R-Factor-Mediated Nuclease Activity Involved in Thymineless Elimination

R-factor 1818 (R-1818) had no effect on the efficiency of plating of ligase-deficient phage T4 mutants on strains of Escherichia coli containing excess, normal, or defective ligase. However, if the R(+) bacterial strain that overproduced ligase was first starved of thymine, its ability to propagate ligase-deficient phage was reduced by as much as fivefold compared with the burst size on the thymine-starved R(-) strain. In contrast, it was found that after ultraviolet irradiation of the host the phage burst size was higher on the R(+) ligase overproducing strain than the R(-) derivative. The maximal level of R-factor elimination produced by thymine starvation was inversely related to the ligase level of the host. Ultraviolet irradiation did not cure the R factor from strains containing wild-type levels of ligase, but did cause elimination from strains with excess or defective ligase. The results suggest that R-1818 codes for a nuclease that is induced by thymine starvation and which, possibly in conjunction with host-mediated nucleases, is responsible for its elimination under these conditions.     

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

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 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/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 mug/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 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.     

Control of the Synthesis of Macromolecules During Amino Acid and Thymine Starvation in Bacillus subtilis

Studies of Maal?e, Lark, and others with amino acid- and thymine-starved cultures revealed successive steps in the biosynthesis of Escherichia coli chromosomes. In this study, the corresponding mechanisms in Bacillus subtilis 168 were examined. Using a strain requiring both thymine and tryptophan, we found that, 3 hr after the start of amino acid starvation, when the deoxyribonucleic acid (DNA) content of the culture had increased 40 to 50%, DNA synthesis ceased. After 4 to 5 hr, 100% of the cells were immune to thymineless death; their chromosomes had presumably been completed. Immune cultures slowly incorporated (3)H-thymine. Thymine incorporation increased 20-fold 30 min after readdition of amino acids, indicating reinitiation of chromosome synthesis. Simultaneous presence of amino acids and thymine was required for reinitiation. If 5-bromouracil (5-BU) was added instead of thymine, newly replicated DNA segments could be separated by centrifugation in CsCl. Analysis of the CsCl fractions by a transformation assay showed that the order in which the markers were synthesized was ade-16, thr-5, leu-8, metB5. Less than half the chromosomes started resynthesis synchronously in 5-BU. Nevertheless, chromosome alignment in the amino acid-starved culture is probably very good: marker frequency analysis of its DNA gives the same normalized frequencies as DNA from "perfectly" aligned spores. Full viability is maintained in the chromosome-arrested culture for 10 hr in thymine-free medium in the absence or presence of amino acids. In the latter condition, protein synthesis proceeds, and the cells filament and become more lysozyme-sensitive. Such cells must be incubated and plated on hypertonic or on slow-growth media; otherwise, they undergo "quasiosmotic" thymineless death. This death is thus apparently not directly attributable to any damage of chromosomal DNA. Further, weakening of the teichoic acid portion of the cell wall is not involved, since (32)P incorporation into teichoic acid is normal. Chloramphenicol prevents quasiosmotic thymineless death and also inhibits (32)P incorporation into teichoic acid. Chromosome-synthesizing cultures suffer thymineless death of two types: quasiosmotic death, and death insusceptible to osmotic rescue.     

Influence of Thymine Starvation on the Integrity of Deoxyribonucleic Acid in Escherichia coli

The influence of thymine starvation on the single-strand molecular weight of deoxyribonucleic acid (DNA) from Escherichia coli was determined by sedimentation through gradients of alkaline sucrose. Growth of cells for as long as 150 min in thymineless medium did not significantly reduce the molecular weight below the control value of 2.4 +/- 0.3 x 10(8) daltons. Incubation of cells in thymineless medium after exposure to 500 ergs/mm(2) of ultraviolet light or 20 krad of (137)Cs gamma rays did not appear to block the rejoining of single-strand breaks associated with irradiation. Thus, DNA repair enzymes, presumably including DNA ligase, are not significantly inhibited by thymine starvation.     

