Effects of Ultraviolet Radiation on Respiration and Growth in Radiation-resistant and Radiation-sensitive Strains of Escherichia coli B

Ultraviolet (UV) irradiation at 254 nm causes different respiration and growth responses in log-phase cultures of Escherichia coli B/r and B(s-1). These differences are correlated with the ability and inability, respectively, of these bacterial strains to repair UV-induced lesions in deoxyribonucleic acid (DNA). After irradiation, B(s-1) cells (radiation-sensitive) exhibit uncoupling of growth and respiration; growth and synthesis cease, whereas respiration continues. B/r cells (radiation-resistant) grown on glycerol exhibit severe temporary inhibition of growth and respiration after UV, and the coupling of these two processes is maintained, except at a very high UV dose. Inhibition begins at about the time DNA synthesis resumes and continues for a period of time that is dependent upon dose. Glucose-grown cells do not exhibit severe respiratory, growth, and synthetic inhibitions; these processes remain coupled in the cells during the postirradiation period. Photoreactivation treatment delays uncoupling of growth and respiration in B(s-1) and prevents inhibition of respiration and growth in B/r. These results indicate that the postirradiation responses result from the presence of pyrimidine dimers in DNA. Ultraviolet irradiation of B/r and B(s-1) cells results in an accumulation of adenosine triphosphate by 30 min after UV. This accumulation decreases with time and does not appear to be related to the inhibition of respiration in glycerol-grown B/r cells. The results on B/r are interpreted in terms of a control mechanism for reestablishment of a balance among macromolecules in the irradiated cells so as to provide them with the potential to survive. The specific steps in such a reestablishment of balance appear to depend upon the substrate oxidized. In B(s-1) cells, which cannot repair UV-induced damage in DNA, some control mechanism that coordinates cellular processes may be inactivated.     

Relation Between Survival and Deoxyribonucleic Acid Replication in Ultraviolet-Irradiated Resistant and Sensitive Strains of Escherichia coli B/r

When arabinose-grown Escherichia coli B/r is ultraviolet (UV) irradiated in the logarithmic phase of growth, the dose inactivation curve for both colony formation and deoxyribonucleic acid (DNA) synthesis (based on the relative rates of synthesis) is exponential in nature. When protein synthesis is inhibited before UV-irradiation, both inactivation curves have a large shoulder. Pre-irradiation inhibition of protein synthesis increases considerably the colony-forming ability of a UV-irradiated Hcr(-) and Rec(-) strain of E. coli B/r. However, with the repair-deficient strains, both the shoulder and slope of the survival curve are affected. We investigated the effect of UV irradiation on DNA synthesis in Hcr(-) bacteria and found that pre-irradiation inhibition of protein synthesis increases UV resistance of DNA replication in this strain also. The results suggest that inhibition of protein synthesis before irradiation increases UV resistance in E. coli B/r by a mechanism which is independent of both the excision and recombination repair systems.     

Effect of dark repair on ultraviolet sensitivity of bacteriophage-infected bacteria

Feiner, R. R. (Columbia University, New York, N.Y.), and R. F. Hill. Effect of dark repair on ultraviolet sensitivity of bacteriophage-infected bacteria. J. Bacteriol. 91:1239-1247. 1966.-Changes in ultraviolet (UV) sensitivity of phage-host complexes during phage development have been studied for the following systems: T1 and Escherichia coli B, T1 and E. coli K-12S, lambda and E. coli K-12S. Complexes were formed with bacterial strains differing in ability to dark-repair UV damage to deoxyribonucleic acid and, after irradiation, were plated on bacteria differing similarly. In the first half of the latent period, the resistance of complexes formed with nonrepairing bacteria increased considerably; with T1 and E. coli B hcr(-), in 4 min the resistance became the same as that of complexes formed with repairing bacteria. The repair ability of plating bacteria affected survival curves only upon irradiation in the second half of the latent period after mature phages were present in the initial complex. Use of nonrepairing bacteria both for initial infection and for plating of late complexes resulted in a series of survival curves showing for all three systems the same pattern of change originally reported for T2-E. coli B complexes. Thus, a hitherto unexplained difference between radiation survival curves for T-even and T-odd phages seems due to repair of T-odd phages by the host.     

