Thymineless Death in Escherichia coli : Deoxyribonucleic Acid Replication and the Immune State

Ten micrograms of 2-nitro-1-butyl pyridinium sulfate (NBPS) per milliliter inhibited the growth of Bacillus subtilis by blocking thymidylate synthetase. The inhibition could be reversed by the addition of thymine or cytosine.2-Nitro-1-butyl pyridinium sulfate (NBPS) is an inhibitor produced by Nalco Chemical Corp. and is used commercially to control crown rot of bananas. We undertook an investigation to determine the mode of action of NBPS, by using Staphylococcus epidermidis and Bacillus subtilis as our test organisms.Growth of S. epidermidis and B. subtilis was inhibited by 10 ,ug of NBPS per ml, but the inhibitory effect could be reversed by dilution to 5 Ag/ml. Respiration of inhibited (100 ,g/ml) and noninhibited cultures was determined in a Warburg respirometer (17). The results ( Fig. 1) indicate that NBPS has relatively little effect on oxygen uptake, and the morphology of the cells did not appear to be affected by NBPS.Saline solution, nutrient broth, and nutrient broth plus 0.03% yeast extract were added to inhibited cultures (10 Mg of NBPS per ml) at different time periods to determine the reversibility of the inhibition. The results (Fig. 2) indicate that the addition of saline solution did not reverse the inhibition. Nutrient broth with 0.03 % yeast extract was more effective in reversing the inhibition than nutrient broth alone.A number of specific products were tested for their ability to reverse the inhibition. The constituents added individually were: 0.03% yeast extract; a basic salts solution (14); hydrolyzed ribonucleic acid, 0.005 and 0.01 %; nicotinamide, 2 and 4 ,g/ml; biotin, 2 and 4 ,ug/ml; p-aminobenzoic acid, 2 and 4 gg/ml; thiamine, 2 and 4 Ag/iml; folic acid, 2 and 4 Mg/ml; pyridoxine, 2 and 6 Ag/ml; nicotinamide adenine dinucleotide, 2 and 4 ,ug/ml; thymine, 25, 50, 100, 150, 200, 250, and 300 ,g/ml; and tryptophan, 50, 100, and 200 MAg/ml. The four I Present address: Botany, Provo, Utah 84601. remaining bases, adenine, guanine, cytosine, and uracil, were added separately in amounts of 25, 50, 100, 200, 250, 300, and 400 Ag/lml. Cytosine and thymine were added together to inhibited cultures in concentrations of 10, 20, 30, 40, 50, 60, and 70 Ag/ml to check for a synergistic reversing action of these two bases. Of the various factors tested for reversibility, only thymine and cytosine reversed the inhibition. When thymine and cytosine were assayed together, no synergistic effect was observed. Thymine, at 50 ,g/ml, effectively reversed the inhibitory action of 10 Mg of NBPS per ml.Thymine-2-'4C was fed to a thymineless mutant of B. subtilis growing in Spizizen's medium (16) containing various concentrations of NBPS (0 to 30 ,g/ml). The results shown in Fig. 3 indicate that NBPS markedly retarded the uptake of thymine into deoxyribonucleic acid (DNA). Thymidine kinase and thymidylate synthetase were isolated and purified by streptomycin sulfate precipitation, differential centrifugation, diethylaminoethyl cellulose, and Sephadex chromatography. The assay mixture for thymidine kinase...

Section: -Nitro-1-butyl Pyridinium Sulfate (Nbps) Is An Inhibitor Prsupporting

confidence: 88%

“…Growth of S. epidermidis and B. subtilis was inhibited by 10 ,ug of NBPS per ml, but the inhibitory effect could be reversed by dilution to 5 Ag/ml. Respiration of inhibited (100 ,g/ml) and noninhibited cultures was determined in a Warburg respirometer (17).…”

Section: -Nitro-1-butyl Pyridinium Sulfate (Nbps) Is An Inhibitor Prmentioning

confidence: 95%

Mode of Action of 2-Nitro-1-Butyl Pyridinium Sulfate

Seale

Weber

1970

“…The lethal event may be associated with the repair of single-strand nicks in DNA. However, the sensitivities of various mutants to TLD, UV irradiation, and nalidixic acid treatment form no consistent pattern which can be explained by a single lesion in the DNA repair mechanism (7,8). The importance of continuing protein and RNA synthesis in TLD was investigated further by Cummings and his co-workers (5, 6) using E. coli B.…”

mentioning

confidence: 99%

“…Whereas most E. coli strains (e.g. B/r, 15, K-12) had a 50-min lag period after thymine deprivation before death was evident, E. coli B began losing colonyforming ability immediately and was therefore 711 on October 2, 2020 by guest http://jb.asm.org/ Downloaded from DANKBERG AND CUMMINGS termed "sensitive" to TLD (7). It was found that inhibition of protein synthesis in this strain led to inhibition of TLD, immunity to TLD, and recovery following TLD (5,6).…”

mentioning

confidence: 99%

Protein Synthesis and Deoxyribonucleic Acid-Membrane Attachment During Thymineless Death in Escherichia coli

Dankberg

Cummings

1973

Self Cite

The proteins synthesized during thymineless death in Escherichia coli B and B/r were analyzed by polyacrylamide gel elctrophoresis. It was found that the amount of a protein of molecular weight 80,000 to 88,000 is greatly increased during thymineless death compared to the amounts of other cell proteins. A technique for the isolation of cell membrane-deoxyribonucleic acid (DNA)-nascent ribonucleic acid (RNA) complex on detergent crystals was used to determine whether DNA might be detached from the cell membrane as a result of thymineless death. It was found that under no conditions of thymineless death or immunity to thymineless death was there any change in the attachment of DNA or pulse-labeled RNA to cell membrane. When thymine-requiring strains of Escherichia coli are deprived of thymine, there is a permanent loss of colony-forming ability. This phenomenon, termed thymineless death (TLD), was first discovered by Barner and Cohen in 1953 (1). Concomitant with cell death, ribonucleic acid (RNA) and protein synthesis continued, and it was possible to induce the synthesis of some enzymes with or without thymine present (4).

“…Both the ribosomal and messenger RNA synthesized during thymine deprivation are normal in terms of composition (6,19), and Hanawalt (8) has argued that thymineless death is associated with the accumulation of singlestrand breaks in DNA resulting from messenger RNA synthesis. However, on the basis of more recent studies with chloramphenicol, Cummings and Kusy (2,3) have suggested that protein synthesis is directly involved in the lethality.…”

mentioning

confidence: 99%

Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine

Deutch

Pauling

1971

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 ,g/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 ug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 ug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 ug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 ' This paper is derived from a dissertation submitted by Charles E. Deutch to the Graduate Dvision, University of California, Riverside, in partial satisfaction of the requirements for the degree of Doctor of Philosophy. A portion of this work was