SOME OBSERVATIONS ON THE LETHAL EFFECTS OF NEAR-ULTRAVIOLET LIGHT ON ESCHERICHIA COLI, COMPARED WITH THE LETHAL EFFECTS OF FAR-ULTRAVIOLET LIGHT

Abstract— Near-ultraviolet light (365.5 nm) reduces the ability of Escherichia coli B/r and B_8-1, to form colonies on nutrient agar after irradiation. This lethal effect is distinct from that obtained after far-u.v. irradiation (253.7 nm) because the far-u.v. sensitive and resistant strains are equally susceptible to near-u.v. Variation in susceptibility to ultraviolet light during growth is more marked for near-u.v. than for far-u.v. The number of survivors after near-u.v. irradiation of log phase cells is affected by several post-irradiation treatments; more cells survive if growth immediately after irradiation occurs at higher temperatures (unlike far-u.v.). Also, the presence of acriflavine and caffeine in the nutrient agar decreases the number of survivors (in common with far-u.v.).     

DNA TURNOVER IN BUFFER-HELD ESCHERICHIA COLI AND ITS EFFECT ON REPAIR OF UV DAMAGE

Abstract— Continuous DNA degradation and resynthesis, without a net change in cellular DNA content, were observed in buffer-held, non-irradiated E. coli B/r. This constant DNA turnover probably involves most of the genome and reflects random sites of DNA repair due to the polA-dependent excision-resynthesis repair pathway. Under these non-growth conditions, it appears that at any given time there is a minimum of one repair site per 6.5 × 10⁶ daltons DNA, each of which is at least 160 nucleotides long.
While the amount of DNA degradation is not influenced by prior exposure to UV radiation, the synthetic activity decreases with increasing UV fluence. We suggest that when sites of DNA turnover occur opposite to cyclobutyl dipyrimidines in UV-irradiated cells, repair of the latter damage can be prevented. This implies that both beneficial and deleterious processes take place in irradiated buffer-held cells, and that cell survival depends on the delicate balance between DNA turnover and repair of UV-damage. Based on these findings, we propose a model to explain the limited repair observed during post-irradiation liquid-holding and to account for the large difference in cell survival between irradiation at low fluence rates (fluence-rate dependent recovery) and at high fluence rates followed by liquid-holding (liquid-holding recovery).     

SYNERGISTIC LETHAL ACTION OF ULTRAVIOLET-VIOLET RADIATIONS AND MILD HEAT IN ESCHERICHIA COLI

Abstract— The lethal interaction of far ultraviolet (254nm), near ultraviolet (334 and 365nm) and violet visible (405nm) radiation treatment with mild heat treatment was studied. Except at 254nm, a strong positive radiation dose-dependent interaction (synergism) was always observed. The efficiency of sensitisation to heat, as a function of dose at each wavelength, was found to be directly correlated with the dose necessary to eliminate the shoulder from the survival curve of a repair proficient strain but was apparently unrelated to the relative near-ultraviolet sensitivities of a repair deficient strain. The interaction was independent of the order of treatments. A radiation dose of 10⁶ Jm^-2 at 365nm slightly sensitised a cell population to 45°C incubation (normally non-lethal) and strongly sensitised the cells to 48°C treatment (normally 80 percent survival after 2 hours). It is proposed that in addition to DNA damage, both heat treatment and near ultraviolet treatment interfere with DNA recovery mechanisms so that the combination of the two agents inevitably leads to a strong positive interaction.     

ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

MEASUREMENT OF DNA CROSSLINKS BY S1 NUCLEASE: INDUCTION AND REPAIR IN PSORALEN-PLUS-360 nm LIGHT TREATED ESCHERICHIA COLI

Abstract—DNA crosslinks in Escherichia coli cells. exposed to 4.5',8-trimethylpsoralen plus 360 nm light, were measured using a rapid and sensitive new approach. The assay is based on the specificity of S₁ nuclease from Aspergillus oryzae to single-stranded DNA. Bacterial cells were lysed and the DNA denatured by alkali. Following acid neutralization. crosslinked DNA undergoes spontaneous renaturation and is rendered S₁-nuclease resistant and therefore acid-precipitable. The single-stranded fraction after denaturation by alkali decreases with increasing near UV light exposure in the presence of TMP following first order kinetics. The kinetics were faster when exposure was at 4°C rather than at 20°C. This suggests that excision of crosslinks occurs during exposure at the higher temperature. Indeed. since the rate of DNA crosslinking in a uvr B mutant which is excision-deficient was higher than in wild type bacteria at 4°C, some excision must have occurred even in the cold. DNA from excision-proficient cells incubated at 37°C following exposure to TMP-plus-near UV at 4° showed a greater single stranded fraction than that from non-incubated cells. This indicates repair of DNA crosslinks. which proceeded with a half-time of 8 min at 37°C and was unaffected by substitution of thymine in DNA by 5-bromouracil.     

