Liquid-holding effects in U.V.-irradiated phage T3

Abstract— Holding complexes of u.v.-irradiated (254 nm) T3 phage in E. coli B/r cells for several hours at 37°C in buffer, or broth with chloramphenicol, affects the phage survival in at least two different ways: (1) by enhancing excision repair, resulting under certain conditions in liquid-holding recovery (LHR), and (2) by destroying the phage (holding inactivation). LHR is most apparent in buffer containing 20 μg ml^-1 chloramphenicol (CAP). It is expressed by as much as a 10–fold increase in the fraction of complexes that display host-cell reactivation (resulting from excision repair), but the percentage of u.v. lesions repaired within repair-proficient complexes is slightly decreased. LHR is not observed if T3 infects the repair-deficient strain B_s-1. Holding inactivation is readily observed with unirradiated phage complexes in broth containing CAP. The response of irradiated-phage complexes to liquid-holding conditions is more complex: holding inactivation is less effective for irradiated than for unirradiated phage DNA (i.e. the irradiated DNA is to some extent ‘protected’), and processes leading to LHR are superimposed. Thus under certain holding conditions one observes the paradoxical phenomenon that the viable titer of irradiated phage is several times higher than that of unirradiated phage. The nature of holding inactivation is not known, nor is the mechanism by which irradiated DNA is partially protected against it. Holding inactivation does not require protein synthesis; it is rather enhanced at high CAP concentration and seems to be favored by otherwise active cell metabolism. At high CAP concentrations (200–400 μg ml^-1, as compared to 20 μg ml^-1) irradiated-phage complexes show neither LHR nor protection against holding inactivation. Likewise they fail to undergo some step by which the phage DNA becomes insensitive to repair inhibition by caffeine.     

HOST-CELL REACTIVATION OF NON-LETHAL ULTRAVIOLET-EFFECTS

Abstract— Delay of intracellular growth of u.v.-irradiated bacteriophage T1 and Λ was compared in host-cell reactivating [HCR(+)] and non-host-cell reactivating [HCR(—)] bacterial strains. At a given phage survival level, intracellular growth delay occurs to the same extent in HCR (+) and HCR (-) strains; at a given absolute u.v.-dose, this delay is considerably more expressed in HCR (-) than in HCR (+) strains. Therefore, it does not reflect the time required for the HCR repair of otherwise lethal U.V. lesions. The results rather suggest that U.V. causes, besides lethal lesions, stable photoproducts in the DNA, which are a priori non-lethal, and which are recognized and efficiently eliminated by the HCR repair system. The HCR enzymes likewise act on (non-lethal) u.v.-photoproducts causing prophage induction in lysogenic cells. Consequently, one obtains the maximum induction effect in a lysogenic HCR (-) strain at a much lower u.v.-dose than in the corresponding lysogenic HCR (+) strain. In contrast, u.v.-damage causing loss of the host cell's capacity to support growth of unirradiated phage is not affected by HCR.     

RECOVERY OF HAEMOPHILUS INFLUENZAE FROM ULTRAVIOLET AND X-RAY DAMAGE

Abstract— Results of experiments on reactivation of ultraviolet (u.v.)-irradiated Haemophilus influenzae and cellular reactivation of u.v.-damaged transforming deoxyribonucleic acid (DNA) and bacteriophage are reported. Liquid-holding recovery (LHR) is small for the u.v.-sensitive mutant BC100 which, relative to the wild type, also has greatly reduced host-cell reactivation (HCR) of u.v.-inactivated phage, and competent cultures show reduced competent cell reactivation (CCR) of u.v.-inactivated transforming DNA. BC100 cells can be transformed with DNA isolated from the wild type strain Rd to a u.v. resistance similar to that of Rd, and irradiation of the DNA reduces the transformation frequency for this marker (uvr). The u.v.-resistant mutant BC200 displays very little LHR under the usual conditions where reactivation occurs after plating. The colony-forming ability (cfa) of irradiated BC200 is greater than that of Rd, but HCR and CCR are the same on this mutant as on the wild type. The major difference between Rd and BC200 is the enhanced u.v. survival of cfa of the latter. It was determined that this difference reflects cell lysis of irradiated Rd and lack of lysis in BC200 cultures. That lysis is closely correlated with damage to the bacterial chromosome is suggested by the finding that the lytic response of Rd (as determined turbidimetrically) can be negated by the liquid-holding procedure, but lysis of BC100 (which lacks comparable DNA-repair ability) can be only partially inhibited by this procedure. LHR occurs when post-plating dark recovery is incomplete, is temperature-sensitive, and occurs unimpeded when post-u.v. protein synthesis is inhibited by chloramphenicol. It is suggested that enzymatically catalyzed reactivation of DNA occurs or is initiated during liquid-holding of u.v.-irradiated H. influenzae Rd and that the necessary enzyme(s) exists prior to appearance of u.v. lesions in the DNA. Results are reported for X-ray inactivation of transforming DNA as assayed on BC100, Rd and BC200 and of the cfa of the three strains.     

