PHOTODYNAMIC AND SUNLIGHT INACTIVATION OF ESCHERICHIA COLI STRAINS DIFFERING IN NEAR-UV SENSITIVITY AND RECOMBINATION PROFICIENCY

Abstract— Stationary cells of four Escherichia coli strains exhibiting all four possible combinations of genes controlling near-UV sensitivity ( nur vs nur ⁺) and recombination proficiency (far-UV sensitivity; recA1 us recA ⁺) have been inactivated by visible light in the presence of acridine orange (AO, 10µg/m l ) and sunlight. The results demonstrate that strains sensitive to near-UV inactivation are also sensitive to inactivation by visible light in the presence of AO and sunlight irrespective of the recA allele carried by the strain. These results may be interpreted to mean that major mechanisms of inactivation of stationary E. coli cells by near-UV, visible light in the presence of AO and sunlight are similar and not closely related to the mechanism of inactivation by far-UV.     

THE INTERACTION OF A GENE (nur) CONTROLLING NEAR-UV SENSITIVITY AND THE polA1 GENE IN STRAINS OF E. COLI K12

Abstract— When stationary cell populations of the Escherichia coli W3110 strain and the polA1 mutant (p3478) derived directly from it were compared for their sensitivity to near-UV (NUV, 300–400 nm) inactivation, the polA1 strain proved to be more sensitive. By appropriate matings and transductions, four essentially isogenic strains have been developed which carry all four possible combinations of genes conferring far-UV (FUV, 200-300 nm) sensitivity ( polA1 vs. polA ⁺) and NUV sensitivity ( nur vs. nur ⁺). Stationary cells of strains carrying either the polA1 or polA ⁺ allele in combination with the nur allele are indistinguishable in their sensitivity to NUV inactivation and are equivalent in their NUV sensitivity to the original polA1 mutant strain (p3478). With the two strains carrying the nur ⁺ allele, stationary cell populations of the polA1 strain are clearly more sensitive to NUV inactivation than is the polA ⁺ strain. The NUV sensitizing effect of the polA1 mutation in a nur ⁺ genetic background is about the same as that of the nur mutation at the 0.37 survival level. This may mean that the polA1 and nur mutations sensitize E. coli stationary cell populations to NUV inactivation by a common mechanism.     

SINGLE-STRAND DNA BREAKS INDUCED BY 365 NM RADIATION IN ESCHERICHIA COLI STRAINS DIFFERING IN SENSITIVITY TO NEAR and FAR UV

The gene mutation nur has been shown specifically to sensitize Escherichia coli stationary phase cells to inactivation by broad spectrum near-UV (NUV) radiation. In the work reported here, E. coli strains RT1. RT2, RT3, and RT4, carrying the 4 possible combinations of recA1, recA⁺, nur , and nur⁺ , were exposed to monochromatic NUV (365 nm). The strains carrying the nur allele (RT1 and RT2) were more sensitive to inactivation by this wavelength and exhibited considerably more single strand break's (SSB's) than the strains carrying the nur⁺ allele (RT3 and RT4). As predicted, following X-irradiation the strains carrying the recA1 allele (RT1 and RT3) were more sensitive than the recA⁺ strains (RT2 and RT4). We conclude that the enhanced SSB's observed in strains RT1 and RT2 following monochromatic NUV irradiation correlated with the nur mutation and are unrelated to the recA1 mutation.     

THE EFFECTS OF LIQUID HOLDING ON NEAR-UV(300–400 nm) IRRADIATED ESCHERICHIA COLI STRAINS DIFFERING IN NEAR AND FAR-UV RADIATION SENSITIVITY

Abstract— Stationary phase cells from four Escherichia coli strains differing in near- (nur vs. nur ₊) and far-UV (recAl vs. recA₊) radiation sensitivity were subjected to near-UV radiation (NUV) in 0.85% saline. Although the NUV-irradiated cultures yielded increased colony numbers following 24 h of liquid holding (LH), a fluctuation test for each experiment showed that the observed increases were not due to recovery but were in fact due to cell multiplication. The decline in viability observed after NUV with liquid holding using the fluctuation test was equivalent in strains RT2, 3 and 4 while the decline observed with RT1 was less marked. The discrepancy between LH involving cell densities of 10₈-10₉ and 1–4 cells/m/ can be resolved by assuming that with dense cell suspensions, NUV-induced membrane damage leads to leakage or lysis, supplying sufficient nutrients to allow growth of undamaged, surviving cells.     

