Mechanism of action of nalidixic acid on Escherichia coli. 3. Conditions required for lethality

Deitz, William H. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), Thomas M. Cook, and William A. Goss. Mechanism of action of nalidixic acid on Escherichia coli. III. Conditions required for lethality. J. Bacteriol. 91:768-773. 1966.-Nalidixic acid selectively inhibited deoxyribonucleic acid (DNA) synthesis in cultures of Escherichia coli 15TAU. Protein and ribonucleic acid synthesis were shown to be a prerequisite for the bactericidal action of the drug. This action can be prevented by means of inhibitors at bacteriostatic concentrations. Both chloramphenicol, which inhibits protein synthesis, and dinitrophenol, which uncouples oxidative phosphorylation, effectively prevented the bactericidal action of nalidixic acid on E. coli. The lethal action of nalidixic acid also was controlled by transfer of treated cells to drug-free medium. DNA synthesis resumed immediately upon removal of the drug and was halted immediately by retreatment. These studies indicate that nalidixic acid acts directly on the replication of DNA rather than on the "initiator" of DNA synthesis. The entry of nalidixic acid into cells of E. coli was not dependent upon protein synthesis. Even in the presence of an inhibiting concentration of chloramphenicol, nalidixic acid prevented DNA synthesis by E. coli 15TAU.     

Mechanism of Action of Nalidixic Acid on Escherichia coli V. Possible Mutagenic Effect

Cook, Thomas M., (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), William A. Goss, and William H. Deitz. Mechanism of action of nalidixic acid on Escherichia coli. V. Possible mutagenic effect. J. Bacteriol. 91:780-783. 1966.-With a streptomycin-dependent organism, Escherichia coli ATCC 11143, it has been shown that exposure to nalidixic acid, under conditions permitting some bactericidal action, results in an increased number of streptomycin-independent bacteria among the survivors. This effect is evident only with proliferating cultures, and is related to the concentration of nalidixic acid and the duration of exposure.     

Bactericidal action of nalidixic acid on Bacillus subtilis

Cook, Thomas M. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), Karen G. Brown, James V. Boyle, and William A. Goss. Bactericidal action of nalidixic acid on Bacillus subtilis. J. Bacteriol. 92:1510-1514. 1966.-Nalidixic acid at moderate concentrations exerts a bactericidal action upon the gram-positive bacterium Bacillus subtilis. The synthesis of deoxyribonucleic acid (DNA) in B. subtilis is selectively inhibited by nalidixic acid at concentrations approximating the minimal growth inhibitory concentration. Higher concentrations (25 mug/ml) result in a 30 to 35% degradation of DNA. After extended exposure to nalidixic acid, protein synthesis is also depressed. Cells of B. subtilis treated with nalidixic acid exhibit characteristic morphological abnormalities including cell elongation and development of gram-negative areas. From the results presented, it can be concluded that the mode of action of nalidixic acid upon susceptible bacteria is similar for both gram-positive and gram-negative species.     

Mechanism of action of nalidixic acid on Escherichia coli. Vi. Cell-free studies

The effects of nalidixic acid in vitro on deoxyribonucleic acid (DNA)- polymerase (deoxyribonucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7), deoxyribonucleotide kinases (ATP: deoxymono- and diphosphate phosphotransferases), and deoxyribosyl transferase (nucleoside: purine deoxyribosyltransferase, EC 2.4.2.6) were examined employing partially purified and crude extracts of Escherichia coli ATCC 11229 and E. coli 15TAU. Nalidixic acid had no inhibitory effect on the DNA-polymerase of the wild-type strain E. coli ATCC 11229 at concentrations of 1.4 x 10(-3) to 2.8 x 10(-3)m. No inhibition of deoxyribonucleotide kinase activity was observed at concentrations of nalidixic acid ranging from 2 x 10(-3) to 8.6 x 10(-3)m. Nalidixic acid (0.43 x 10(-4) to 0.43 x 10(-3)m) had no inhibitory effect on the deoxyribosyl transferase activity of crude extracts obtained from E. coli ATCC 11229 or E. coli 15TAU. Analytical CsCl density gradient centrifugation demonstrated that the DNA obtained after treatment of E. coli 15TAU with nalidixic acid was not cross-linked. These results suggest that the prevention of DNA synthesis in vivo by nalidixic acid is not attributable to inhibition of DNA polymerase, deoxyribonucleotide kinase, deoxyribosyl transferase, or to cross-linking of the DNA of treated cells.     