Thymineless Death in Escherichia coli in Various Assay Systems: Viability Determined in Liquid Medium

Thymineless death has been studied in four different Thy(-) strains of Escherichia coli by using various assay methods including conventional plating techniques as well as one performed entirely in liquid medium. Plating on L agar resulted in a greater loss in viability than the other assay methods, but this extrasensitivity of starved cells to L-agar plating quickly disappeared upon readdition of thymine to the starved cultures. This indicated that cellular damage responsible for the additional killing on L agar is reversible. The results obtained by three other assay methods, the liquid assay, plating on nutrient agar, or plating on tris(hydroxymethyl)aminomethane-minimal agar, did not differ significantly from each other with all strains tested except strain JG 151. In this strain thymineless death was much faster when assayed in the liquid system than by plating. It is suggested that thymineless death detected on nutrient or minimal agar is not a result of plating, but that the lethal event actually occurs during the period of thymine starvation.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Properties of Thymineless Strains of Bacillus megaterium

Both Bacillus megaterium KM:T(-)R(1), a strain partially resistant to thymineless death, and strain KM:T(-), the parent strain, can satisfy their thymine requirement with either thymidine, 5-methyldeoxycytidine, or 5-methyluridine. Neither strain can use 5-methylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, or 5-aminouracil for this purpose. Strain KM:T(-)R(1) requires as little as 0.01 mM thymine for maximum growth, whereas strain KM:T(-) requires 0.10 to 0.20 mM thymine. Lysogenic KM:T(-)R(1) dies more rapidly in the presence of mitomycin C than the corresponding phage-sensitive strain. Unexpectedly, the lysogenic strain was found to be less sensitive to thymineless death than the phage-sensitive strain. Lysogenic KM:T(-)R(1) is induced by exposure to mitomycin C and by thymineless incubation. It is concluded that thymineless death occurs by a mechanism which is unrelated to phage induction and that a major lethal effect of mitomycin C is probably a consequence of phage induction.     

Effect of Thymine Limitation on Chromosomal Deoxyribonucleic Acid Synthesis in Proteus mirabilis

The effects of thymine limitation on the rates of growth, deoxyribonucleic acid (DNA) synthesis, and increase in viable cell number for a thymine auxotroph of Proteus mirabilis were investigated. At thymine concentrations of 1.0 mug/ml and below, these rates were markedly decreased. After a reduction in thymine concentration from 10 mug/ml to 0.2 mug/ml, mass synthesis continued at the preshift rate for several hours. In contrast, the rate of DNA synthesis immediately decreased, resulting in a decrease in the DNA to mass ratio to about one-half of its normal level. Viable counts remained constant for several hours after the reduction in thymine concentration, and enlarged cells and multicellular "snakes" were formed. The rate of DNA synthesis was reduced at thymine concentrations below approximately 1.7 mug/ml. The addition of thymine to cultures which had been completely starved for thymine increased the rate of DNA synthesis to at least twice its normal value; this suggests that extra rounds of chromosome replication can be induced in P. mirabilis as previously observed in Escherichia coli.     

Control of Cell Growth in Bacteria: Experiments with Thymine Starvation

When cells of Escherichia coli THU were starved for thymine, they continued to grow without division for at least two successive volume doublings at their initial rate. Within experimental error this average rate of volume increase, 0.21 mum(3) per hr, was identical with that observed in control cultures during two generations of growth in the presence of thymine. This growth rate was also independent of the age of the cells at the time of starvation. These results are consistent with the hypothesis, proposed earlier, that growth rates are controlled by uptake sites for binding, transport, or accumulation of compounds into the cell, that the number of these sites is constant throughout most of the cell cycle, and that this number doubles near or at cell division.     

