Nalidixic acid was shown to inhibit specifically the synthesis of deoxyribonucleic acid (DNA) in Escherichia coli. Slight effects on protein and ribonucleic acid (RNA) synthesis were observed only at higher levels of drug or after prolonged incubation. The inhibition of DNA synthesis in E. coli 15TAU, as measured by incorporation of C14-labeled thymine, was observed after exposure to nalidixic acid for 10 min. Inhibition of the incorporation of C14-labeled uracil into RNA and C14-labeled L-arginine into protein (21 and 28% inhibition, respectively) was observed only after 60 min of exposure. When cultures of E. coli 15TAU were exposed to 3.0 ,g/ml of nalidixic acid (slightly greater than the minimal growth inhibitory concentration), the incorporation of C'4-labeled thymidine was inhibited 30 to 40% after 90 min. Nalidixic acid at 10,ug/ml, a lethal concentration, inhibited thymidine incorporation 72% during this period.

of action of nalidixic acid on Escherichia coli. J. Bacteriol. 88:1112-1118. 1964.-Nalidixic acid was lethal for proliferating cultures of Escherichia coli. Associated with this lethal effect was the forimiation of elongated, serpentine forms. Cultures treated with nalidixic acid were osmotically stable; lethality was observed in the presence of stabilizers. Although it was possible to demiionstrate leakage of intracellular components fromii treated cells, this effect occurred only after 99%of the cells were nonviable. Nalidixic acid had little or no effect on respiration with glucose as substrate. If cellular growth was restricted by suboptimal temperature or nutritional deficiencies, the drug was not lethal. Chemnical analysis of cellular constituents revealed that lipid, protein, and ribonucleic acid levels were of the samle order of magnitude in control and drug-treated cells. Only deoxyribonucleic acid (l)NA) levels were imiarkedlv lowered in drug-treated cells. These facts are consistent with the view that nalidixic acid interferes with the synthesis of E. coli D)NA. Nalidixic acid (1-ethyl-1 ,4-dihydro-7-methyl-4-oxo-1 ,8-nal)hthyridine-3-carloxylic acid) is a new antibacterial agent, the synthesis of which was reported by Lesher et al. (1962). This naphthyridine has a broad spectrum of antibacterial activity; is especially active against gramnegative species, including those associated with urinary-tract infections (Deitz, Bailey, and Froelich, 1964); and is very well tolerated by laboratory animals and man (Lishmnan and Swinney, 1963). This report plresents the results of preliminary studies to deterimiine the mechanism of action of nalidixic acid. We examined the effect of this compound on the growth, motility, respiration, integrity of the cell membrane and cell wall, an(l chemical composition of Eseherichia coli. MIATEIRIALS AND MAI}THODS Cultures. F. coli strain 198 (AT'CC 11229) and E. coli strain 15 (T-A-U-; obtained fromIDepartment of Bacteriology, Rutgers U'niversity, New B3runswick, N.J.; hereafter referred to as E. coli 15 TAPT) were maintained by periodic on October 5, 2020 by guest http://jb.asm.org/ Downloaded from ACTION OF NALIDIXIC ACID ON E. COLI 1956). Control cultures and cultures treated with penicillin G (1,000 units per ml) were also studied. After 4 hr, samples were diluted in 1% peptone water and in 1 %, peptone water plus stabilizers, and viability was determined in Tryptone Glucose Extract Agar containing stabilizers. Cell suspensions also were examined microscopically.

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

COOK, THOMAS M. (Sterling-Winthrop Research Institute, Rensselaer, N.Y.), WILLIAM H. DEITZ, AND WILLIAM A. Goss. Mechanism of action of nalidixic acid on Escherichia coli. IV. Effects on the stability of cellular constituents. J. Bacteriol. 91:774-779. 1966.-Treatment of Escherichia coli 15TAU with nalidixic acid resulted in degradation of the nucleic acids of the cells, whereas protein was unaffected. Deoxyribonucleic acid (DNA) degradation appeared to be more extensive than ribonucleic acid degradation during periods of comparable bactericidal action. The onset of DNA degradation was evident prior to a measurable bactericidal effect. However, within the range of 2 to 20%, DNA degradation was accompanied by a decrease in viable cell numbers. Degradation of DNA to acid-soluble material occurred only under conditions permitting the bactericidal action of nalidixic acid. Arrest of the bactericidal action of nalidixic acid by the addition of dinitrophenol or chloramphenicol also inhibited DNA degradation. The acid-soluble products, which were excreted into the medium, have not been characterized completely, but probably were not phosphorylated.

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.