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. 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.     

Two pathways of division inhibition in UV-irradiated E. coli

Molecular Genetics and Genomics - We have investigated the mechanism of division inhibition in E. coli following UV-irradiation or nalidixic acid treatment. After UV, two separate mechanisms, both...     

Two pathways of division inhibition in UV-irradiated E. coli

We have investigated the mechanism of division inhibition in E. coli following UV-irradiation or nalidixic acid treatment. After UV, two separate mechanisms, both dependent upon recA+, appear to block division. One mechanism is dependent upon sfiA and sfiB, is inhibited by low levels (4 micrograms/ml) of rifamycin and is expressed in tif mutants at 42 degrees C. The second mechanism is independent of sfiA, and sfiB, is resistant to rifamycin and does not occur in cells lacking DNA replication forks. We suggest that this second mechanism is the result of the failure to terminate DNA replication in inhibited cells. Nalidixic acid inhibition of cell division also appears to involve both mechanisms but as found previously replication forks are also necessary to induce the sfi pathway.     

Regulation of murein biosynthesis and septum formation in filamentous cells of Escherichia coli PAT 84.

Both the beta-lactam antibiotic, cephalexin, and the deoxyribonucleic acid synthesis inhibitor, nalidixic acid, are known to inhibit cell division in Escherichia coli and induce the formation of filaments. The biosynthesis of murein was investigated in these filaments and compared with the murein synthesized by the normally dividing rods of E. coli PAT 84. Differences were found in the extent of peptide side-chain cross-linkage. Filamentous cells had higher extents of cross-linkages in their newly synthesized murein. Quantitative analyses of the D-alanine carboxypeptidase and transpeptidase reactions in the different cells revealed that the carboxypeptidase activity of the filamentous cells was partially inhibited. These results were similar to those previously found with filaments that were obtained after growth of the thermosensitive division mutant at its restrictive temperature. We conclude that the formation of new cell ends (septa) depends on the proper balance between the activities of the D-alanine carboxypeptidase that regulates the availability of precursor doners and the transpeptidase, which catalyzes cross-linking and attachment of newly synthesized murein.     

Generation of buds, swellings, and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti.

Inhibition of cell division in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis results in elongation into long filaments many times the length of dividing cells. As a first step in characterizing the Rhizobium meliloti cell division machinery, we tested whether R. meliloti cells could also form long filaments after cell division was blocked. Unexpectedly, DNA-damaging agents, such as mitomycin C and nalidixic acid, caused only limited elongation. Instead, mitomycin C in particular induced a significant proportion of the cells to branch at the poles. Moreover, methods used to inhibit septation, such as FtsZ overproduction and cephalexin treatment, induced growing cells to swell, bud, or branch while increasing in mass, whereas filamentation was not observed. Overproduction of E. coli FtsZ in R. meliloti resulted in the same branched morphology, as did overproduction of R. meliloti FtsZ in Agrobacterium tumefaciens. These results suggest that in these normally rod-shaped species and perhaps others, branching and swelling are default pathways for increasing mass when cell division is blocked.     

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(+).     

Modulation of cell wall synthesis by DNA replication in Escherichia coli during initiation of cell growth.

Resting cells of Escherichia coli are able to initiate growth and murein biosynthesis in the presence of beta-lactam antibiotics binding to penicillin-binding proteins (PBPs) 1a and 1b (E. J. de la Rosa, M. A. de Pedro, and D. Vázquez, Proc. Natl. Acad. Sci. USA 82:5632-5635, 1985). Under these conditions, cells elongate normally until they approach the first doubling in mass, the time at which cell lysis starts. Assuming that coupling between DNA replication and cell division both in cells starting growth and in growing cells is essentially similar, triggering of the lytic response in the beta-lactam-treated cells coincides with the termination of the first round of DNA replication. This coincidence suggests that both events are interrelated. We investigated this possibility by studying the initiation of growth in cultures of wild-type strains and in cell division mutants treated with beta-lactams inhibiting PBPs 1a and 1b and with the DNA replication inhibitor nalidixic acid. Addition of nalidixic acid, even late in the first cell cycle, prevented the lytic response of the cells to the blockade of PBPs 1a and 1b. The effect of nalidixic acid is more likely due to its action on DNA replication itself than to its indirect inhibitory effect on cell division or to its ability to induce the SOS system of the cell. These observations favor the idea that the cell wall biosynthetic machinery might be modulated by DNA replication at precise periods during cell growth.     

Accumulation of cyclic GMP in filaments of Escherichia coli BUG6

Experiments with Escherichia coli BUG6, a temperature-sensitive cell division mutant, have shown that at the restrictive temperature (42 degrees C) the loss of cell division potential (filamentation) was accompanied by an unusual increase in intracellular cyclic GMP (cGMP). At the permissive temperature (30 degrees C), cell division proceeded normally, and cGMP did not accumulate. Increasing the osmotic strength of the medium with NaCl suppressed filamentation in BUG6 at 42 degrees C and also suppressed the temperature-sensitive accumulation of cGMP. The addition of nalidixic acid to BUG6 at 30 degrees C induced filamentation but failed to cause cGMP accumulation. A similar accumulation of cGMP has not been observed in other E. coli strains.     

Effects of some platinum IV complexes on cell division of Escherichia coli.