DNA Repair in Human/Embryonic Chick Heterokaryons: Ability of Each Species to Aid the Other in the Removal of Ultraviolet-Induced Damage

Cultured human and embryonic chick fibroblasts possess different enzyme-mediated processes to repair cyclobutyl pyrimidine dimers induced in their deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. While dimers are corrected in human cells by excision repair, a photoenzymatic repair process exists in embryonic chick cells for the removal of these potentially deleterious UV photoproducts. We have utilized a sensitive enzymatic assay to monitor the disappearance, i.e. repair, of dimer-containing sites in fused populations of human and chick cells primarily consisting of multinucleate human/chick heterokaryons. Fused cultures were constructed such that UV photoproducts were present only in chick DNA when evaluating excision repair and only in human DNA when evaluating photoenzymatic repair. Based on the kinetics of site removal observed in these cultures we are led to conclude the following: Within heterokaryons per se the photoreactivating enzyme derived from chick nuclei and at least one excision-repair enzyme (presumably a UV endonuclease) derived from human nuclei act on UV-damaged DNA in foreign nuclei with an efficiency equal to that displayed toward their own nuclear DNA. Hence, after cell fusion these chick and human repair enzymes are apparently able to diffuse into foreign nuclei and once therein competently attack UV-irradiated DNA independently of its origin. In harmony with the situation in nonfused parental cultures, in heterokaryons the chick photoenzymatic repair process rapidly removed all dimer-containing sites from human DNA including the residual fraction normally acted upon slowly by the human excision-repair process.     

Rejoining of radiation-induced single-strand breaks in deoxyribonucleic acid of Escherichia coli: effect of phenethyl alcohol.

Single-strand breaks in deoxyribonucleic acid of Escherichia coli B/r cells exposed to 20 krads of gamma radiation could be rejoined by incubation of irradiated cells in growth medium. In the presence of 0.25% phenethyl alcohol, this repair was completely inhibited although deoxyribonucleic acid and protein syntheses were suppressed only partially.     

Chromosome Replication and the Division Cycle of Escherichia coli B/r

The average amount of deoxyribonucleic acid (DNA) per cell was measured in steady-state cultures of Escherichia coli B/r grown at 37 C in glucose-limited chemostats or in batch cultures in the exponential growth phase as maintained with one of several carbon sources. Within experimental errors, DNA content was dependent only on growth rate and independent of the type of culture, the carbon source, or the addition of growth factors. The amount of DNA per cell increased continuously with growth rate over the range of 0.02 to 3 divisions per hour. The data over the entire range of growth rates are in agreement with a constant time for a single replication point to traverse the entire genome, 47 min, and with cell division following 25 min after termination of replication. The measured amount of DNA per genome was 4.2 x 10(-15) g (or 2.5 x 10(9) daltons).     

Efficiency of Thymidine Incorporation in Escherichia coli B/r as a Function of Growth Rate

Short-term labeling with radioactive thymidine led to inaccurate estimates of deoxyribonucleic acid synthesis at some growth rates in steady-state cultures of Escherichia coli B/r. Estimates were corrected in chemostat cultures by adding adenosine, a known inhibitor of thymidine phosphorylase.     

Growth in cadmium-containing medium induces resistance to heat in E. coli

We have shown that 10 microM Cd2+ in the growth medium can induce resistance to subsequent heat treatment in E. coli B/r. Resistance was shown by cells during an extended lag phase and, especially, during log phase. The results contrast with the effect of Cd2+ exposure on radiation lethality, for which sensitization was previously reported in cells from lag and stationary phase cultures.     

Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains.

The period of DNA synthesis C during the cell cycle was determined over a broad range of generation times in slowly growing, steady-state batch cultures in the exponential phase and in chemostat cultures of three strains of Escherichia coli, strains B/r A, B/r K, and B/r TT, utilizing measurements of average amounts of DNA per cell and cell survival after radioactive decay of 125I incorporated into the DNA of synthesizing cells. At each growth rate, values for cell survival and for C periods were the same within experimental errors for the three strains. The length of the DNA synthesis period increased linearly with generation (doubling) time T of the culture and approached a limiting value of C = 0.36T at very long generation times. In very slowly growing cultures, DNA replication was limited almost entirely to the final third of the cell cycle. D periods, between termination of DNA replication and cell division, were found to be relatively short at all growth rates for each strain. Average amounts of DNA per cell measured in slowly growing cultures of strains B/r A and B/r TT were indistinguishable from results for strain B/r K at the same growth rates. Amounts of DNA per cell calculated from the cell survival values alone are completely consistent with the measured DNA per cell.     