PHOTOBIOLOGY OF RNA BACTERIOPHAGES—I. ULTRAVIOLET INACTIVATION AND PHOTOREACTIVATION STUDIES*

Abstract— Biologically active f2-RNA, Obtained from bacteriophage f2, was inactivated by ultraviolet (u.v) light (2537 Å) with a quantum yield of 3.3 ± 0.3 times 10^-3 when assayed in the dark with protoplasts of an F- strain of E. coli k12. Assay under “black light” gave a quantum yield of 2.7 ± 0.5 times 10^-3 which was just enough lower to suggest that 17 per cent photorecovery of the u.v. lesions has taken place. Intact phage f2 was inactivated by u.v. radiation with a quantum yield of 0.7 ± 0.12 times 10^-3, Thus the whole phage is much less sensitive than the free RNA. No evidence of photorecovery was found in u.v.-irradiated RNA phage 7S assayed in its host Pseudomonas aeruginosa.     

THE EFFECTS OF HOST-CELL REACTIVATION ON ASSAY OF U.V.-IRRADIATED HAEMOPHILUS INFLUENZAE TRANSFORMING DNA

Abstract— The host cell reactivation (HCR) mechanism in Haemophilus influenzae cells is inhibited by sub-microgram concentrations of acriflavine (as is already known to be true for Escherichia coli ). Exposure of these cells to similar concentrations of the drug during bacterial transformation increases the apparent ultraviolet light (u.v.) sensitivity of previously irradiated transforming DNA, indicating a repair of this DNA after uptake by the cells under normal conditions. Repair is inhibited by applying acriflavine at any time up to one hour after competent cells contact the irradiated transforming DNA. The fraction of the u.v. damage repaired by HCR is very different for different genetic markers. Those markers which are most u.v. sensitive when assayed in the absence of acriflavine are most poorly repaired, suggesting that this is the reason for their higher sensitivity. For all markers the fraction of the damage repairable by in vitro photoreactivation is approximately constant, and strongly overlaps the damage repairable by HCR. The degree of HCR achieved can be altered by experimental treatment of the H. influenzae DNA prior to transformation. Thus treatment of irradiated DNA with an enzyme from Micrococcus lysodeikticus –known to attack u.v. damaged, but not undamaged DNA–prevents subsequent intracellular repair of the same u.v. lesions whose repair is inhibited by acriflavine. Similarly, partial replacement of the thymine in transforming DNA by 5-bromouracil (BU) strongly inhibits repair of subsequent u.v. damage. As in bacteriophage, the BU effect is relieved if the u.v. exposure occurs in the presence of cysteamine. It is clear that intracellular repair must be considered in interpreting experiments with u.v.-irradiated transforming DNA.     

The effect of ultraviolet light on RNA metabolism in bromouracil deoxyriboside-grown HeLa cells

Abstract— The presence of 5-bromouracil deoxyriboside (BrUdR) in the DNA of HeLa cells has profound effects on RNA metabolism after u.v. irradiation. In normally grown cells 200 ergs/mm² depresses RNA synthesis by about 30 per cent while in BrUdR-grown cells the same exposure to u.v. depresses RNA synthesis by 95 per cent. When BrUdR-grown cells are u.v. -irradiated after being labeled with ³H-uridine, the normal autoradiographic pattern, where label shifts from nucleus to cytoplasm, fails to occur. Also, in lieu of the increase in RNA specific activity that is observed in unirradiated cells for a few hours after 20-min pulse-labeling, there occurs a constant decrease in specific activity after the irradiation.     

KINETICS OF ENZYMATIC PHOTOREACTIVATION STUDIED WITH PHAGE T1 AND ESCHERZCHZA COLI Bs-1*

Abstract— The kinetics of enzymatic photoreactivation (PR) of u.v.-induced killing was compared among E. coli B_s-1, phage T1 in B_s-1 and phage T1 in irradiated B_s-1. The PR action spectrum showed no substantial difference between PR of B_s-1 and PR of T1 in B_s-1. The PR D₃₇ (i.e. the PR dose required to reactivate all but 37 per cent of the reactivable lethal lesions) was found to decrease linearly with decreasing U.V. dose whether U.V. was given to produce pyrimidine dimers in B_s-1 DNA, which then compete with irradiated T1 DNA for PR enzyme, or to B_s-1 or T1 DNA to produce dimers serving as substrate for the PR enzyme. A generalized Michaelis-Menten formula was used to analyze the data and the following conclusions were drawn. (1) The number of PR enzyme molecules per cell available for PR of T1 DNA inside the B_s-1 host is only a quarter of the number available for PR of the B_s-1 host itself. (2) The Michaelis constant K_m for reaction of host-DNA-damage and PR-enzyme becomes larger when the host damage acts as competitive inhibitor to PR of T1 DNA than when it is the substrate for PR enzyme. (3) PR enzyme retains almost all its initial catalytic efficiency even after about two-hundred rounds of catalytic functioning. Conclusions (1) and (2) suggest that PR enzyme is concentrated within the nuclear area surrounding the host DNA.     

THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

OVERLAPPING ACTION OF HOSTCELL REACTIVATION AND PHOTOREACTIVATION IN BACTERIA

Abstract— The photoreactivation rate of U.V. irradiated phages is decreased in u.v. irradiated bacteria. In contrast, the normal photoreactivation rate is observed if the irradiated bacteria are photoreactivated before phage infection. The decrease of the photoreactivation ratc is understood as a competing effect of the u.v. lesions in the bacterial nucleic acids for the photoreactivation enzyme. This competitive inhibition can be diminished not only by photoreactivation of the bacteria before phage infection but also by hostcell reactivation of the u.v. lesions in the bacterium. The results provide strong evidence that hostcell reactivation and photoreactivation revert the same u.v. photoproducts in bacterial nucleic acids. The experiments show that the hostcell reactivation enzyme is not induced by phage infection or by irradiation, but is normally present in the bacterial cell.     

The effect of γ-irradiation on the thermal decomposition of magnesium bromate

The thermal decomposition of -irradiated magnesium bromate was studied by dynamic thermogravimetry. The reaction order, activation energy, frequency factor and entropy of activation were calculated by the Coats-Redfern equation and were compared with those of the unirradiated salt. Irradiation enhances the decomposition and the effect increases with irradiation dose. The activation energy decreases on irradiation. The mechanism for the decomposition of unirradiated and irradiated magnesium bromate follows the Avrami model equation, 1-/1-/1/3=kt, and the rate-controlling, process is a phase boundary reaction assuming spherical symmetry.     

Photobiology of RNA bacteriophages. II. U.V.-irradiation of f2: effects on extracellular stages of infection and on early replication

Abstract— The effect of u.v. irradiation (2537 Å) on the RNA bacteriophage f2 has been studied with respect to the adsorption of f2 to E. coli K12 (male strain), the penetration of f2-RN A into the host cell and the conversion of the phage nucleic acid to the double-stranded replicative intermediate. The biological parameter most sensitive to u.v. was the plaque-forming ability of the phage. Its loss could be attributed to several factors. (1). A binding of capsid protein to phage nucleic acid interfering with host penetration by the f2-RNA. (2). Desorption of some irradiated phage at 37° from their attachment sites on the host. (3). Molecular alterations in the RNA preventing formation of the replicative intermediate within the host. The relationship of these factors to the lack of photoreactivability of irradiated f2 is discussed.     

A mutant of Escherichia coli K12 exhibiting varying ultraviolet sensitivities depending on the temperature of incubation after irradiation

Abstract— A mutant, URT-43, was isolated from E. coli C600 dar⁺. The mutant has a characteristic feature in that its sensitivity to ultraviolet (u.v.) light is greatly influenced by the temperature at which irradiated bacteria are incubated. On the basis of dose-reduction factor, URT-43 is approximately ten times more sensitive at 42° than at 30°C, even though unirradiated bacteria are not thenno-sensitive, The mutant could not repair u.v.-irradiated bacteriophage Λ_vir in the dark either at 30° or at 42°C, indicating that it is defective in host-cell reactivation. In contrast, the same bacteriophage was reactivated in preirradiated URT-43 if the host-bacteriophage complex was plated at 30° but there was no reactivation at 42°C. Therefore u.v.reactivation was positive at 30° but negative at 42°C. The induction of prophage by URT-43(Λ_h) was achieved by much lower doses of U.V. light than that required for the induction of lysogenic wild type bacteria. Experiments were performed in which irradiated URT-43 was first incubated for various periods in liquid media and plated both at 30° and 42°C. It was found that irradiated bacteria came to be resistant to subsequent plating at 42° only when they were preincubated in the liquid medium containing necessary amino acids and at 30°C. Since this phenomenon was completely inhibited by chloramphenicol, the process seemed to require de novo protein synthesis. An hypothesis was proposed that there are at least two independent dark-repair mechanisms in E. coli; one is responsible for host-cell reactivation and the other is responsible for U.V. reactivation.     

CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.     