QUANTITATIVE EVIDENCE FOR ENZYMATICALLY-INDUCED DNA DOUBLE-STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K-12

Abstract— We have recently reported that DNA double-strand breaks arise enzymatically during the course of excision repair in uvr ⁺ strains of Escherichia coli K-12. Survival curves for ultraviolet (UV) irradiated E. coli K-12 pol⁺ (JG139) and polA1 (JG138) strains have a pronounced shoulder region. The regions of the survival curves at which killing approaches exponential correspond to the fiuences at which DNA double-strand breaks (assumed to be lethal events) accumulate linearly. Reducing the number of UV photoproducts either by photoreactivation or fluence fractionation results in an increase in survival and a decrease in the yield of DNA double-strand breaks in both strains. These data support the hypothesis that enzymatically-induced DNA double-strand breaks may be the lesion ultimately responsible for UV-induced cell killing in the pol⁺ strain of E. coli K-12. and perhaps also in the polA1 strain.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

PHOTOREACTIVATION OF ESCHERICHIA COLI Bs-3 AFTER IN ACTIVATION BY 313 nm RADIATION IN THE PRESENCE OF ACETONE

Abstract— The colony-forming ability of E. coli B_s-3 can be inactivated by light of 313 nm wavelength in an acetone-sensitized photochemical reaction. This ability can subsequently be restored quantitatively by illumination with photoreactivating light. A small fraction of the population cannot be inactivated; this is assumed to be due to a complete dark repair of the lesions, whatever the dose of radiation has been. Thus, such triplet energy-transfer experiments can successfully be applied to whole cells. Since thymine dimers are formed almost exclusively, this suggests a new way of studying these lesions in relation to the biologically observable effects.     

EFFECT OF LIQUID HOLDING ON THE REPAIR OF PHOTOINDUCED DAMAGES IN ACRIFLAVINE SENSITIZED ESCHERICHIA COLI CELLS

Abstract— Damage caused by visible light in the presence of acriflavine can be repaired in various strains of Escherichia coli possessing one or the other repair mechanism. The number of viable cells of the irradiated E. coli cultures increases on holding in buffer. The results of liquid holding suggest that the major role played by holding in liquid medium is the removal of dye molecules from inside the cells; this would create a favourable condition for recovery during subsequent incubation.     

EFFECT OF DIETARY LIPID ON UV LIGHT CARCINOGENESIS IN THE HAIRLESS MOUSE

Abstract— Isocaloric feeding of diets varying in lipid content to albino hairless mice has shown that their susceptibility to skin tumorigenesis induced by simulated solar UV light was not affected by the level of polyunsaturated fat, 5% or 20%. However a qualitative effect of dietary lipid was demonstrated. Mice fed 20% saturated fat were almost completely protected from UV tumorigenesis when compared with mice fed 20% polyunsaturated fat. Multiple latent tumours were detected in the saturated fat-fed mice by subsequent dietary replenishment, suggesting that a requirement for dietary unsaturated fat exists for the promotion stage of UV-induced skin carcinogenesis.     

EFFECT OF BLUE/UV LIGHT ON ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS IN THE ABSENCE OF BULK CAROTENOIDS

Abstract Anthocyanin synthesis in the hypocotyl of tomato ( Lycopersicon esculentum ) seedlings responds strongly and specifically to blue/UV light while the response to red and far-red light, operating through phytochrome, is weak. The herbicide Norflurazon (SAN 9789) was used to inhibit synthesis of colored carotenoids almost completely without affecting growth and development measurably. Even though carotenoid content was reduced to less than 2% of normal and the fluence rate response function for blue and UV light was linear within the experimental range, Norflurazon treatment did not reduce seedling sensitivity toward blue/UV light. It was concluded that at least'bulk'carotenoids are not the photoreceptor chromophore of the blue/UV photoreceptor pigment.     

ELIMINATION OF THE EFFECT OF CONTAMINATING CO2 IN THE 18O-LABELING OF THE CO2 PRODUCED IN BIOLUMINESCENT REACTIONS

Abstract— Although the mechanism of bioluminescent reactions in various species, such as fireflies, ostracod crustaceans ( Cypridina ), sea pansies ( Renilla ), and the deep-sea shrimp Oplophorus , are thought to involve dioxetanone intermediates, studies reported in the past from different laboratories have included widely different experimental results, most likely due to various factors including the effects of contaminating CO₂. With the improved technique employed in the present study, bioluminescent reactions of the firefly and Cypridina in ¹⁸O₂ gas resulted in an incorporation of over 75% of ¹⁸O into one oxygen of the product CO₂. with a reproducibility within a few per cent. When ¹³CO₂. instead of the product CO₂ of the bioluminescent reaction, was studied in an H₂¹⁸O medium, the exchange of one oxygen of ¹³CO₂ with H₂O was 64%. and the effect of contaminant CO₂ amounted to 1418% of the total CO₂. These results suggest that every molecule of CO₂ formed in the bioluminescent reactions of the firefly and Cypridina had intially contained 1 oxygen atom derived from O₂.     