Deoxyribonucleic Acid Repair Replication after Ultraviolet Light or X-Ray Exposure of Bacteria

A comparison of repair synthesis after ultraviolet light (UV) or X-ray exposure was made in Escherichia coli strains 15T(-) (555-7) and B/r by use of a D, (15)N, (13)C density labeling system. During the initial 15 min of incubation after UV irradiation, both a "repair" synthesis and a reduced semiconservative deoxyribonucleic acid (DNA) synthesis occurred. In the so-called "physiological" dose range used, the latter was greater than the former. X-irradiation of cells, at doses producing similar levels of cell death as in the UV-exposed cultures, did not lead to a similar repair replication process. However, a density heterogeneity of the DNA synthesized in the initial 10 min after exposure was observed. This is interpreted in terms of X ray-induced DNA degradation. Normal cells showed only a semiconservative type of replication and, therefore, within the limits of resolution of the system used (the incorporation of 1,000 to 5,000 nucleotides per replicating chromosome could be measured), DNA in normal cells did not appear to undergo a repair synthesis involving thymine exchange. These results indicate that not all repair mechanisms mimic that found after UV exposure.     

Growth rate-dependent control of chromosome replication initiation in Escherichia coli.

The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria. In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h). A low-thymine-requiring (thyA deoB) derivative of E. coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations. It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication. The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.     

Thymineless Death in Escherichia coli: Inactivation and Recovery

The effects of chloramphenicol (CAP) on the progress of thymineless death (TLD), nalidixic acid (NA) inactivation, ultraviolet (UV) irradiation, and mitomycin C (MC) inactivation were studied in Escherichia coli B, B(s-1), B(s-3), B(s-12), and B/r. This was done before, during, and after inactivation. During the progress of inactivation, it was found that at 10 to 20 mug of CAP per ml, up to 50% of the UV-sensitive bacteria survived TLD and about 10% survived NA. In E. coli B/r, at these concentrations of CAP, about 10 to 15% of the cells survived TLD and about 20 to 25% survived NA. Concentrations of CAP greater than 25 mug/ml actually increased the sensitivity of E. coli B, B(s-1), B(s-3), and B(s-12) to inactivation by either TLD or NA; at 150 mug of CAP per ml, the sensitivity of E. coli B/r to inactivation also increased. When E. coli B cells were incubated in CAP prior to inactivation, the longer the preincubation the longer onset of TLD was delayed; NA inactivation was also affected in that the rate of inactivation after CAP incubation was greatly decreased. Preincubation of E. coli B/r with CAP had much less effect on the progress of inactivation. After thymineless death, incubation in CAP plus thymine led to a rapid and almost complete recovery of E. coli B and B(s-12). Lesser recoveries were observed after inactivation due to UV, NA, or MC inactivation. E. coli B(s-1) and B/r did not recover viability after any mode of inactivation, and E. coli B(s-3) and B(s-12) recovered from UV to about 20% of the initial titer. It was suggested that protein synthesis, in particular proteins involved in deoxyribonucleic synthesis, was a determining factor in these inactivating and recovery events.     

Some features of the reaction of intracellular bacteriophages T1 to UV irradiation.

The reaction of complexes pf phage T1-cells of E. coli B or E. coli Bs-1 to UV irradiation was investigated. The complexes were irradiated at various stage of infection, and their survival, extent of Hcr and Phr, were evaluated. It was found that the UV resistance of phage DNA in the second half of the latent period fluctuates. Hcr after UV exposure at these stages of infection operates in a small volume. The ability of intracellular phage to photoreactivate when cells of E. coli B were infected is constant after irradiation at many stages of infection, except the early ones. In the complexes of phage T1-bacteria of E. coli Bs-1 this ability declines while infection is promoted. The daughter phage particles released from UV irradiated complexes undergo Phr and Hcr only after irradiation at the late stages of infection. This was not the cases when complexes of phage-bacteria were irradiated during the first half of the latent period. A possible tole of UV-damaged phage DNA in propagation of infection and in maturation of phage particles is discussed.     

Influence of Ribosides on Ultraviolet Resistance of Escherichia coli: Role of Deoxyribonucleic Acid Synthesis in the Ultraviolet Resistance Enhancement After Amino Acid Prestarvation

Changes in the resistance of cells of Escherichia coli B/r Hcr(+)thy(-)trp(-) to ultraviolet radiation were investigated after the following pretreatments: (i) amino acid starvation which, according to previous conclusions, enabled the cells to complete replication cycles of deoxyribonucleic acid (DNA); (ii) amino acid starvation during which the synthesis of DNA was arrested by the addition of 50 mug of cytidine per ml. The results showed that the enhancement of resistance observed after amino acid prestarvation was in correlation with the amount of DNA which was synthesized during the amino acidless period. The enhancement of resistance can be abolished by the addition of the riboside at any phase of the starvation period. This shows that the enhancement of resistance was not a consequence of the total inhibition of metabolism but of unbalanced growth which evoked the completion of replication cycles of DNA.     

