Hydrogenase synthesis in Bradyrhizobium japonicum Hupc mutants is altered in sensitivity to DNA gyrase inhibitors.

In the Hupc mutants of Bradyrhizobium japonicum SR, regulation of expression of hydrogenase is altered; the mutants synthesize hydrogenase constitutively in the presence of atmospheric levels of oxygen. The DNA gyrase inhibitors nalidixic acid, novobiocin, and coumermycin were used to inhibit growth of wild-type and mutant cells. For each inhibitor tested, growth of mutant and wild-type strains was equally sensitive. However, in contrast to the wild type, the Hupc mutants synthesized hydrogenase in the presence of high levels of any inhibitor. Cells were incubated with the drugs and simultaneously labeled with 14C-labeled amino acids, and hydrogenase was immunoprecipitated with antibody to the large subunit of the enzyme. Fluorograms of antibody blots then were scanned to determine the relative amount of hydrogenase (large subunit) synthesized in the presence or absence of the gyrase inhibitors. The amount of hydrogenase synthesized by the Hupc mutants in the presence of 300 micrograms of nalidixic acid per ml was near the level of enzyme synthesized in the absence of the inhibitor. No hydrogenase was detected in antibody blots of wild-type cultures which were derepressed for hydrogenase in the presence of 100 micrograms of coumermycin or novobiocin per ml. In contrast, hydrogenase was synthesized by the Hupc mutants in the presence of 100 micrograms of either drug per ml. The amount synthesized ranged from 5 to 32% and 20 to 49%, respectively, of that in the absence of those inhibitors, but nevertheless, hydrogenase synthesis was detected in all of the mutants examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen

The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.     

Reduction of Chloroplast DNA Content in Solanum nigrum Suspension Cells by Treatment with Chloroplast DNA Synthesis Inhibitors

Suspension cell cultures of Solanum nigrum were grown in the presence of six different chloroplast DNA synthesis inhibitors in order to determine whether the pool size of chloroplast DNA (cpDNA) could be selectively reduced relative to the nuclear DNA content. One of the effects of the inhibitors was a reduction in cell growth and viability. Cell growth (fresh weight) was reduced 50% (in 8 day cultures) by: 100 micromolar bisbenzimide, 8 micromolar ethidium bromide, 0.3 micromolar 5-fluordeoxyuridine (Fudr), 200 micromolar nalidixic acid, 30 micromolar novobiocin, or 10 micrograms per milliliter rifampicin. At these concentrations, three of the inhibitors, ethidium bromide, Fudr, and rifampicin, also substantially reduced the viability of the cultures. Analyses of the chloroplast and nuclear DNA content per gram fresh weight by dot blot hybridizations indicated that the reduction of cpDNA content was greatest at inhibitor concentrations which reduced cell growth by more than 50% but this depended on the culture conditions. For example, the two DNA gyrase inhibitors, nalidixic acid and novobiocin, were more effective in lowering cpDNA content in cultures which were transferred (2 × 4 days) once during the eight day incubation. Because several inhibitors were toxic to cell growth, the DNA content of treated cells was also determined on the basis of cell (protoplasts) number. Analyses of nuclear and cpDNA content per cell for each treatment indicated that only the DNA gyrase inhibitors, nalidixic acid, and novobiocin reduced cpDNA content. Neither inhibitor reduced nuclear DNA content. These results suggest that DNA gyrases participate in cpDNA replication. The selective reduction of cpDNA content in regeneratable cultures may facilitate the generation and selection of cpDNA mutants or transformants from higher plants.     

Increased expression of biodegradative threonine dehydratase of Escherichia coli by DNA gyrase inhibitors

The synthesis of inducible biodegradative threonine dehydratase of Escherichia coli increased several-fold in the presence of the DNA gyrase inhibitors, nalidixic acid and coumermycin. Temperature-sensitive gyrB mutants expressed higher levels of dehydratase as compared to an isogenic gyrB+ strain. Immunoblotting experiments showed increased synthesis of the dehydratase protein in the presence of gyrase inhibitors; addition of rifampicin and chloramphenicol to cells actively synthesizing enzyme preventing new enzyme production. Increased expression of dehydratase by gyrase inhibitors was accompanied by relaxation of supercoiled DNA.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.     

Effect of DNA gyrase inhibitors and urea on the expression of cysB, the regulatory gene of the cysteine regulon

cysB, the regulatory gene of the cysteine regulon, is autoregulated. Inhibitors of both gyrase subunits, nalidixic acid and novobiocin, affect the expression of cysB, as monitored by beta-galactosidase activity in cysB::lac fusion strains. In gyrA mutants that are resistant to nalidixic acid, this drug does not affect cysB expression. The amount of mRNA transcribed from the cysB promoter isolated from cultures grown in the presence of gyrase inhibitors was significantly lower than that from the control culture without inhibitors. Urea also decreased cysB expression. These results suggest that DNA topology could play a role in cysB expression.     

Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli.

The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro. Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed. No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors. In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures. Thus, a functional DNA gyrase is required for rRNA synthesis. Purified DNA gyrase had no effect on rRNA synthesis in a purified system. However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins. Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.     

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.     

DNA gyrase activities from Rhodobacter capsulatus: analysis of target(s) of coumarins and cloning of the gyrB locus

Bacterial DNA gyrase is composed of two subunits, gyrase A and B, and is responsible for negatively supercoiling DNA in an ATP-dependent manner. The coumarin antibiotics novobiocin and coumermycin are known inhibitors of bacterial DNA gyrase in vivo and in vitro. We have cloned, mapped, and partially sequenced Rhodobacter capsulatus gyrB which encodes the gyrase B subunit that is presumably involved in binding to coumarins. DNA gyrase activities from crude extracts of R. capsulatus were detected and it was shown that the R. capsulatus activity is (1) inhibited by novobiocin and coumermycin, (2) ATP-dependent and, (3) present in highly aerated and anaerobically grown cells. We previously observed that when R. capsulatus coumermycin-resistant strains are continuously recultured on media containing coumermycin they sometimes acquired mutations in hel genes (i.e., cytochromes c biogenesis mutations). We discuss the possibility that coumarins may inhibit cytochromes c biogenesis as a second target in R. capsulatus via hel (i.e., a putative ATP-dependent heme exporter).     

Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

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.     

Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme.

The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile. Most strains were sensitive to doses of coumarins (0.02 to 10 micrograms/ml) which specifically inhibit DNA gyrase in eubacteria. Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri. In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa). Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically. Topological analysis has shown that the 1.7-kilobase plasmid from Halobacterium sp. strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment. These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase.     

The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.     

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.     

The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [³H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a ‘swivelase’ role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, d-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.     

DNA-damaging agents and DNA-synthesis inhibitors induce luminescence in dark variants of luminous bacteria

The DNA-damaging agents mitomycin C and UV irradiation, as well as the DNA-synthesis inhibitors nalidixic acid, novobiocin and coumermycin, induce the de novo synthesis of luciferase and in vivo luminescence in dark variant cells of the luminous bacteria Photobacterium leiognathi. Mitomycin C and nalidixic acid also cause the induction of luminescence in wild-type cells in the absence of its natural inducer. In spite of the high level of in vivo luminescence of the treated dark-variant cells, none of these agents result in the appearance of genetically luminous revertants. The possibility is discussed that these agents phenotypically induce luminescence through their ability to trigger ‘SOS functions’, which in turn leads to the transitory inactivation of certain repressors.     

cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin

Summary Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Nov^r) mutants: 10% of Nov^r mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.