THE EXPRESSION OF LIQUID HOLDING RECOVERY IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI REQUIRES A DEFICIENCY IN GROWTH MEDIUM-DEPENDENT DNA REPAIR

Ultraviolet (UV) irradiated Escherichia coli K-12 recA cells (but not rec⁺ cells) show enhanced survival if they are held in buffer prior to plating for viability. To understand the role of the recA mutation in this liquid holding recovery (LHR) phenomenon, we have studied LHR in a temperature sensitive recA 200 mutant. The detection of LHR requires that the irradiated cells be recA when they are plated on growth medium, but the recA deficiency plays no role during liquid holding (LH). We conclude that it is the extreme sensitivity of recA cells in growth medium to unrepaired DNA daughter-strand gaps that magnifies the beneficial effects of the excision repair of DNA lesions during LH. Furthermore, we demonstrate a correlation between a strain's inability to perform growth medium dependent repair and its ability to express LHR. The relative amount of LHR was: recA > recF > lexA > recB > wild type (with the recB and wild-type strains showing negative LHR). Two strains did not show this correlation; the uvrD strain showed less LHR than expected from its UV radiation sensitivity, while the polA strain showed more. The molecular bases for these exceptions are explored.     

THE ROLE OF DNA POLYMERASE I IN LIQUID HOLDING RECOVERY OF UV-IRRADIATED ESCHERICHIA COLI

Abstract. –Excision of cyclobutyl dipyrimidines from, and accumulation of strand interruptions in, DNA of different strains of E. coli K12 were determined during liquid holding recovery after UV irradiation. The extent of Pyr <> Pyr excision was the same (20–25%) for both a pol A mutant ( E. coli P3478) and its parental wild type strain ( E. coli W3110); however, single strand interruptions accumulate during liquid holding of polA cells, but not in the parental strain. In contrast, excision was greatly reduced in a mutant (KMBL 1789) which is defective in the 5'→3' exonucleolytic function of DNA polymerase I. These data suggest that excision and resynthesis during liquid holding are carried out primarily, if not entirely, by DNA polymerase I. We further conclude that excision alone is both a necessary and sufficient condition to elicit liquid holding recovery, and that this excision requires a functional polymerase I 5'→ 3' exonuclease.     

PHOTOPROTECTION OF E. COLI B/r: RESPIRATION,GROWTH, MACROMOLECULAR SYNTHESIS AND REPAIR OF DNA*

Abstract— Photoprotecting effects of near UV radiations (300–400 nm, maximum at 360 nm) against far UV radiations (primarily 254 nm) have been studied in Escherichia coli B/r cells in minimal medium with glycerol as a carbon source. Near UV light (10⁵ Jm^-2) has a negligible effect on survival, but causes transitory inhibition of respiration, growth, DNA, RNA, and protein syntheses and cell division. Far UV (52 J m^-2) reduces survival to about 0.5 per cent; respiration, growth and RNA and protein syntheses proceed for about 60 min, after which they nearly cease for several hours. Near UV given before this fluence of far UV increases survival 10-fold and the above processes resume at times and with kinetics characteristic of those produced by lower fluences of far UV. Single-strand breaks appear in the DNA of both unprotected and photoprotected cells; repair of the breaks is essentially complete in protected but not unprotected cells. The viability kinetics for far-UV-irradiated cells with and without photoprotecting treatment are identical except that the curve for the latter is displaced upward about 1 log; exponential increases (cell division) begin at 120 min in each case. The data suggest that, in B/r cells grown under our particular conditions, namely in minimal medium with glycerol, photoprotection is not the result of growth or division delays, but reflects an increased repair capability due to continued respiration.     

POSTREPLICATION REPAIR IN uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 IS INHIBITED BY RICH GROWTH MEDIUM

Abstract Escherichia coli K-12 uvrA or uvrB strains grown to logarithmic phase in minimal medium showed higher survival after ultraviolet (UV) irradiation (254 nm) if plated on minimal medium (MM) instead of rich medium. This'minimal medium recovery'(MMR) was largely blocked by additional recA56 (92% inhibition) or lexA101 (77%) mutations, was partially blocked by additional recB21 (54%), uvrD3 (31%) or recF143 (22%) mutations, but additional polA1 or polA5 mutations had no effect on MMR. When incubated in MM after UV irradiation, the uvrB5 and uvrB5 uvrD3 strains showed essentially complete repair of DNA daughter-strand gaps (DSG) produced after UV radiation fluences up to ∼ 6 J/m² and ∼1 J/m², respectively, and then they accumulated unrepaired DSG as a linear function of UV radiation fluence. However, when they were incubated in rich growth medium after UV irradiation, they did not show the complete repair of DSG and unrepaired DSG accumulated as a linear function of UV radiation fluence. The fluence-dependent correlation observed for the uvrB and uvrB uvrD cells between UV radiation-induced killing and the accumulation of unrepaired DSG, indicates that the molecular basis of MMR is the partial inhibition of postreplication repair by rich growth medium. Rich growth medium can be just MM plus Casamino Acids or the 13 pure amino acids therein in order to have an adverse effect on survival, regardless of whether the cells were grown in rich medium or not before UV irradiation.     

