EFFECTS OF ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT ON THE OUTER MEMBRANE OF ESCHERICHIA COLI

Abstract The hydrophobic photosensitizer acridine plus near-ultraviolet light damages both plasma membranes and outer membranes in Escherichia coli. Two lines of evidence are presented that outer membrane proteins are affected by acridine plus near-ultraviolet light treatment and that the effect is selective for certain proteins. First, analysis of outer membrane proteins on sodium dodecylsulfate polyacrylamide gels revealed that some protein bands are diminished upon treatment while others remain unaltered. New bands also appear, suggesting degradation or crosslinking reactions. Second, bacteriophage adsorption studies showed that treatment of E. coli F cells with acridine plus near-ultraviolet light causes a loss in functionality of the receptor for phage T5. Treatment of E. coli ABU57 cells under comparable conditions has no discernable effect on the functionality of the receptor for phage BF23.     

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.     

PROTECTION FROM VISIBLE LIGHT DAMAGE TO ENZYMES AND TRANSPORT IN ESCHERICHIA COLI*

Abstract— Cysteine, anthranilic acid and adenine, in decreasing order of effectiveness, protected flavo-enzymes of E. coli against inactivation with high-intensity, heterochromatic light above 400 nm. Cell suspensions illuminated for 40 min in the presence of cysteine retained 30, 40 and 55% of their succinate, l -α-glycerophosphate and d -lactate dehydrogenase activities, whereas less than 10% activity could be detected in the absence of this protective agent. Different effects on respiration and transport systems were obtained with these protective agents. Adenine and anthranilic acid provided substantial protection of the glycine and methylthio-ß- d -galactoside transport systems but failed to attenuate adverse light effects on respiration and phenylalanine uptake. Cysteine did not protect respiration and potentiated damage to selected transport systems. Our results suggest that flavins act as photosensitizers.     

MEMBRANE DAMAGE CAN BE A SIGNIFICANT FACTOR IN THE INACTIVATION OF ESCHERICHIA COLI BY NEAR-ULTRAVIOLET RADIATION

Abstract A DNA repair competent strain of Escherichia coli K-12 showed sensitivity to inorganic salts (at concentrations routinely used in minimal media) after irradiation with broad spectrum near–UV radiation, at fluences that caused little inactivation when plated on complex growth medium. This effect was not observed with cells that had been exposed to 254 nm radiation. This sensitivity to minimal medium was increased by increasing the salt concentration of the medium and by increasing the pH of the medium. This sensitivity was greatly increased by adding to the medium a low concentration of commercial glassware cleaning detergent that had no effect on unirradiated cells or far-UV irradiated cells. These findings may explain the large variability often observed in near-UV radiation survival data, and demonstrate that, at least on minimal medium plates, membrane damage contributes significantly towards cell killing. This phenomenon is largely oxygen dependent.     

GROWTH DELAY INDUCED IN ESCHERICHIA COLI BY NEAR-ULTRAVIOLET RADIATION: RELATIONSHIP TO MEMBRANE TRANSPORT FUNCTIONS*

Abstract— Rates of leucíne transport, oxygen utilization, and glucose and succinate uptake were determined in cultures of Escherichia coli B/r before and after exposure to near-UV light. Within experimental errors, rates of uptake of glucose and of succinate were proportional to growth rate at all times during the recovery or growth delay period following near-UV exposure and the same proportionality was maintained in unexposed cultures. However, rates of leucine uptake and incorporation and of oxygen utilization were not related to growth rate in a simple fashion. The results suggest that inhibition of carbon source transport is a fundamental component, and may be a primary mechanism, in growth delay induced by near-UV radiation.     

INDUCED REPAIR IN ESCHERICHIA COLI: INDUCTION BY ULTRAVIOLET LIGHT AT-79°C

Two of the phenomena associated with induced (S.O.S.) repair, namely induced inhibition of post radiation DNA degradation and induced radioresistance have been shown to be elicited by 245 nm radiation applied to E. coli cells in the frozen state at -79°C. The effect of radiation in this condition is considerably less photoreactivable than similar effects produced by exposure in the wet state. Since protein-DNA crosslinks are believed to be formed under these conditions, such consequences of UV radiation appear to be a potent inducer of induced repair.     