SENSITIVITY OF HemA MUTANT Escherichia coli CELLS TO INACTIVATION BY NEAR-UV LIGHT DEPENDS ON THE LEVEL OF SUPPLEMENTATION WITH δ-AMINOLEVULINIC ACID

Abstract— Four strains carrying all four possible combinations of the alleles nur, nur⁺, uvr A6 and uvr A ⁺ were transduced to hemA8 . The hemA8 mutation blocks the synthesis of δ-aminolevulinic acid (δ-ALA), one of the first steps in the synthesis of porphyrin and, ultimately, cytochromes essential for aerobic respiration. The cells were grown either with or without δ-ALA and treated with broad-spectrum near-ultraviolet light (NUV; 300–400 nm). hemA8 defective cells grown without δ-ALA were resistant to inactivation by NUV while hemA8 cells were sensitive to such inactivation when supplemented with δ-ALA. The sensitivity to NUV inactivation conferred by the nur gene was retained in the hemA8 derivatives. The sensitivity of such cells to NUV inactivation can be controlled by varying the level of δ-ALA supplementation. The level of δ-ALA supplementation did not influence the sensitivity of the cells to inactivation by far-UV light (FUV; 200–300 nm). The near-UV sensitivity of hemA⁺ cells was not significantly altered when grown with δ-ALA suppiementation suggesting that endogenously formed δ-ALA supports the normal, regulated level of porphyrin synthesis. These results can be interpreted to mean that porphyrin components of the respiratory chain in E. coli represent chromophores involved specifically in broad-spectrum NUV inactivating events.     

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.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

FLUENCE-RATE DEPENDENCE OF MONOPHOTONIC REACTIONS OF NUCLEIC ACIDS IN VITRO AND IN VIVO

The Bunsen-Roscoe law, also known as the reciprocity law ( E = f(F) with F = I t ) has only limited validity for monophotonic reactions of nucleic acids. Especially at low fluence rates, the extent of in vitro and in vivo photoreactions of nucleic acids in the far-UV and near-UV range is a function of the fluence and of the fluence rate ( E = f (F;I)). In vitro experiments with poly(dA)poly(dT) clearly show that the far-UV (254 nm) response, indicated by the changes of the ellipticity at 315 nm, does not obey the Bunsen-Roscoe law at low fluence rates in the range between 1 W m^-2 and 20 W m^-2. In vivo experiments with Escherichia coli revealed very similar anomalies. Studying the growth delay after irradiation with far-UV light at 280 nm or near-UV light at 334 nm, we have confirmed the lack of reciprocity in both spectral ranges. The failure of the Bunsen-Roscoe law for the 280 nm and 334 nm UV irradiation effect at low fluence rates was in the range O < I < 40 W m^-2. In both cases reciprocity occurred at higher fluence rates (40 < I < 100 W m^-2).     

GENETIC CONTROL OF NEAR-UV SENSITIVITY INDEPENDENT OF EXCISION DEFICIENCY (uvrA6) IN ESCHERICHIA COLI K12

By appropriate matings, recombinant strains carrying all four possible combinations of genes controlling near-UV (nur vs nur⁺) and far-UV (uvrA6 vs uvrA⁺, excision repair function) sensitivity have been constructed. Near and far-UV inactivation experiments with the four recombinant strains reveal that inactivating events induced by near and far-UV do not appear to overlap. These results are analogous to our previously reported experiments (Tuveson and Jonas, 1979) with recombinant strains carrying all four possible combinations of genes controlling near-UV sensitivity (nur vs nur⁺) and recombination proficiency (far-UV sensitivity, recA1 vs recA⁺). The results of these two sets of experiments taken together may mean that any recA⁺ or uvrA⁺ repairable lesions induced by near-UV are repaired equally well by either system and do not require the simultaneous participation of both repair systems.     