Effects of ultraviolet light irradiation in biotreatment of organophosphates

Excess biomass accumulation in reactor biodegradation processes is undesirable: it increases the disposal cost and upsets the operation of biological reactors if not properly controlled. In this study, we investigated the feasibility of using ultraviolet (UV) light irradiation to reduce biomass accumulation and increase the specific biodegradation activity. UV irradiation has been widely used to introduce DNA damage in bacteria. Here we apply this technology to the biodegradation of organophosphates by recombinantEscherichia coli strains that contain arecA mutation and a cloned organophosphate hydrolase gene. We show that therecA negative strains after UV irradiation reduce the growth rate but increase the specific organophosphate hydrolase activity. This increase in specific enzyme activity is not owing to continued protein synthesis from the plasmid after the damage of chromosomal DNA by UV irradiation. Rather, it is likely to be caused by an increase in membrane permeability to the substrate. Kinetic analysis suggests that the membrane transport of paraoxon is the rate-limiting step in its biodegradation.     

Role of Ozone in UV‐C Disinfection,Demonstrated by Comparison between Wild‐Type and Mutant Conidia of Aspergillus niger

This study aimed to investigate the tolerance of a melanized wild‐type strain of Aspergillus niger (CON1) and its light‐colored mutant (MUT1) to UV–C light and the concomitantly generated ozone. Treatments were segregated into four groups based on whether UV irradiation was used and the presence or absence of ozone: (?UV, ?O₃), (?UV, +O₃), (+UV, ?O₃) and (+UV, +O₃). The survival of CON1 and MUT1 conidia under +UV decreased as the exposure time increased, with CON1 showing greater resistance to UV irradiation than MUT1. Ozone induced CON1 conidium inactivation only under conditions of UV radiation exposure. While, the inactivation effect of ozone on MUT1 was always detectable regardless of the presence of UV irradiation. Furthermore, the CON1 conidial suspension showed lower UV light transmission than MUT1 when examined at the same concentration. Compared with the pigment in MUT1, the melanin in CON1 exhibited more potent radical‐scavenging activity and stronger UV absorbance. These results suggested that melanin protected A. niger against UV disinfection via UV screening and free radical scavenging. The process by which UV–C disinfection induces a continual decrease in conidial survival suggests that UV irradiation and ozone exert a synergistic fungicidal effect on A. niger prior to reaching a plateau.     

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.     

Inactivation by Pulsed Light of Bacillus subtilis Spores with Impaired Protection Factors

The resistance to pulsed light (PL) of spores of Bacillus subtilis strain 168 and of strains with mutations increasing sensitivity to UV‐C or affecting spore structure was evaluated and compared to resistance to continuous UV‐C and moist heat, in order to reveal original mechanisms of inactivation by PL. Spores of B. subtilis strain 168 (1A1) and eight mutant strains (sspA, sspB, sspAB, cotA, gerE, cotE, uvrA and recA) were exposed to PL (up to 1.77 J cm^?2), continuous UV‐C (up to 147 mJ cm^?2) and moist heat at 90°C. Spores of the strains lacking proteins linked to coat formation or structure (cotA, gerE and cotE) were markedly more sensitive to PL than 1A1, while their sensitivity to continuous UV‐C or to moist heat was similar to the one of strain 1A1. Coat proteins had a major contribution to the resistance of B. subtilis spores to PL irradiation characterized by short‐time and high‐energy pulses of white light in the wavelengths 200–1100 nm. In contrast the role of coat proteins to UV‐C or to moist heat resistance was marginal or null.     

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.     

POSTIRRADIATION PROPERTIES OF A UV-SENSITIVE VARIANT OF CHO

Abstract A UV-hypersensitive mutant of Chinese hamster ovary (CHO) cells, termed 43-3B, has been used in a comparative study with the wild type CHO in order to determine the involvement of repair in several postirradiation phenomena. 43-3B has the same growth rate and chromosome number as the wild type CHO-9. It is hypersensitive to UV irradiation (D₀ of 0.3 J/m² as compared to 3.2 J/m² for the wild type). 43-3B shows only about 17% of the UV-stimulated unscheduled DNA repair synthesis of CHO-9 as measured by autoradiography. When breaks in supercoiled chromatin are measured after UV by the nucleoid sedimentation method, the mutant appears to be capable of carrying out only limited incision. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line, suggesting that the removal of UV-induced replication blocks by excision repair is the most important factor in allowing recovery of UV-inhibited DNA synthesis. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild type CHO-9, but little recovery was seen in 43-3B. This indicates that excision repair capability can also be important in split-fluence recovery.     

