the 4-hydroxyl-3-(3-methyl-2-butenyl)benzoic acid moiety, found in novobiocin and clorobiocin, and the reduplication of the coumarin-noviose-5-methylpyrrole, found in coumermycin A1, were most effective in gyrase inhibition. In vivo, the ability of these compounds to inhibit the growth of E. coli varied greatly. The enhanced inhibition of gyrase in vitro conferred by a 5-methylpyrrole relative to an amide in the 3'-noviose position was reflected in inhibition of bacterial multiplication. Several substitutions at the 3 position of the coumarin core conferring similar antagonism of gyrase in vitro resulted in substantially different inhibitory activities for E. coli, suggesting that these moieties at the 3 position affect drug access to the intracellular target. This target was shown for isobutyryl PNC-NH2 (PNC-NH2 is 3-amino-4-hydroxy-8-methyl-7-[3-O-(5-methyl-2-pyrrolylcarbonyl)noviosyloxy] coumarin) and confirmed for novobiocin, coumermycin A1, and clorobiocin to be in the B subunit of DNA gyrase.

Aminocoumarins↗
PubMed1986

Effect of 4-quinolones and novobiocin on calf thymus DNA polymerase alpha primase complex, topoisomerases I and II, and growth of mammalian lymphoblasts.

The influence of ciprofloxacin, nalidixic acid, norfloxacin, novobiocin, and ofloxacin on elements of eucaryotic DNA replication was investigated in vitro. Each of the 4-quinolones, when present in amounts of more than 100 micrograms/ml, reversibly inhibited the DNA synthesis performed by the 95 DNA polymerase alpha primase complex from calf thymus. Novobiocin at 500 micrograms/ml or at higher concentrations irreversibly inactivated DNA polymerase alpha primase complex. The accuracy of in vitro DNA synthesis in the absence of repair mechanisms was determined from amber-revertant assays with phi X174am16(+) DNA as template. The antimicrobial agents did not significantly increase the frequencies of base pairing mismatches during the course of replication, indicating that the basal mutation rate is not affected by novobiocin and the 4-quinolones. The Ki values of 50% inhibition of DNA topoisomerases from calf thymus by ciprofloxacin, norfloxacin, novobiocin, nalidixic acid, and ofloxacin were 300, 400, 1,000 or more, 1,000 or more, and 1,500 or more micrograms/ml, respectively, in the case of topoisomerase I, and the Ki values were 150, 300, 500, 1,000, and 1,300 micrograms/ml, respectively, in the case of topoisomerase II. The procaryotic topoisomerase II is approximately 100-fold more sensitive to inhibition by ciprofloxacin, norfloxacin, and ofloxacin than is its eucaryotic counterpart. Growth curves of lymphoblasts were recorded in the presence of ofloxacin and ciprofloxacin. Neither 1 nor 10 micrograms of ciprofloxacin or of ofloxacin per ml affected cell proliferation. Ofloxacin and ciprofloxacin at 100 micrograms/ml inhibited cell growth; 1,000 micrograms/ml led to cell death. No correlation exists between the antimicrobial and cytotoxic activities of the 4-quinolones.

Cell Division↗
PubMed2003

Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance.

DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP. PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

Adenosine Triphosphatases↗
PubMed2004

Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons): a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase.

Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-A resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-A resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 micro M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K(m) increased approximately 5-fold and the V(max) decreased approximately 30%. In contrast, the topoisomerase IV ATP K(m) decreased by a factor of 6, and the V(max) increased approximately 2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.

Adenosine Triphosphatases↗
PubMed1961

Continuous fermentation of novobiocin.

Continuous fermentation trials with Streptomyces niveus in a nine-stage fermentation system (7-liter reaction volume per stage) indicated that the cultures used gradually lost their ability to produce novobiocin when cultured over periods from 10 to 25 days. It was found that mycelial degeneration could be circumvented by operational means during continuous culture using the following technique: Two interchangeable 24-liter stages were installed at the front end of the nine-stage system and connected in parallel with the latter. Alternatingly one of these two tanks was then used as first stage of the continuous fermentation system. The holdup time in the first vessel was adjusted to limit cell growth chiefly to this stage so that most of the antibiotic production took place in subsequent stages. The first stages were switched at approximately weekly intervals. Each of the new tanks was prepared as a batch, inoculated with a high-producing cell population, and allowed to grow for 3 days before it was connected to the remaining system for continuous operation. Using this technique no evidence of culture degeneration was encountered in subsequent novobiocin production stages over a period of 33 days. In conventional runs without periodic replacement of the first stage, culture degeneration with the novobiocin fermentation occurred within a period of 10 to 25 days of continuous operation. This observation indicates that the described technique offers a solution to the problem of maintaining high steady-state titers in continuous novobiocin fermentations. Extension of this technique to other continuous fermentations where culture degeneration is a problem is indicated.

Anti-Bacterial Agents↗
PubMed1999

tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin.