Role of the product of recB-gene in the thymine-less death and characteristics of this phenomenon in a thy-recB--mutant of Escherichia coli K-12]

Thymine requiring mutants of rec+ and recB- Escherichia coli strains have been tested for their response to thymine deprivation. Exonuclease V-deficient mutant is less sensitive to thymine deprivation than the wild type strain, because there is no lag period at thymineless death of recB- thy- cells. However, the mechanism of thymineless death of thy- rec+ and thy- recB- cells may be different. Two types of thymineless death are proposed to exist. The first type is due to DNA primary structure damages (single-strand breaks or gaps), accompanied by DNA degradation. The restoration of the balance disturbed by the thymine deprivation between DNA and protein synthesis rates by their balanced inhibition promotes a complete repair of structural damages in DNA and prevents the death of rec+ cells. The second type of thymineless death is not linked with the formation of DNA damages, and this is observed in recB- thy- mutant, defective in exonuclease V.     

Nucleic Acid and Protein Synthesis During Thymineless Death in Lysogenic and Nonlysogenic Thymine Auxotrophs

Thymine deprivation causes the non-phage-associated component of thymineless death in Escherichia coli 15T(-). Addition of thymine causes the phage-associated component to occur.     

Thymineless death in Bacillus megaterium

Wachsman, J. T. (University of Illinois, Urbana), S. Kemp, and L. Hogg. Thymineless death in Bacillus megaterium. J. Bacteriol. 87:1079-1086. 1964.-Strain KM:T(-), a thymine auxotroph of Bacillus megaterium strain KM, rapidly loses the ability to multiply when incubated in the absence of thymine, on an otherwise sufficient medium. At 37 C, there is a lag of approximately 60 min, prior to the onset of exponential death (decrease of 1 decade per 50 min). The extent of the decrease in viable count varies from 4 to 5 decades after 5 hr of starvation. The cells die more slowly at 30 C (decrease of 1 decade per 120 min) after a lag of approximately 90 min. Thymine starvation permits substantial net ribonucleic acid (RNA) and protein synthesis, but only slight deoxyribonucleic acid synthesis. In contrast with the changes occurring at 30 C, thymineless death at 37 C is eventually accompanied by a rapid hydrolysis of RNA and by cell lysis. Chloramphenicol inhibits thymineless death at 37 C. Strain T(-)R(1), a derivative of strain KM:T(-), undergoes a very low rate of thymineless death at 37 C (decrease of 1 decade per 240 min). Neither hydrolysis of RNA nor cell lysis occurs during 8 hr of thymine starvation. Strain KM:T(-)H(-) (doubly auxotrophic for thymidine and histidine) requires histidine for maximal thymineless death at 37 C. Preincubation of this strain on the basal medium supplemented with thymidine alone enables the population to become increasingly immune to subsequent thymineless death.     

Phases of thymineless death inEscherichia coli 15 TAU

On the basis of the course of loss of colony-forming ability it was possible to disting guish at least three phases of thymineless death in a culture ofEscherichia coli 15 TAU starved in a thymine-free medium enriched with arginine and uracil. The main differences between the individual phases were: (a) half-time of the loss of colony-forming ability; (b) only in the first phase was thymineless death reversible; (c) beginning in the second phase, part of the cells lysed after thymine was added to the liquid medium or when cells were plated on agar with complete medium; (d) lysis was much slower in the third phase than in the second; (e) in the presence of caffeine the course of the first and second phase was not affected but cells did not die in the third phase. Cells surviving in the third phase in the presence of caffeine are probably those which did not die even in medium T^?A^?U^? (Maaløe & Hanawalt, 1961). Transition from one phase to another was caused neither by change in the composition of medium during thymine starvation nor by heterogeneity of this culture from a genetical point of view.     