A comparison was made of the effects of cis-tetrachlorodiaminoplatinum (IV) (cis-TCDPt), rans-TCDPt), and hexachloroplatinum (HCP) on growth and cell division of Escherichia coli strains D21 and D22. At or below 40 microgram/mL, cis-TCDPt inhibited cell division but not growth, DNA, or protein synthesis, although areas of increased electron density could be demonstrated in treated cells. In contrast, 40 microgram/mL of trans-TCDPt or HCP inhibited growth. Trans-TCDPt-treated cells developed condensed nucleoids; HCP-treated cells showed no obvious cytological changes to correlate with growth inhibition. Combination of cis-TCDPt with nalidixic acid, both at one-half the lowest filament-forming concentrations, resulted in formation of filaments, suggesting an additive effect. Combination of cis-TCDPt followed by ampicillin on E. coli B/r resulted in single bulges near the center of the filaments. Cis-TCDPt could therefore inhibit an initial step in the septation sequence, possibly at the level of the regulation of the hydrolytic enzymes. Whether cis-TCDPt exerts its effect by interreaction with DNA or with a membrane target is still uncertain.     

Plasmid Replication and the Induced Synthesis of Colicins E1 and E2 in Escherichia coli

Induction of colicins E1 and E2 in Escherichia coli occurs when plasmid synthesis has been inhibited either by nalidixic acid or by lack of deoxyribonucleic acid polymerase I. Moreover, colicin E1 and E2 synthesis induced by mitomycin C and exposure to chloramphenicol is not associated with a large increase in circular plasmid deoxyribonucleic acid. The mean plasmid content of cells in populations having a low spontaneous frequency of colicin-producing cells because of growth at low temperature or because of the presence of recA(-) or crp(-) alleles, is not significantly different to that in wild-type cells grown at 37 C.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Cell Division During Inhibition of Deoxyribonucleic Acid Synthesis in Escherichia coli

When cultures of Escherichia coli B/r growing at various rates were exposed to ultraviolet light, mitomycin C, or nalidixic acid, deoxyribonucleic acid (DNA) synthesis stopped but cell division continued for at least 20 min. The chromosome configurations in the cells which divided were estimated by determining the rate of DNA synthesis during the division cycle. The cultures were pulse-labeled with (14)C-thymidine, and the amount of label incorporated into cells of different ages was found by measuring the radioactivity in cells born subsequent to the labeling period. The cells which divided in the absence of DNA synthesis were those which had completed a round of chromosome replication prior to the treatments. It was concluded that completion of a round of replication is a necessary and sufficient condition of DNA synthesis for cell division.     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.     

The adhesive properties of a nonenteropathogenic strain of Escherichia coli in systems in vitro on isolated rat enterocytes and in vivo

The adhesive properties and colonizing capacity of E. coli strain O83, isolated from feces of healthy humans and marked according to its resistance to rifampicin and nalidixic acid, were studied. In vivo experiments on germ-free rats revealed that these bacteria were capable of colonizing intestinal mucosa; colonization increased from the small to large intestine and E. coli cells were mainly concentrated in the intestinal lumen and in mucin. In vitro studies showed that this nonenteropathogenic E. coli strain possessed pronounced adhesive properties with respect to the colonic cells of germ-free rats; these properties were considerably less pronounced with respect to the enteric cells of the small intestine. The electron microscopic study of E. coli cells revealed the presence of fimbriae and fibrillae on their surface.     

Transcription but not translation is required for EDTA-induced autolysis in Escherichia coli

Rifampicin, but not chloramphenicol or other inhibitors of translation, inhibited EDTA-induced autolysis in Escherichia coli. Inhibition of EDTA-induced autolysis in E. coli was also observed with nalidixic acid and novobiocin, inhibitors of topoisomerase II. Rifampicin or nalidixic acid-resistant mutants of E. coli were resistant to the inhibitory effect of the respective antibiotic on EDTA-induced autolysis. The implications of these studies in regard to our understanding of the regulation of autolysis in E. coli are discussed.     

Induction of cell division in a temperature-sensitive division mutant of Escherichia coli by inhibition of protein synthesis.

Synchronous cells of the thermosensitive division-defective Escherichia coli strain MACI (divA) divided at the restrictive temperature (42 degrees C) if they were allowed to grow at 42 degrees C for a certain period before protein synthesis was inhibited by adding chloramphenicol (CAP) or rifampicin. The completion of chromosome replication was not required for such divA-independent division. Synchronous cells of strain MACI divided in the presence of an inhibitor of DNA synthesis, nalidixic acid, if they were shifted to 42 degrees C and CAP or rifampicin was added after some time; cells of the parent strain MC6 (div A+) treated in the same way did not divide. These data suggest that coupling of cell division to DNA synthesis depends on the divA function. The ability to divide at 42 degrees C, whether or not chromosome termination was allowed, was directly proportional to the mean cell volume of cultures at the time of CAP addition, suggesting that cells have to be a certain size to divide under these conditions. The period of growth required for CAP-induced division had to be at the restrictive temperature; when cells were grown at 30 degrees C, in the presence of nalidixic acid to prevent normal division, they did not divide on subsequent transfer to 42 degrees C followed, after a period, by protein synthesis inhibition. A model is proposed in which the role of divA as a septation initiator gene is to differentiate surface growth sites by converting a primary unregulated structure, with the capacity to make both peripheral wall and septum, to a secondary structure committed to septum formation.     

The influence of nalidixic acid on Escherichia coli growth in milk

The high antibacterial activity of nalidixic acid against Escherichia coli, cultivated in raw and pasteurized milk has been shown. The low oxygen reduction potential had no influence on the antibacterial activity of this drug. The natural antibacterial agents in active milk from an inflamed udder have reduced the efficacy of nalidixic acid inhibition of the growth of E. coli.