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.     

HYDROGEN PEROXIDE INDUCED RESISTANCE TO BROAD-SPECTRUM NEAR-ULTRAVIOLET LIGHT(300–400 nm) INACTIVATION IN Escherichia coli

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur⁺, uvrAb, and uvrA ⁺ were either untreated or pretreated with a sublethal dose of H₂0₂ prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H₂O₂ did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H₂O₂ pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H₂0₂ leads to resistance of such cells to inactivation by H₂O₂ and NUV, survival of H₂O₂ treated bacteriophage PI cml clr100 is not enhanced when assayed on H₂O₂ pretreated E. coli host cells.     

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.     

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.     

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.     

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.     

GENETIC CONTROL OF NEAR-UV (300–400nm) SENSITIVITY INDEPENDENT OF THE recA GENE IN STRAINS OF ESCHERICHIA COLI K12

Abstract— Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function have been inactivated with near-UV (300–400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV ( recA ) sensitivity can be explained hy assuming that a different chromosomal gene ( nur ) controls near-UV sensitivity. Support for this hypothesis comes from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV ( recA 1 vs recA ⁺ ) and near-UV sensitivity ( nur vs nur⁺ ). Transduction with phase P1 has shown that introduction of the recA 1 allele into a recA⁺ recipient does not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggest that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58).     

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.     

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.     

Induction of Cell Killing, Mutation and umu Gene Expression by 6-Mercaptopurine or 2-Thiouracil with UVA Irradiation

Abstract— When Escherichia coli cells were irradiated by UVA in the presence of 6-mercaptopurine (6-MP) or 2-thiouracil (S²Ura), two kinds of repair-deficient strains of recA ⁻ and uvrA ⁻ were killed more efficiently than the parental wild-type strain having normal repair capacities. In addition, these agents with UVA exposure greatly induced the incidence of mutations in the uvrA ⁻ strain as compared with the wild-type strain but not the uvrA ⁻ strain. Furthermore, the induction of expression of umuDC genes was investigated in two Salmonella typhimurium strains, TA1S35 and TA1538, carrying a pSK1002 plasmid. In these systems, it is easy to measure β-galactosidase activities for the induced activities of SOS responses. These agents with UVA exposure also induced expression of the umuDC genes. These results suggest that 6-MP and S²Ura with UVA induce DNA damage which is repairable by the excision repair mechanism.     

CHEMILUMINESCENT REACTIONS OF SO

Abstract— The rapid bimolecular reaction SO + O₃= SO₂+O₂+ 106 kcal/mole
yields electronically excited SO₂ in the ³B ₁ and ¹B₂ states with some vibrational excitation, as well as SO₂ in its electronic ground state. It is shown that k₁ = 1.5 x 10¹² exp (-2100/ RT ) cm² mole^-1 s-¹ and that the formation of electronically excited SO₂ involves higher activation energies.     

LIGHT-DEPENDENT CYTOTOXIC REACTIONS OF ANTHRACENE

Anthracene is a photodynamic compound in vitro. In the presence of oxygen, it is known to generate singlet oxygen and participate in Type II reactions. In aqueous solution, it also participates in Type I reactions, such as in the photoreduction of cytochrome c, which can be suppressed by superoxide dismutase. In argon, direct photoreduction of cytochrome c also takes place. Anthracene induces the photodynamic hemolysis of human erythrocytes and inactivates Escherichia coli cells photodynamically. By using a series of E. coli strains differing in DNA repair capabilities and catalase proficiency, sensitivity to inactivation by anthracene plus NUV was correlated with catalase deficiency rather than with particular repair deficiencies. The fact that carotenoid genes cloned and expressed in E. coli offered partial protection suggests that the membrane may be one possible target for inactivation by anthracene plus NUV. Anthracene plus NUV inactivated Haemophilus influenzae transforming DNA and led to nicking of supercoiled pBR322 DNA in vitro. In vivo, therefore, anthracene is a phototoxic molecule whose cytotoxicity could be the result of damage to more than one target.     