EVIDENCE AGAINST A PHOTOPROTECTIVE COMPONENT OF PHOTOREACTIVATION IN SACCHAROMYCES CEREVISIAE

Abstract— Photoreactivation-deficient ( phr ⁻) mutants of Saccharomyces cerevisiae were shown to lack in vitro DNA-photolyase activity. A phr ⁻ mutant was then compared with a phr ⁺ strain for near-UV induced photoprotection from far-UV irradiation. Neither strain exhibited a photoprotective effect.     

Alteration of uracil-DNA glycosylase activity by uracil dimers in DNA

Abstract The formation of colonies in solid medium was used as a criterion of viability to determine the effect of ultraviolet radiation on Trichomonas vaginalis. Both viability (colony) counts and total cell (hemocytometer) counts were used to estimate physiological ages of cell populations to be irradiated. Washed-cell suspensions in 0.6% saline were exposed to far- (254 nm) and near-UV (300–400 nm) radiation and dose-response survival curves were constructed from colony counts. The effect of far-UV was found to be independent of growth phase with the D₀ for exponential, early stationary, and late stationary cells 2.6, 2.7, and 2.7 J/m², respectively. Survival to near-UV increased with the age of cells with the estimated D₅₀ being 216 J/m² for exponential cells, 1360 J/m² for early stationary cells, and 4200 J/m² for late stationary cells. Exponential cells of Trichomonas gallinae irradiated with near-UV had a D₅₀ of 340 J/m². T. vaginalis is highly sensitive to far-UV relative to protozoa. T. vaginalis and T. gallinae are highly sensitive to near-UV relative to other microorganisms.     

SENSITIVITIES TO MONOCHROMATIC 254-nm AND 365-nm RADIATION OF CLOSELY RELATED STRAINS OF Saccharomyces cerevisiae WITH DIFFERING REPAIR CAPABILITIES

Abstract— Sensitivity to monochromatic 254- and 365-nm radiation was compared in closely related yeast strains with defects in one or more of the excision-repair ( rad1 ), error-prone repair ( rad18 ), or recombinational-repair ( rad51 ) pathways. At 254 nm, mutants defective in a single repair pathway exhibited slight to moderate UV sensitivity; those defective in two separate pathways were somewhat more UV sensitive, while triple mutants defective in all three pathways exhibited extreme UV sensitivity with a lethal event corresponding to 0.05 J m⁻². Repair defects also rendered mutants sensitive to 365-nm radiation; strains with single defects exhibited slight sensitivity, mutants with two defective pathways were more sensitive, and triple mutants exhibited maximal sensitivity with a lethal event corresponding to 2.4 times 10⁴ J m⁻². In the triple mutant ( rad1, rad18, rad51 ) at both 254 and 365 nm, the dose per lethal event was almost identical with comparable values in a repair-deficient double mutant ( uvrA, recA ) of Escherichia coli. In the E. coli mutant pyrimidine dimers are believed to be the primary cause of lethality at both wavelengths. Evidence for dimer involvement in the yeast mutant was obtained by demonstrating that lethality at both 254 and 365 nm was photoreactivated by light at 405 nm.     

THE PHOTOTOXICITY OF PHENYLHEPTATRIYNE: OXYGEN-DEPENDENT HEMOLYSIS OF HUMAN ERYTHROCYTES AND INACTIVATION OF Escherichia coli