In previous studies we demonstrated that mutations in the genes cysB, cysE, and cls (nov) affect resistance of Escherichia coli to novobiocin (J. Rakonjac, M. Milic, and D. J. Savic, Mol. Gen. Genet. 228:307-311, 1991; R. Ivanisevic, M. Milic, D. Ajdic, J. Rakonjac, and D. J. Savic, J. Bacteriol. 177:1766-1771, 1995). In this work we expand this list with mutations in rpoN (the gene for RNA polymerase subunit sigma54) and the tRNA synthetase genes alaS, argS, ileS, and leuS. Similarly to resistance to the penicillin antibiotic mecillinam, resistance to novobiocin of tRNA synthetase mutants appears to depend upon the RelA-mediated stringent response. However, at this point the overlapping pathways of mecillinam and novobiocin resistance diverge. Under conditions of stringent response induction, either by the presence of tRNA synthetase mutations or by constitutive production of RelA protein, inactivation of the cls gene diminishes resistance to novobiocin but not to mecillinam.

Alanine-tRNA Ligase↗
PubMed2002

The baeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate.

Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible (Delta)acrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the (Delta)acrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.

Anti-Bacterial Agents↗
PubMed2003

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.

Bacterial Proteins↗
PubMed1966

Genetic modifiers of the phenotypic level of deoxyribonucleic acid-conferred novobiocin resistance in Haemophilus.

Leidy, Grace (Columbia University, New York, N.Y.), Iris Jaffee, and Hattie E. Alexander. Genetic modifiers of the phenotypic level of deoxyribonucleic acid-conferred novobiocin resistance in Haemophilus. J. Bacteriol. 92:1464-1468. 1966.-An apparent increase in novobiocin resistance in Haemophilus aegyptius after a second exposure to a particular H. influenzae transforming deoxyribonucleic acid was shown to be the result not of multi-step transformation but of the action of a gene functioning as an enhancement modifier. The modifier is very closely linked to a streptomycin resistance gene (which is linked to a novobiocin resistance marker); it affects the natural degree of resistance to both novobiocin and kanamycin to a measurable degree. Evidence of a repressor of the enhancement modifier is reported.

DNA, Bacterial↗
PubMed1983

Effect of different media on determination of novobiocin resistance for differentiation of coagulase-negative staphylococci.

Species identification of coagulase-negative staphylococci often requires the determination of novobiocin susceptibility. Although previous investigators have recommended the use of P agar for this purpose, most clinical laboratories do not routinely utilize this medium. For this reason, studies were performed to compare novobiocin susceptibility results obtained with 11 different species of staphylococci (10 isolates of each species), using P agar, Trypticase soy agar with 5% sheep blood, and Mueller-Hinton agar. Tests performed on 70 susceptible isolates (minimal inhibitory concentration less than 1.6 micrograms/ml) resulted in zones of inhibition around 5-micrograms novobiocin disks ranging from 19.6 to 33.9, 16.2 to 26.6, and 21.3 to 36.4 mm on P agar, Trypticase soy agar with 5% sheep blood, and Mueller-Hinton agar, respectively. Forty resistant isolates (minimal inhibitory concentration greater than or equal to 1.6 micrograms/ml) exhibited zones of inhibition ranging from 6.0 to 11.3 mm on P agar, 6.0 to 11.6 mm on Trypticase soy agar with 5% sheep blood, and 6.0 to 13.5 mm on Mueller-Hinton agar. Using the established cut off of 16 mm to define novobiocin resistance for the identification of coagulase-negative staphylococci, we correctly identified 100% of the strains tested, regardless of the media utilized.

Coagulase↗
PubMed1984

Effect of novobiocin on mycoplasma virus L2 replication.

L2 is a temperate mycoplasma virus containing 11.8 kilobase pairs of negatively superhelical double-stranded DNA. We observed L2 DNA with less superhelicity in novobiocin-treated cells than that in untreated cells. However, although no change in viral DNA superhelicity could be found in novobiocin-treated novobiocin-resistant cells, L2 production decreased in these novobiocin-treated cells.

Acholeplasma laidlawii↗
PubMed1990

The F1 ATP synthetase beta-subunit: a major yeast novobiocin binding protein.

Novobiocin affects DNA metabolism in both prokaryotes and eukaryotes, resulting in cell death. In prokaryotes, the drug is a specific inhibitor of DNA gyrase, a type II topoisomerase that can be purified on a novobiocin-Sepharose column. The yeast type II topoisomerase is neither the biochemical, nor the genetic target of the antibiotic. We have purified the major yeast novobiocin binding proteins and identified one of them as the beta-subunit of the yeast mitochondrial F1 ATP synthetase, a protein highly conserved throughout evolution. The inactivation of this protein might explain the toxic effects of novobiocin on higher eukaryotic cells.

Amino Acid Sequence↗
PubMed1990

Treatment of Staphylococcus aureus mastitis with penicillin and novobiocin: antibiotic concentrations and bacteriologic status in milk and mammary tissue.