RELATIONSHIP BETWEEN SURVIVAL AND THYMINE-DIMER EXCISION IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr ⁺ thy- trp ^-, E. coli B/r hcr ^-thy^- trp ^-, and E, coli 15 T^- 555–7 thy ^- meth ^- trp ^- arg ^- has been studied.
The prestarvation increased the resistance of all the strains but reasonably inhibited excision of thymine dimers. Thus the enhancement of u.v. resistance after amino acid prestarvation was not due to more complete excision of thymine dimers.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

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.     

MECHANISM OF PHYSICAL QUENCHING OF SINGLET MOLECULAR OXYGEN BY CHLOROPHYLLS and RELATED COMPOUNDS OF BIOLOGICAL INTEREST

Abstract— The rate constant k₅/ > for physical quenching of singlet oxygen O₂(δ₁;) by the sensitizer in dye-sensitized photooxygenations is determined in the case of chlorophylls a and b (7.3 times 10⁸, 4.2 times 10⁸ M^-1 s^-1 respectively), pheophytins a and b (7.4 times 10⁷, 3.0 times 10⁷ M^-1 s^_1 respectively), tetraphenylporphyrin (4.4 times 10⁷ M^-1 s^_1), magnesium tetraphenylporphyrin (5.0 times 10⁸ M^-1 s^_1), zinc tetraphenylporphyrin (1.5 times 10⁸ M^-1 s^_l) and protoporphyrin IX-dimethylester (9.1 times 10⁷ M ^-1 s^_1) in benzene. These sensitizers show a linear correlation between log k_s^O , and their half-wave oxidation potentials and the value of the slope is similar to that observed for various compounds such as phenols. It is concluded that (i) the interaction between chlorophylls and related compounds with singlet oxygen may involve an exciplex as for phenols, and (ii) physical quenching may be envisaged as a spin-orbit-induced intersystem crossing within the exciplex.     

LETHALITY IN REPAIR-PROFICIENT ESCHERICHIA COLI AFTER 365 nm ULTRAVIOLET LIGHT IRRADIATION IS DEPENDENT ON FLUENCE RATE

Abstract Reciprocity (total applied fluence produces the same response, regardless of the fiuence rate) for the lethal effects caused by 365 and 254 nm ultraviolet light (UV) was studied for repair-proficient and -deficient Escherichia coli strains. In the repair-proficient strain, E. coli WP2 uvrA * recA *, reciprocity after 365 nm UV was only observed at fluence rates of about 750 Wm^-2 and above. Below this rate, the cells became increasingly sensitive as the fluence rate was decreased. Similar lack of reciprocity was obtained whether the cells were exposed at 0 or 25°C. The double repair-defective mutant, E. coli WP100 uvrA recA , showed complete reciprocity after 365 nm UV over the same range of fluence rates measured for the repair-proficient strain. For 254 nm UV, complete reciprocity occurred in both strains over a range of fluence rates differing by an order of magnitude.     

INACTIVATION OF PHAGE BY NEAR-ULTRAVIOLET RADIATION AND HYDROGEN PEROXIDE

A series of phage with different genomes (both single-stranded and double-stranded RNA and DNA) was inactivated with hydrogen peroxide (H₂O₂) in various combinations with far-ultraviolet (FUV) and near-ultraviolet (NUV) radiations. In every case but one (a lipid-coated phage), a sublethal H₂O₂ concentration greatly enhanced killing by NUV but not FUV. Moreover, this NUV/H₂O₂ synergism was oxygen independent and there was little if any host cell reactivation upon NUV plus H₂O₂ inactivation. These results suggest that these phage are inactivated by a common mechanism irrespective of nucleic acid composition, but that some phage genomes may be more vulnerable to NUV/H₂O₂ inactivation than others.