Inhibition of RNA polymerase from Bacillus thuringiensis and Escherichia coli by beta-exotoxin.

The characteristics of exotoxin inhibition of deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase isolated from Escherichia coli and Bacillus thuringiensis were investigated. RNA polymerase isolated from a variety of growth stages was partially purified and assayed using several different native and synthetic DNA templates, and exotoxin inhibition patterns were recorded for each. Although 8 to 20-h RNA polymerase extracts of E. coli retained normal sensitivity to exotoxin (50% inhibition at a concentration of 7.5 X 10(-6) M exotoxin), RNA polymerase isolated from late exponential and ensuing stationary-phase cultures of B. thuringiensis were nearly 50% less sensitive than exponential RNA polymerase activity. Inhibition patterns relating culture age at the time of RNA polymerase extraction to exotoxin inhibition suggested a direct correlation between diminishing exotoxin sensitivity and sporulation. Escherichia coli RNA polymerase could be made to mimic the B. thuringiensis exotoxin inhibition pattern by removal of sigma from the holoenzyme. After passage through phosphocellulose, exotoxin inhibition of the core polymerase was 30% less than the corresponding inhibition of E. coli holoenzyme. Heterologous enzyme reconstruction and assay were not possible due to loss of activity from the B. thuringiensis preparation during phosphocellulose chromatography, apparently from the removal of magnesium. In enzyme velocity studies, inhibition with exotoxin was noncompetitive with respect to the DNA template in the RNA polymerase reaction.     

Deoxyribonucleic Acid Synthesis During the Division Cycle of Escherichia coli: a Comparison of Strains B/r, K-12, 15, and 15T− Under Conditions of Slow Growth

The rates of deoxyribonucleic acid (DNA) synthesis during the division cycles of the Escherichia coli strains B/r, K-12 3000, 15T(-), and 15 have been measured in synchronous cultures, under several conditions of slow growth. These synchronous cultures were obtained by sucrose gradient centrifugation of exponentially growing cultures, after which the smallest cells were removed from the gradient and allowed to grow. Sucrose gradient centrifugation did not adversely affect the cell cycle, since an experiment in which an exponentially growing culture was pulsed with [(3)H]thymidine prior to the periodic separation and assay of the smallest cells resulted in the same conclusions, as given below. In the strains of E. coli that were studied, a decreased rate of [(3)H]thymidine incorporation was seen late in the cell cycle, prior to cell division. No decrease in the rate of [(3)H]thymidine incorporation was seen at or near the beginning of the cell cycle. Thus, all these strains appear to regulate DNA synthesis in a similar fashion during slow growth. In addition, a correlation between the appearance of cells with visible cross-walls and the start of a new round of DNA synthesis was seen, indicating that these two events might be related.     

Isolation, Characterization, and Genetic Analysis of Mutator Genes in Escherichia coli B and K-12

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut(+)) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA(+) transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.     

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.     

Requirement for Protein Synthesis in rec-Dependent Repair of Deoxyribonucleic Acid in Escherichia coli after Ultraviolet or X Irradiation

Deprivation of amino acids required for growth or treatment with chloramphenicol or puromycin after irradiation reduced the survival of Rec(+) cells of Escherichia coli K-12 which had been exposed to either ultraviolet (UV) or X radiation. In contrast, these treatments caused little or no reduction in the survival of irradiated recA or recB mutants. The effect of chloramphenicol on the survival of X-irradiated cells was correlated with an inhibition of repair of single-strand breaks in irradiated deoxyribonucleic acid (DNA), previously shown to be controlled by recA and recB. In UV-irradiated cells no effect of chloramphenicol was detected on the repair of single-strand discontinuities in DNA replicated from UV-damaged templates, a process controlled by recA but not by recB. From this we concluded that inhibiting protein synthesis in UV or X-irradiated cells may interfere with some biochemical step in repair dependent upon the recB gene. When irradiated Rec(+) cells were cultured for a sufficient period of time in minimal growth medium before chloramphenicol treatment their survival was no longer decreased by the drug. After X irradiation this occurred in less than one generation time of the unirradiated control cells. After UV irradiation it occurred more slowly and was only complete after several generation times of the unirradiated controls. These observations indicated that replication of the entire irradiated genome was probably not required for rec-dependent repair of X-irradiated cells, although it might be required for rec-dependent repair of UV-irradiated cells.