ACTION SPECTRA FOR LETHALITY IN RECOMBINATION-LESS STRAINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI*

Abstract— Action spectra for lethality of both stationary and exponentially growing cells of recombinationless (recA) mutants of Salmonella typhimurium and Escherichia coli were obtained. Maximum sensitivity was observed at 260nm which corresponds to the maximum absorbance of DNA. However, a shoulder occurred in the 280–300 nm range that departed significantly from the absorption spectrum of DNA. At wavelengths longer than 320nm, the shapes of inactivation curves departed significantly from those at wavelengths shorter than 320nm and survival curves at wavelengths longer than 320nm had a large shoulder. A small peak or shoulder occurred in the 330–340nm region of the action spectra. The special sensitivity of recA mutants to broad spectrum near-UV radiation may be due to synergistic effects of different wavelengths. Parallels between the inactivation of recA mutants and the induction of a photoproduct of l -tryptophan toxic for recA mutants (now known to be H₂O₂) suggest that H₂O₂ photoproduct from endogenous tryptophan may be involved in the high sensitivity of these strains to broad spectrum near-UV radiation.     

EFFECT OF UV IRRADIATION ON LETHAL INFECTION OF MICE WITH Candida albicans

Exposure of mice to UV radiation inhibits the induction and elicitation of the delayed-type hypersensitivity (DTH) response to Candida albicans. To determine whether UV irradiation also affects the pathogenesis of systemic C. albicans infection, C3H mice were exposed to a single dose of 48 kJ/m² UV-B radiation from FS40 sunlamps 5 days before or 5 days after sensitization with formalin-fixed C. albicans and challenged intravenously (i.v.) with a lethal dose of viable fungi 6 days after sensitization (11 or 1 days after UV irradiation). Exposing unsensitized mice to UV radiation 11 days before lethal challenge had no effect on survival, but the survival time of mice exposed to UV radiation 1 day before challenge was reduced by more than 50%. In the latter group, decreased survival time correlated with persistence of C. albicans in the brain and progressive growth of C. albicans in the kidneys. Sensitization of unirradiated mice with formalin-fixed C. albicans extended their survival time following lethal i.v. challenge with viable C. albicans. Exposing the mice to UV radiation 5 days before sensitization did not abrogate this beneficial effect of sensitization on survival, even though it significantly reduced the DTH response. Thus, immunity to systemic infection did not depend on the ability of the mice to exhibit a DTH response to C. albicans. The beneficial effect of sensitization on survival after lethal infection was abrogated, however, in mice exposed to UV radiation 1 day before lethal challenge with C. albicans. Furthermore, these mice were unable to contain the progressive growth of C. albicuns in the kidneys, in contrast to sensitized, unirradiated mice. The induction of cutaneous inflammation with turpentine had no effect on the survival rate of mice lethally infected with C. albicans, suggesting that inflammation alone is not sufficient to decrease the survival time of C. albicans-infected mice.     

Degradation of the DNA of recA mutants of Escherichia coli K-12 after irradiation with ultraviolet light. II. Further studies including a recA uvrA double mutant

Abstract— The u.v.-induced degradation of the DNA of the recA1 mutant of Escherichia coli K-12 has been further investigated. The extensive degradation was mostly prevented by an additional mutation in the uvrA gene. Furthermore, when amino acid-starved recA bacteria were irradiated and held under a condition preventing DNA replication, the DNA was degraded very little. The mode of u.v.-induced DNA degradation was investigated by comparing the time course of release of label from the acid-insoluble fraction of two growing cultures of a recA uvrA double mutant; the one was pulse-labeled with ³H-thymidine and the other was pulse-labeled and chased thereafter for 12 min. It was found that, in spite of very little degradation of the bulk of DNA. the label incorporated into the former culture was lost rapidly from the acid-insoluble fraction. while the label incorporated into the latter culture was not. It was concluded. therefore. that breakdown of the replicating point is characteristic of recA mutants and that degradation of the bulk of DNA is induced in some way by the function of the uvrA gene. Therefore, the degradation of the major part of DNA which follows the degradation of the replicating point appears to be a secondary phenomenon.     