Abstract— Phenylheptatriyne (PHT) plus near-ultraviolet light(320–400 nm; NUV) hemolyzed human erythrocytes in an oxygen dependent manner. When the phototoxicity of PHT plus NUV was tested with a series of Escherichia coli strains carrying all four possible combinations of genes controlling excision proficiency ( uvrA6 vs uvrA ⁺) and catalase activity (HPII, katF vs katF *), the membrane was found to be an important lethal target. Consistent with this observation. PHT plus NUV did not induce histidine independent ( his-4 ⁺) mutations in the four tester strains (RT7h-RT10h). Using tester strain RT10h, it was shown that there was no inactivation by PHT plus NUV in nitrogen. Results of experiments with an E. coli fatty acid auxotroph (K1060) treated with PHT plus NUV are also consistent with membrane proteins being the chief targets for attack. Radicals were formed during the photolysis of PHT plus NUV in aqueous solutions, both in the presence of air and under nitrogen. Since PHT plus NUV did not hemolyze erythrocytes or inactivate E. coli cells under nitrogen, these radicals are not cytotoxic.     

REDUCTION BY NEAR-ULTRAVIOLET (334 nm) LIGHT OF E. COLI CAPACITY FOR PHAGE GROWTH DEPENDS UPON THE rel GENE AND 4-THIOURIDINE

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) has long been known to produce a transient reduction of the capacity of bacteria to support phage growth. The present work shows that, at high fluenœs (40–100 kJ/m²), 85% of 334-nm-induced reduction of capacity in Escherichia coli B/r requires the rel gene; that is, it results from rel -gene activity caused by the near-UV treatment. This rel -gene activity leads to (1) a bacterial growth delay and concomitantly lowered bacterial metabolism, and (2) a parallel delay in phage development, with a considerable depression of burst size. We propose that the observed effects on phage development are a consequence primarily of the lowered bacterial metabolism, but they may also result partly from a direct inhibition of phage DNA synthesis by the rel gene product, these effects together leading to the observed reduction of capacity in a rel ⁺ strain. The remaining 15% of capacity reduction, observed in a rel strain, has an unknown mechanism, but does appear to involve a delay in phage development.
At least 95% of the total capacity reduction observed in the rel ⁺ strain in the range 40–100 kJ/m² requires the presence of 4-thiouridine, an unusual base in E. coli transfer RNA, which is presumably both the chromophore and the target for near-UV-induced capacity reduction.     

REPAIR OF NEAR (365 nm)- AND FAR (254 nm)-UV DAMAGE TO BACTERIOPHAGE OF ESCHERICHIA COLI

Abstract: Intact bacteriophage have been irradiated at 365 nm or at 254 nm and then analysed for DNA photoproducts or injected into their bacterial host to test susceptibility of the damage to both phage and host-cell mediated repair systems. Both thymine dimers and single-strand breaks are induced in the phage DNA by 365 nm radiation. The dimers appear to be the major lethal lesion (approximately 2 dimers per lethal event) in both repair deficient bacteriophage T4 and bacteriophage λ. after irradiation with either 254 nm or 365 nm radiation. Damage induced in T4 by either wavelength is equally susceptible to x -gene reactivation (repair sector approximately 0.5). v -gene reactivation acts on a larger fraction of the near-UV damage (repair sector of 0.82 at 365 nm as against 0.66 at 254 nm). The host-cell mediated photoreactivation system is only slightly less effective for near-UV damage but host-cell reactivation (as measured by comparing survival of phage λ. on a uvr⁺ and a uvr^- host) is effective against a far smaller sector of near-UV damage (0.35) than far-UV damage (0.85). Weigle-reactivation (far-UV induced) of near-UV damage to phage λ is not observed. The results suggest that unless the near-UV damaged phage DNA is repaired immediately after injection. the lesions rapidly lose their susceptibility to repair with a consequent loss of activity of the phage particles.     