Absence of pyrimidine salvage and prevention of thymineless radiosensitization in Escherichia coli thyA cells fed dihydrothymine or thymine glycol

Little information is available concerning the metabolic fate of radiation-induced thymine base damage products once they have been excised from DNA. The present study was an attempt to determine whether or not thymine-requiring mutants of Escherichia coli could grow on dihydrothymine (DHT) and thymine glycol (TG) by "salvaging" the altered thymines. A second test of thymine product utilization was prevention of thymineless radiosensitization. Results showed that very low growth of Thy- cells on DHT or TG could be explained by the presence of less than or equal to 1% contaminating thymine in the mixtures. Radiation dose-modification factors (DMFs) for thyA cells fed DHT or TG for 3 h were 1.38 +/- 0.28 and 1.26 +/- 0.24, respectively, whereas the DMF for 3 h thymine-starved cells was 1.63 +/- 0.05. The small (approximately 25%) amelioration of thymineless radiosensitization observed in DHT- or TG-fed cells could probably be explained by contaminating thymine in the medium. Although DHT is a normal metabolite in some cells, neither DHT nor TG could be used efficiently by thymine-requiring cells in the protocol presented.     

Thymineless Mutagenesis in Escherichia coli

To clarify the relationship between thymineless death and thymineless mutagenesis, the induction of arginine revertants of Escherichia coli TAU-bar by thymine starvation was examined in physiological terms. Induced revertants were detectable both on minimal medium lacking arginine and minimal medium supplemented with 1 mug of arginine per ml. Substantial thymineless mutagenesis occurred during the period before the onset of thymineless death. Mutagenesis and loss of viability were observed upon incubation in medium lacking thymine and arginine, and both were inhibited upon incubation in medium lacking thymine and uracil. Mutagenesis also occurred during thymine starvation at 25 C, where there was relatively little loss of viability. At 37 C thymineless mutagenesis did not require complete thymine starvation, and the induction of revertants appeared to be initiated at the same suboptimal thymine concentration at which lethality was first detectable. Mutagenesis was found not to occur preferentially at the growing point of deoxyribonucleic acid replication. These results suggest that thymineless mutagenesis does not involve simply errors in base pairing due to the absence of thymine. The data also suggest that the induction of mutations and thymineless death are due to the same primary event but that mutagenesis is the more sensitive response.     

Thymineless Death and Ultraviolet Sensitivity in Micrococcus radiodurans

Thymine-requiring mutants of Micrococcus radiodurans have been isolated by selection on solid medium containing trimethoprim. Strains requiring either high concentrations of thymine (50 μg/ml) or low concentrations (2 μg/ml) for normal growth were obtained. The Thy⁻ mutant requiring low thymine concentrations has been characterized. It was shown to retain the high ultraviolet light (UV) resistance typical of wild-type M. radiodurans, but it was not resistant to thymineless death. Preliminary exposure of the cells to thymineless conditions resulted in enhanced UV sensitivity, and this interaction occurred under conditions where “unbalanced growth” was inhibited by the addition of chloramphenicol. Upon addition of thymine to deprived cells, UV resistance was gradually restored, and this recovery took place in the absence of protein synthesis. A model is proposed to account for the similarity of thymineless death in bacteria whose deoxyribonucleic acid repair efficiencies differ widely.     

Thymineless Death in Escherichia coli 15T− and Recombinants of 15T− and Escherichia coli K-12

Thymineless death was examined in Escherichia coli 15T(-) and recombinants of 15T(-) and E. coli K-12. Those strains that were very sensitive to thymine deprivation were also very sensitive to a variety of inducing agents (mitomycin C, ultraviolet light, hydroxyurea, and nalidixic acid). Those strains that were relatively resistant to thymineless death were also relatively resistant to the inducing agents. After exposure to thymineless death and the inducing agents, sensitive strains lysed, produced colicin, and had phage particles in their lysates. These strains also showed an increase in the 6-methyladenine content of their deoxyribonucleic acid (DNA) and an increase in the DNA methylase activity of their crude extracts under these conditions. None of these effects was noted in the strains relatively resistant to thymineless death and the inducing agents. These data indicate that there are two types of thymineless death. One is represented by the strains that are very sensitive to thymine deprivation and other inducing agents and is secondary to the induction of phage psi. The strains more resistant to thymine deprivation and the other inducing agents undergo a non-phage-mediated thymineless death. The mechanism of this latter process is currently under study.