ULTRAVIOLET INACTIVATION OF THE ABILITY OF E. COLI TO SUPPORT THE GROWTH OF PHAGE T7: AN ACTION SPECTRUM

Abstract— Ultraviolet (UV)-irradiated E. coli K-12 wild-type cells were sensitized by a post-irradiation treatment with 10^-2 M 2, 4-dinitrophenol (DNP). This effect was not seen in strains carrying a uvr mutation, suggesting that DN P interferes with the excision repair process. The polA strain was sensitized to the same extent as the wild-type strain, while the exrA strain was not affected by DNP treatment.
Recombination deficient strains ( recA, recB and recA recB ) were protected by DNP treatment after UV irradiation. This protection was abolished by the addition of a uvr mutation (i.e., in strains recA uvrB and recB uvrB ).
Alkaline sucrose gradient sedimentation studies showed that DNP treatment interfered with the rejoining of DNA single-strand breaks induced by the excision repair process. This interference was apparently specific for the exr gene-dependent branch of the uvr gene-dependent excision repair process, since the uvr and exr strains were not sensitized while the wild-type and polA strains were sensitized.     

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH-PIGMENT MUTANT OF TOMATO

Abstract— A high-pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light-labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence-response relationships for anthocyanin synthesis after a blue-light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far-red light (FR)-absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6-fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild-type and hp-mutant seedlings, despite the latter accumulating 9-fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence-rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24-h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.     

Synergistic Photobactericidal Activity Based on Ultraviolet‐A Irradiation and Ferulic Acid Derivatives

Ultraviolet‐A (UV‐A)‐mediated bactericidal activity was enhanced by combined treatment with trans‐ferulic acid (trans‐FA, compound 1 ) or its derivatives. Derivative compounds 4 and 10 contain a phenyl group or an l ‐tyrosine HCl tert‐butyl ester, respectively, linked to the carboxyl group of trans‐FA. Of the three compounds, 10 exhibited the highest synergistic activity in a photobactericidal assay based on treating Escherichia coli with a derivative compound and UV‐A irradiation (wavelength 350–385 nm). Inactivation of viable cells at a 4.9 J cm^?2 UV‐A fluence increased from 1.90 to 5.19 logs in the presence of 10 (100 μm ); a 4.95‐log inactivation was achieved with 10 (5 μm ) and a 7.4 J cm^?2 UV‐A fluence. Addition of antioxidants significantly suppressed photosynergistic bactericidal activity, suggesting that reactive oxygen species (ROS) are involved in the combined bactericidal mechanism. Flow cytometry revealed that combined treatment with UV‐A and compound 10 , which showed the highest photobactericidal activity, generates an excess of oxidative radicals in bacterial cells. The bactericidal activity of compound 10 may be due to electrostatic interaction between the molecule's cationic moiety and the cell surface, followed by amplification of ROS generation in the cells.     

Lethal Effect Induced in Pseudomonas aeruglnosa Exposed to Ultraviolet-A Radiation

Ultraviolet-A (365 nm, 120 kJ/m²/h) exposure caused cell death in Pseudomonas aeruginosa at doses at which Escherichia coli cell viability was not affected. We have not found that UVA induced growth delay or any other sublethal effect. Irradiated suspensions of P. aeruginosa showed a marked reduction in membrane-bound succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activities. Succinate-driven respiration and several nutrient transport systems were also inhibited. Whereas SDH and LDH activities were independent of the irradiation conditions, cell viability, respiration and transport systems were protected when irradiation was performed in an N₂ atmosphere. A similar protective effect was observed when cells were grown in media containing glycerol or when preirradiation bacterial growth was carried out at 30°C (instead of 37°C). Results suggest that UVA induces a differential damaging effect on several biochemical functions of P. aeruginosa. The UVA induced photodamage may fall into two categories: indirect damage mediated by oxygen (cell killing and inhibition of respiration and transport systems) and direct damage to SDH and LDH (apparently not oxygen dependent). These enzymes and leucine transport appear not to be involved in the lethal effect described herein because they were altered despite viability-preserving conditions.     

THE SYNERGISTIC ACTION OF ULTRAVIOLET AND X RADIATION ON MUTANTS OF ESCHERICHIA COLI K-12

Abstract— Prior UV irradiation increased the X-ray sensitivity of wild-type E. coli K-12. This synergistic effect of combined UV and X irradiation was also observed, but to a reduced extent, in uvrA, uvrB, uvrC , and polA mutants, but was absent in exrA, recA, recB , or recC mutants of E. coli K-12. Alkaline sucrose gradient studies demonstrated that the wand err gene-controlled, growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks was inhibited by prior UV irradiation. This inhibition probably explains the synergistic effect of these two radiations on survival.