Effect of Nalidixic Acid and Hydroxyurea on Division Ability of Escherichia coli fil+ and lon− Strains

Short periods of incubation in medium containing nalidixic acid or hydroxyurea, followed by a return to normal growth conditions, induced filament formation in Escherichia coli B (fil(+)) and AB1899NM (lon(-)) but not in B/r (fil(-)) and AB1157 (lon(+)). These drugs reversibly stopped deoxyribonucleic acid (DNA) synthesis with little or no effect on ribonucleic acid (RNA) synthesis or mass increase. The initial imbalance caused by incubation in these drugs was the same for B and B/r as was macromolecular synthesis following a return to normal growth conditions. DNA degradation caused by nalidixic acid was measured and found to be the same for B and B/r. Hydroxyurea caused no DNA degradation in these two strains. Survival curves as determined under various conditions by colony formation suggested that the property of filament formation was responsible for the extrasensitivity of fil(+) and lon(-) strains to either nalidixic acid or hydroxyurea. E. coli B was more sensitive to either drug than was B/r or B(s-1). Pantoyl lactone or liquid holding treatment aided division and colony formation of nalidixic acid-treated B but had no effect on B/r. Likewise, the filament-former AB1899NM was more sensitive to nalidixic acid than was the non-filament-former AB1157. The sensitivity of B/r and B(s-1) to nalidixic acid was nearly the same except at longer times in nalidixic acid, when B(s-1) appeared more resistant. Even though nalidixic acid, hydroxyurea, and ultraviolet light may produce quite different molecular alterations in E. coli, they all cause a metabolic imbalance resulting in a lowered ratio of DNA to RNA and protein. We propose that it is this imbalance per se rather than any specific primary chemical or photochemical alterations which leads to filament formation by some genetically susceptible bacterial strains such as lon(-) and fil(+).     

Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.     

Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.     

Induction of radioresistance in Escherichia coli.

The effect of prior treatment by inducing agents on the radioresistance of cells of Escherichia coli has been studied. In order to separate the induction process from the radiation-damage process, cells were first treated with inducing agents such as ultraviolet light, ionizing radiation, or nalidixic acid, allowed to become induced by incubation for 50 min and then given rifampin to prevent further induction. They were then tested for radiation sensitivity. It was found that all strains tested except recA-, lex-, and recB showed very apparent protection. Induction by UV had the most effect and by nalidixic acid the least. The time course of development of protection was observed in one case: it is 50% established in 15 min. The absence of effect in recA- and lex- is explainable by the fact that these cells cannot be induced, for example, for prophage or the inducible inhibitor of post-irradiation DNA degradation. We suggest that the inducible inhibitor of postirradiation DNA degradation is one factor in a recovery system possessed by E. coli cells.     

Chilling cells enhances the bactericidal action of fatty acids on Escherichia coli.

Preincubation at 0 C considerably increased the bactericidal action of 0.4% nonanoic and decanoic acids on Escherichia coli K-12 154. This lethal effect seemed to be dependent on the media used to grow the bacteria. Stationary-phase cells were more sensitive than those from exponential cultures. A mutant (FA31) resistant to the bactericidal action of "cold shock" and 0.4% deconoic acid was isolated from E. coli FA23 (AN E. coli 154 derivative able to grow on 0.1% decanoic acid) by a recycling selection procedure. Other E. coli strains tested showed behavior similar to that of strain K-12 154. The chilling of cells as a tool to improve the bactericidal action of fatty acids in foods is discussed.     

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.     

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.     

Evidence for bactericidal activity of polymorphonuclear leukocytes without phagocytosis.

The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with [3H]thymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.     

Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.     

Effect of Nalidixic Acid on the Growth of Deoxyribonucleic Acid Bacteriophages

The effect of nalidixic acid on the growth of various deoxyribonucleic acid (DNA) bacteriophages has been investigated by one-step growth experiments. The Escherichia coli bacteriophages T5, lambda, T7 and phiR are strongly inhibited by nalidixic acid, whereas T4 and T2 are only partially inhibited. The Bacillus subtilis bacteriophages SP82, SP50, and phi29 are relatively unaffected by nalidixic acid. There is no correlation between those bacteriophages which can grow in the presence of nalidixic acid and the presence of an unusual base in the phage DNA.     

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.     

Replication of the deoxyribonucleic acid of multiple-drug-resistance factor in Escherichia coli.

1. It was shown that a system previously described for labelling R-factor DNA during transfer to an irradiated recipient strain of Escherichia coli did not allow high selectivity in the incorporation of thymine into R-factor DNA. 2. Lack of selectivity was shown to be due to cross-feeding from recipient to donor strain. 3. An improved system using a nalidixic acid-resistant recipient strain is described in which incorporation of thymine into the DNA of donor cells is minimized by addition of nalidixic acid after completion of transfer of the plasmid during conjugation.