COMPARISON OF THE LETHAL EFFECT OF 5-IODOURACIL INCORPORATED INTO BACTERIOPHAGE T4 IN THE PRESENCE AND ABSENCE OF NEAR-VISIBLE LIGHT

Abstract— Bacteriophages T2 or T4 containing 5-iodouracil (IUra) substituted for thymine in their DNA are inactivated by near-visible light, with fluorescent lamps as the source of near-visible light. Inactivation increases with the dose of near-visible light and follows first order kinetics. Relative inactivation rates are linearly proportional to percent substitution. Equivalent per cent substitution by IUra or 5-iodo-2'-deoxyuridine (IdUrd) results in equivalent sensitization to inactivation with both T2 and T4. However, incorporation of IUra into T4 and T2 also is lethal in the absence of light. The lethal effect of IUra substitution differs from the lethal effect of IUra substitution plus near-visible light irradiation in at least three respects: (1) IUra substitution is lethal for T4 under conditions where the residual viability is stable and where environmental light cannot account for the inactivation. (2) The hit curve for IUra lethality, as a function of per cent IUra substitution, has a large shoulder while the hit curve for sensitization to inactivation by near-visible light, as a function of per cent IUra substitution, has no shoulder. (3) At equivalent extents of inactivation. IdUrd substitution in the absence of light has a greater effect on phenotypic expression of T4 than either near-visible light irradiation alone or IUra substitution plus near-visible light irradiation, as measured by either delay in appearance or decrease in total amount of two induced enzyme activities (dihydrofolate reductase and deoxycytidylate hydroxymethylase).     

DIFFERENTIAL CAUSES OF MUTATION AND KILLING IN ESCHERICHIA COLI AFTER PSORALEN PLUS LIGHT TREATMENT: MONOADDUCTS AND CROSS-LINKS

Abstract— On treatment with 8-methoxypsoralen plus near UV light, an excisionless ( uvrB^- ) strain of Escherichia coli showed about 3– and 10 times higher sensitivities to killing and mutation, respectively, than its parental strain. On re-irradiation with near UV in the absence of unbound psoralen, the uvrB^- strain pretreated with psoralen plus near UV showed a decrease in both survival and mutation. After treatment with psoralen plus near UV, re-irradiation of T7 DNA in the absence of unbound psoralen caused an increase in the cross-linked fraction with an equivalent decrease in the non-cross-linked fraction. From these and previous results, we conclude that monoadducts produced by treatment with psoralen plus near UV are converted to cross-links by further irradiation and that, in E. coli , monoadducts are responsible for the mutation induced by psoralen-plus-light whereas cross-links are the major cause of its lethal action.     

DEGRADATION OF THE DNA OF ESCHERICHIA COLI K12 REC- (JC1569b) AFTER IRRADIATION WITH ULTRAVIOLET LIGHT*

Abstract— Degradation of the DNA of a rec^- mutant of Escherichia coli K12 (JC1569 b) induced by u.v. light was investigated. The rate of degradation was much larger by growing bacteria than by stationary cells. When growing bacteria were starved for amino acids, their DNA became resistant to irradiation. The mode of u.v.-induced degradation was investigated by comparing the time course of release from the acid-insoluble fraction of the label for two growing cultures; the one was pulse-labeled with ³H-thymidine and the other was pulse-labeled and chased thereafter for 12 min. It was found that the label incorporated into the former culture begins to be lost from the acid-insoluble fraction prior to the loss of the label incorporated into the latter culture. It was concluded that breakdown of the replicating point precedes degradation of the bulk of the DNA. This result suggested that the replicating point is a sensitive site to irradiation and the u.v.-induced degradation of DNA seemed to be influenced by the state of chromosome at the time of irradiation. Experiments of centrifugation of lysed spheroplasts of bacteria uniformly labeled with ³H-thymidine in alkaline sucrose demonstrated that DNA of low molecular weight appeared after irradiation with only 5 ergs/ mm², and that the molecular weight could not be restored by post-irradiation incubation. Considering these results, an hypothesis is proposed concerning the initiation of induced degradation of the DNA of the rec^- mutant.     

SIMILAR SPECTRA FOR THE INACTIVATION BY MONOCHROMATIC LIGHT OF TWO DISTINCT LEUCINE TRANSPORT SYSTEMS IN ESCHERICHIA COLI

Abstract—Kinetics of inactivation of two separate leucine transport systems (leucine specific and LIV) in E. coli by seven wavelengths of monochromatic light have been studied. Loss of leucine uptake is not due to generalized membrane damage causing non-specific leucine leakage. Inactivations are usually exponential but some wavelengths show shoulders at low doses. Two-component spectra between 254 and 435 nm occur for both transport systems. Inactivation is most efficient at 290 nm and a second peak occurs at 365 nm. Both leucine transport systems are inactivated similarly.