THE EFFECT OF 1,4-DIAZABICYCLO[2.2.2]OCTANE ON THE RADIOSENSITIVITY OF BACTERIA

Abstract— Hydroxyl radicals ('OH) are scavenged by 1,4-diazabicyclo[2.2.2]octane (DABCO) at a diffusion-controlled rate of 1.25 ± 0.1 × 10⁹ M ^-1s^-1. Unlike other efficient 'OH scavengers which exhibit protection of bacteria against irradiation both in oxic and hypoxic conditions, DABCO has been shown to protect Serratia marcescens and various strains of Escherichia coli only in oxic conditions.
DABCO appears to eliminate a component of the sensitization afforded by oxygen in all strains of E. coli tested. The level of this protection increases from ∼15% in the wild type AB 1157 to ∼100% in the recA uvrA mutant AB 2480. It is suggested that DABCO protects against lethal events that can occur on macromolecules other than DNA such as the cell membrane.
Results with added glycerol, as well as work in D₂O solution, indicate that DABCO is more likely to be acting by scavenging radicals rather than by quenching ¹O₂. If ¹O₂ is a component of the sensitization afforded by oxygen, then it is unlikely to be formed in a hydrophilic environment in the cell.     

PROTECTION OF ESCHERICHIA COLI CELLS AGAINST THE LETHAL EFFECTS OF ULTRAVIOLET AND X IRRADIATION BY PRIOR X IRRADIATION: A GENETIC AND PHYSIOLOGICAL STUDY

Abstract— When log phase cells of wild-type E. coli K-12 were maintained in growth medium after X irradiation, they became progressively more resistant to a subsequent exposure to UV or X radiation. The time to achieve maximum resistance was about 60 min. The uvrB, uvrD, polA and certain exrA strains (W3110 background) also demonstrated this X ray-induced resistance to subsequent UV or X irradiation but recA, recB, lex (AB1157 or W3110 backgrounds) and other exrA strains (AB1157 background) did not. The resistance induced in wild-type, uvrB and uvrD cells was characterized by the production or enhancement of a shoulder on the survival curves obtained for the second irradiation, while the resistance induced in the W3110 exrA strains was expressed only as a change in slope. The induction of resistance in the W3110 exrA strain was not inhibited by the presence of chloramphenicol, but that in the wild-type cells appeared to be. The production or enhancement of a shoulder on the survival curves of the rec ⁺ lex ⁺ exr ⁺ cells is consistent with the concept of the radiation induction of repair enzymes. Alternative explanations, however, are discussed.     

NEAR-UV INDUCTION OF SISTER CHROMATID EXCHANGES UNDER AEROBIC AND ANAEROBIC CONDITIONS

Abstract— The induction of sister chromatid exchanges (SCE) and cell sensitivity in mouse myeloma cells (66.2 subclone of MPC11) by irradiation with monochromatic near-UV (365 nm) light were studied under aerobic and anaerobic conditions. Sister chromatid exchanges were studied using the fluorescence-plus-Giemsa technique, and sensitivity was determined by the ability of irradiated and nonirradiated control cells to form colonies in soft agar. Cells were found to be 16 times more sensitive to near-UV light under aerobic exposure, producing an F₃₇ value of 7 × 10⁴ J/m² compared to the F₃₇ value of 11.5 × 10⁵ J/m² under anaerobic conditions. The induction of SCE was also 12 times more efficient for aerobic irradiation than for anaerobic irradiation. The data suggest that the SCE-inducing potential of DNA lesions differs when near-UV irradiation is performed in the presence or absence of air. In addition, the DNA lesions responsible for lethality and also those lesions leading to SCE induction may differ under the two irradiation conditions.     

LIPID PEROXIDATION AND OTHER MEMBRANE DAMAGE PRODUCED IN Escherichia coli K1060 BY NEAR-UV RADIATION AND DEUTERIUM OXIDE

Abstract— With accumulating evidence that the membrane is an important site for near-UV inactivating events, we have investigated the effects of near-UV radiation on the unsaturated fatty acid auxotroph E. coli K1060 following incorporation into the membrane phospholipids of fatty acids with varying degrees of unsaturation. Sensitivity, lipid peroxidation and membrane damage, as determined by ⁸⁶Rb⁺ leakage, have been found to increase with increasing unsaturation in log-phase cells. Similar experiments carried out in D₂O also show an increase in sensitivity, lipid peroxidation and membrane damage, indicating that singlet oxygen may be responsible for such near-UV-radiation-induced damage     

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.