RECOVERY OF Escherichia coli K-12 FROM NEAR-ULTRAVIOLET RADIATION-INDUCED MEMBRANE DAMAGE

Abstract A wild-type Escherichia coli K-12 strain was irradiated with broad-band near-ultraviolet radiation (from Black-Light Blue fluorescent lamps) and after holding at 37°C for various times in a complex recovery medium, was assessed for viability on either complex medium (YENB) or minimal medium containing a high inorganic salt content. A near-ultraviolet radiation fluence was used which reduced the surviving fraction to approximately 10% when assessing for viability on the complex medium plates. A near-ultraviolet radiation induced sensitivity to inorganic salt was observed which was largely recoverable by holding treated cells in a complex recovery medium. The majority of the recovery process occurred in the initial 2 h post-irradiation holding period. No inhibition of the recovery process was produced by adding chloramphenicol (40 μg/m l ) or penicillin G (11 units/m l ) to the recovery medium, indicating that neither protein synthesis nor cell wall synthesis, respectively, were required for recovery. However, by adding bacitracin, an antibiotic which acts in part by inhibiting membrane synthesis, to the recovery medium, an effect on recovery from salt sensitivity was observed. At the concentrations of bacitracin used (0.6 and 0.2 units/m l ), little or no effect was observed on unirradiated cells, but both concentrations decreased the amount of recovery of irradiated cells. These results demonstrate that recovery from near-ultraviolet radiation-induced salt sensitivity occurs, it is independent of cell growth and the effect of bacitracin suggests that membrane synthesis may be required for recovery.     

PYRIMIDINE DIMERS INDUCED IN ESCHERICHIA COLI DNA BY ULTRAVIOLET RADIATION PRESENT IN SUNLIGHT

Abstract— Escherichia coli DNA was irradiated with various wavelengths of monochromatic UV light from 254 to 320 nm, and the relative yields of the different cyclobutane pyrimidine dimers determined. Cytosine–thymine dimers (C < > T) were more frequent than thymine dimers (T < > T) at low fluences of 300 and 313 nm light, whereas the reverse was true at either longer or shorter wavelengths. Thus, in the solar UV range deemed responsible for skin cancer (i.e. 295–315 nm), C < > T are probably more important than T < > T.     

DELAY IN GROWTH AND DIVISION INDUCED BY NEAR ULTRAVIOLET RADIATION IN ESCHERICHIA COLI B AND ITS ROLE IN PHOTOPROTECTION AND LIQUID HOLDING RECOVERY

Abstract. Microscopic observations show that growth delay and division delay occur on nutrient agar after Escherichia coli B has been irradiated at 3341 Å. These effects also occur in nutrient broth.
A near u.v. action spectrum for growth delay in nutrient broth has been obtained. It shows a single peak at 3380 Å and is indistinguishable from the action spectrum for photo-protection from far u.v. (2537 Å) killing in the same organism. Furthermore, photoprotected cells show a much greater growth delay than cells that have not been photoprotected. These, as well as kinetic data, suggest that the essential action of a photoprotection treatment consists in the induction of a growth-division delay. This delay would presumably permit more time for intracellular recovery systems to operate on the far u. v. damage to nucleic acids.
Liquid holding recovery (effected by holding cells in phosphate buffer after far u. v. irradiation) shows complete overlap with photoprotection. It is concluded that photoprotection and liquid holding recovery operate on the same far u. v. damage. As with photoprotection, it is probable that the essential action of a liquid holding treatment is the induction of a growth-division delay.
No photoprotection is observed of intracellular T2 bacteriophage or of E. coli B_s-l (Hill).     

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.     

REPAIR OF UV DAMAGE IN ESCHERICHIA COLI UNDER NON-GROWTH CONDITIONS

Abstract. –A large difference in survival occurs between buffered suspensions of E. coli irradiated with UV radiation at a low fluence rate and those irradiated at a high fluence rate. For sufficiently large fluences, the extent of this fluence rate dependent recovery (FRR) is about two orders of magnitude greater than that which can be brought about by liquid holding recovery (LHR) following high fluence rate irradiation in most of the E. coli strains studied. LHR and FRR occur in excision resynthesis repair proficient (ERR⁺) but not ERR^- strains of E. coli , although its observation can be masked in strains with complete repair potential upon subsequent growth on nutrient plates. Accumulation of DNA strand interruptions and excision of cyclobutyl dipyrimidine occur during LHR and FRR but are more extensive for the latter. Our data suggest mat events beyond incision and excision occur during LHR and FRR, but differences in the extent of ERR during LHR and FRR cannot account for the difference in cell survival between these two phenomena.     