Cows with Staphylococcus aureus mastitis received either one or two intramammary infusions containing 100,000 U penicillin G and 150 mg novobiocin. Milk and tissue samples were obtained from each quarter. Peak mean penicillin and novobiocin concentrations from antibiotic-positive tissue samples were .013 U/mg and .06 microgram/mg, respectively, for quarters treated once. Quarters treated twice had peak mean penicillin and novobiocin concentrations of .057 U/mg and .06 microgram/mg, respectively. Viable Staphylococcus aureus were isolated intermittently from milk and tissue samples of quarters positive for penicillin and novobiocin for both treatment groups. Histological analysis of mammary parenchyma demonstrated marked decreases in luminal area and increases in connective tissue area and leukocyte infiltration in S. aureus-infected quarters compared with uninfected controls, suggesting that reduction in milk collecting space and presence of inflammatory changes may be responsible for poor drug distribution.

Animals↗
PubMed1975

[Changes in the liver transport of bile pigments in certain forms of jaundice under the influence of novobiocin and luminal].

The novobiocin and luminal test application increases the possibilities to differentiate premicrosome from postmicrosome jaundice and extends the knowledge on hyperbilirubinemia pathobiochemistry. Novobiocin (an antibiotic) inhibits the conjugation and glucuronidizing of bile pigments. Accordingly, the novobiocin test is strongly positive in any premicrosome jaundice form--Gilberts's disease, posthepatitis hyperbilirubinemia (PHHB) and hemolytic jaundice. It is negative in hepatocellular and hepatocanalicular jaundice, and in Dubin-Johnson's syndrome. In premicrosome jaundice with fluctuating bilirubinemia and normal bilirubin level at the time of examination, the novobiocin test may bring about manifestation of a latent or temporary compensated defect which proves to be most helpful in diagnosing. It is assumed that hemolytic jaundice is caused, apart from bile pigments hyperproduction, also by concomitant disorders in the intrahepatal transport of bile pigments. The latter hypothesis is corroborated by the data of the study. The luminal test is likewise markedly positive in premicrosome jaundice, and negative--in chronic hepatocellular and hepatocanalicular jaundice. In some instances with slight defect in the intrahepatal transport of pigments, the luminal test accounts for normalization of bilirubinemia and of the direct to total bilirubin ratio. The test is moderately positive in the syndrome of Dubin-Johnson. The latter fact supports the hypothesis that luminal, alongside the effect on transport mechanisms, exerts influence on bile pigments excretion also.

Bile↗
PubMed1991

Novobiocin treatment reverses radiation-induced alterations in higher-order DNA structure in L5178Y nucleoids.

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.

Animals↗
PubMed1986

Effect of the bacterial DNA gyrase inhibitors, novobiocin, nalidixic acid, and oxolinic acid, on oxidative phosphorylation.

When incubated with isolated intact rat liver mitochondria, novobiocin and nalidixic acid act as uncouplers of oxidative phosphorylation; they stimulate oxygen uptake and inhibit ATP synthesis. Novobiocin is about as powerful an uncoupler as is 2,4-dinitrophenol, nalidixic acid is somewhat less powerful, and oxolinic acid exerts no inhibition whatsoever at the concentrations used. The three inhibitors are without effect on oxidative phosphorylation in Escherichia coli nor does novobiocin affect this process in a novobiocin-permeable mutant of yeast. While it would appear that oxolinic acid may be a relatively specific tool for the manipulation of the superhelicity of DNA in complex systems such as mammalian mitochondria and intact mammalian cells, the specificity of each of these inhibitors may depend upon the particular conditions and species used and such experiments require adequate controls on oxidative phosphorylation.

Animals↗
PubMed1979

The use of induced mammary infections for evaluating dry cow treatment products. II. Trial of a proposed method to compare three levels of novobiocin.

Infections were induced at the end of lactation in all udder quarters of 19 cows by the infusion of 0.2 mL of a 10(-4) dilution in milk of a six hour milk culture of Staphylococcus aureus, strain 305 (A.T.C.C No. 29740). Two right or two left udder quarters were infected at 15 days and the opposite two five days before the last milking of lactation. Following the last milking all four udder quarters of eight cows were treated with 400 mg novobiocin in 10 mL of 2% aluminum monosterate in peanut oil, gelled. All udder quarters of eight other cows were treated with 50 mg novobiocin in the same vehicle and the udder quarters of three cows were treated with the vehicle only. At calving, eight of 32 quarters treated with 400 mg novobiocin were still infected, as were 18 of 32 treated with 50 mg of novobiocin and all those quarters treated with vehicle only. Results were identical from udder quarters infected 15 and five days before drying off. No significant differences were found between quarters in milk yield on the last day of lactation, nor the length of the dry period. An increasing number of udder quarters were infected at calving with increase in lactation age of the cow, although the small number of cows would not allow a firm conclusion. A significant difference in results was found between front and hind udder quarters, only five of 32 front quarters were infected at calving as compared to 21 to 32 hind quarters. The method proposed was found to give essentially the same results as those from a large field trial using the same antibiotic. It should therefore be useful in evaluation trials of new antibiotic products for dry cow treatment.

Animals↗
PubMed1978

[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.

Antifungal Agents↗