ON THE MECHANISM OF RADIOSENSITIZATION BY 5-BROMOURACIL. THE OCCURRENCE OF DNA STRAND BREAKS IN U.V.-IRRADIATED PHAGE T4 AS INFLUENCED BY CYSTEAMINE

Abstract— U.V.-irradiation of phage T 4Bo^r results in a decrease in sedimentation rate of BU-DNA which is attributed to single- and double-strand breaks. No breaks could be observed in unsubstituted DNA. Cysteamine present during u.v.-irradiation is able to prevent double-strand breaks but does not influence the production of single-strand breaks measured by alkaline sucrose gradient centrifugation. The biological importance and nature of DNA strand breaks due to BU-incorporation as well as the action of the protective agent on these breaks and on the biological activity are discussed.     

WEIGLE REACTIVATION IN ACINETOBACTER CALCOACETICUS

Abstract— Weigle (W)-reactivation was demonstrated in Acinetobacter calcoaceticus for the UV-irra-diated lysogenic phage P78. The reactivation factor (survival of irradiated phage on irradiated bacteria/ survival on unirradiated bacteria) reached a maximum value of 20. This was obtained at UV-doses giving phage and host survivals of about 5 times 10_-6 and 1 times 10_-1, respectively. Intracellular development of W-reactivated P78 was followed by one-step growth experiments. Conditions which allowed maximal W-reactivation also extended the period of phage production and yielded a somewhat reduced burst size.     

MISCHKLONE U.V.-INDUZIERTER MUTANTEN BEIM PHAGEN KAPPA

Abstract— Clear-plaque mutations of phage k of Serratia are induced by extracellular u.v.-irradiation in a 2–hit process. The 2–hit-nature cannot be due to induction of one hit in each of the two DNA-strands since replating the contents of 97 wildtype plaques of u.v.-survivors (u.v.-dose 4.5 min) revealed only 1 case of heterozygosity; at least 20 cases would have been expected if phage with 1–hit-mutations formed phages looking like wildtype. On the other hand, only 1 case of heterozygosity was observed among replatings of 94 c -mutants induced by the u.v.-dose 4.5 min (survival 10^-3); most of the plaques contained pure c -type. The pure c -mutant clones are very probably due to 'recessive' lethal lesions in the nonpremutated DNA-strand. This is indicated by the dose dependence of the frequency of heterozygotes; at a dose of 3 min u.v. (survival 1.2 × 10^--²) 9 heterozygotes were observed among 95 mutants tested. From these numbers the rate of induction by u.v. of the recessive lesions's can be calculated. The data at the higher dose are in satisfactory agreement with the calculated rate. Also other types of plaque mutations (e, t, b ) showed heterozygosity. Two cases of abnormal heterozygosity were observed; one contained 2 stable mutant types ( c and b ), another one wildtype, c -tm and l -type.     

Thermal decomposition of gamma-irradiated potassium bromate by dynamic thermogravimetry

The thermal decomposition of γ-irradiated KBrO₃studied by dynamic thermogravimetry. The reaction order, activation energy, frequency factor and entropy of activation were computed by means of the Coats-Redfern, Freeman-Carroll and modified Horowitz-Metzger methods and were compared with those for the unirradiated salt. Irradiation enhances the decomposition and the effect increases with the irradiation dose. The activation energy is decreased on irradiation. The mechanism for the decomposition of unirradiated and irradiated KBrO₃ follows the Avrami model equation, [1-(1-α)^1/3] = kt, and the rate-controlling process is a phase boundary reaction assuming spherical symmetry.     

Mutagenic effects of near ultraviolet and visible radiant energy on continuous cultures of escherichia coli

Abstract— –The induction of mutation to phage T5 resistance by near ultraviolet (u.v.) and visible light was studied in chemostat cultures of Escherichia coli strains B/r and B/r/1, trp. The visible light mutation rate to phage T5 resistance was independent of growth rate over the range studied. This result is consistent with a photochemical mechanism of mutagenesis. Changeovers, in which a faster growing subpopulation takes over the culture, usually causing the mutant frequency to decline sharply, occur more frequently in chemostat cultures irradiated with visible light than in cultures treated with far u.v. or caffeine. A preliminary action spectrum was obtained with aerated chemostats that revealed effective wavelengths to be between 330 nm and 500 nm. Wavelengths longer than 500 nm were not effective. Wavelengths longer than 340 nm were not mutagenic in anaerobic chemostats. This oxygen requirement for mutagenesis between 340 nm and 500 nm is consistent with a photodynamic mechanism of action. In aerated cultures, wavelengths between 400 nm and 500 nm were as effective as wavelengths between 330 nm and 400 nm. A number of naturally occurring compounds, including riboflavin and vitamin K, are consistent with the data as candidates for the chromophore responsible for near u.v. and visible light mutagenesis.     

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.