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.).     

EFFECTS OF 8-METHOXYPSORALEN PLUS NEAR-ULTRAVIOLET LIGHT ON THE NEMATODE

Abstract— Effects of 8-methoxypsoralen plus near-UV light on the nematode Caenorhabditis elegans were studied as a novel example of photosensitized actions at the individual level. Either the eggs or the worms were illuminated with near-UV light in the presence of 8-methoxypsoralen. The treatment decreased hatchability of the eggs depending on light fluence and concentration of the sensitizer. Inhibition of growth and premature death were observed when the larvae in the second stage were illuminated in the presence of 8-methoxypsoralen. When young adults were treated before the beginning of egg-laying, they grew to lay eggs, but the total number of eggs deposited per hermaphrodite was decreased and the life span was shortened.     

INACTIVATION OF THE LACTOSE PERMEASE OF ESCHERICHIA COLI BY MONOCHROMATIC ULTRAVIOLET LIGHT

Abstract— The lactose permease of E. coli is inactivated exponentially by seven wavelengths of monochromatic UV light. An action spectrum reveals that the shorter wavelengths (243, 290 and 313 nm) are much more efficient than longer wavelengths. Inactivation at 290 nm is most efficient and is not due to generalized membrane damage. The rate of counterflux of intracellular β-galactoside in response to externally added β-galactoside was slowed by 290 nm irradiation, indicating destruction of the facilitated diffusion mechanism. The induction of β-galactosidase and β-galactoside permease was co-ordinate both with and without pre-irradiation by 290 nm light. The β-galactosidase is approximately 26-fold more resistant to 290 nm than the permease. These results are discussed in terms of a greater sensitivity of membrane proteins to 290 nm light, which may be due to the role of aromatic amino acids in conferring stability to the permease in the membrane.     

LIQUID HOLDING RECOVERY OF PHOTODYNAMIC DAMAGE IN E. COLI

Escherichia coli cells lose viability when irradiated with visible light in the presence of acridine dyes. There has been some controversy about whether such photodynamic damage can be repaired. Several investigations on different E. coli strains failed to reveal any significant difference in the sensitivity to photodynamic damage between radiation-resistant E. coli B/r and radiation-sensitive E. coli B_s-1 strains (Uretz, 1964; Janovska et al. , 1970) suggesting the absence of repair. On the contrary, other investigators (Rupp, 1966; Harm, 1968) strongly predicted the existence of a dark-repair mechanism for such damage. No systematic study of liquid-holding recovery of photodynamic damage has yet been reported and knowledge of repair capacity for photodynamic damage is still incomplete. In the present communication, liquid-holding-repair capability of E. coli B/r and B_s-1 for photodynamic inactivation in the presence of acridine orange or acriflavine has been investigated. These studies show that B/r can repair photodynamic damage while B_s-1 is deficient in this ability.     

PHOTOREACTIVATING ENZYME FROM ESCHERICHIA COLI

Abstract— Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E. coli strain lysogenic for a defective Λ bacteriophage carrying the phr gene. The resulting enzyme has a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consists of an apoprotein and cofactor, both of which are necessary for catalytic activity. The apoprotein has a monomer molecular weight of 35 ,200 and shows stable aggregates under denaturing conditions. The amino acid analysis of the E. coli enzyme is very similar to that of the photoreactivating enzyme from orchid seedlings ( Cattelya aurantiuca ). Both have arginine at the amino terminus. The cofactor, like the holoenzyme, shows absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme has an action spectrum which peaks at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme shows any detectable absorption between 300 and 400 nm.     

ACTION SPECTRUM FOR GROWTH DELAY INDUCED BY NEAR-ULTRAVIOLET LIGHT IN E. coli B/r K*

Abstract— The action spectrum for growth delay induced by near-uv light was determined for Escherichia coli B/r growing in a defined medium. This spectrum agrees with and extends that determined earlier by Jagger and his co-workers for E. coli B growing in nutrient broth. The extended spectrum is indistinguishable from the absorption spectrum for 4-thiouridine above 320 nm, but deviates significantly at wavelengths shorter than this from the spectrum for 8–13 link formation in transfer RNA containing 4-thiouridine at position 8. These results extend the evidence that 4-thiouridine in transfer RNA is the chromophore for near-UV induction of growth delay, but weaken the case for linkage of a pyrimidine at position 13 in transfer RNA as the mechanism of growth delay.     

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.     

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.