Efficiency of pyrimidine dimer formation in Escherichia coli across UV wavelengths

AIMS: Inactivation of Escherichia coli as a function of ultraviolet (UV) wavelength was investigated by using the endonuclease-sensitive site (ESS) assay to quantify pyrimidine dimer formation. METHODS AND RESULTS: Ultraviolet dose-response curves were determined based on both log reduction in colony-forming units (CFU) and endonuclease-sensitive sites per kb DNA (ESS/kb) for monochromatic 254-nm low-pressure (LP) UV, polychromatic medium-pressure (MP) UV, 228 and 289-nm UV irradiation. UV irradiation from LP and MP UV sources were approx. equal in both CFU reduction and pyrimidine dimer formation at all UV doses studied; 228-nm irradiation was less effective than LP or MP, and 289-nm irradiation was the least effective in both CFU reduction and pyrimidine dimer formation. These results are in qualitative agreement with the absorption spectrum of pyrimidine bases in DNA. Results indicated an approx. linear relationship between ESS/kb and log CFU reduction. CONCLUSIONS: Formation of pyrimidine dimers in genomic DNA is primarily responsible for UV inactivation of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributed to fundamental understanding of UV disinfection and aids in UV reactor design.     

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Assessment of DNA damage and repair in Mycobacterium terrae after exposure to UV irradiation

AIM: Ultraviolet (UV) irradiation for drinking water treatment was examined for inactivation and subsequent dark and photo-repair of Mycobacterium terrae. METHODS AND RESULTS: UV sources tested were low pressure (monochromatic, 254 nm) and medium pressure (polychromatic UV output) Hg lamps. UV exposure resulted in inactivation, and was followed by dark or photo-repair experiments. Inactivation and repair were quantified utilizing a molecular-based endonuclease sensitive site (ESS) assay and conventional colony forming unit (CFU) viability assay. Mycobacterium terrae was more resistant to UV disinfection compared to many other bacteria, with approximately 2-log reduction at a UV fluence of 10 mJ cm(-2) ; similar to UV inactivation of M. tuberculosis. There was no difference in inactivation between monochromatic or polychromatic UV lamps. Mycobacterium terrae did not undergo detectable dark repair. Photo-repair resulted in recovery from inactivation by approximately 0.5-log in less than 30 min for both UV lamp systems. CONCLUSIONS: Mycobacterium terrae is able to photo-repair DNA damage within a short timeframe. The number of pyrimidine dimers induced by UV light were similar for Escherichia coli and M. terrae, however, this similarity did not hold true for viability results. SIGNIFICANCE AND IMPACT OF THE STUDY: There is no practical difference between UV sources for disinfection or prevention of DNA repair for M. terrae. The capability of M. terrae to photo-repair UV damage fairly quickly is important for wastewater treatment applications where disinfected effluent is exposed to sunlight. Finally, molecular based assay results should be evaluated with respect to differences in the nucleic acid content of the test micro-organism.     

Molecular indications of protein damage in adenoviruses after UV disinfection

Adenoviruses are resistant to monochromatic, low-pressure (LP) UV disinfection--but have been shown to be susceptible to inactivation by polychromatic, medium-pressure (MP) UV--when assayed using cell culture infectivity. One possible explanation for the difference between UV lamp types is that the additional UV wavelengths emitted by MP UV enable it to cause greater damage to viral proteins than LP UV. The objective of this study was to examine protein damage in adenoviruses treated with LP and MP UV. Results show that MP UV is more effective at damaging viral proteins at high UV doses, though LP UV caused some damage as well. To our knowledge, this study is the first to investigate protein damage in UV-treated adenovirus, and the overview presented here is expected to provide a basis for further, more detailed work.     

Influence of photoreactivating light intensity and incubation temperature on photoreactivation of Escherichia coli following LP and MP UV disinfection

Aims:  To investigate the effects of fluorescent light intensity, sunlight intensity and temperature on photoreactivation of Escherichia coli after low-pressure (LP) and medium-pressure (MP) ultraviolet (UV) disinfection.
Methods and Results:  Two E. coli strains were irradiated with LP and MP UV lamps, and exposed to various fluorescent light (0–23 kLux) and sunlight intensities (1–80 kLux), and temperatures (4–50°C). Escherichia coli concentrations were enumerated at hourly intervals to determine photoreactivation rates and final photoreactivation levels. Higher photoreactivation rates and levels were observed with increasing fluorescent light intensities, while high sunlight intensity (>12 kLux) caused a one-log decrease in E. coli concentrations. When exposed to near-optimum growth temperatures (23–37°C), photoreactivation levels were higher than those with too high (50°C) or too low (4°C) temperatures. Overall, photoreactivation following MP UV disinfection was lower than that following LP UV disinfection.
Conclusions:  Photoreactivation of bacteria following UV disinfection can be a problem in tropical countries where sunlight is abundant and temperatures are high, unless high sunlight intensity is present or if MP UV disinfection is employed.
Significance and Impact of the Study:  With the increased use of UV disinfection, it is imperative that photoreactivation be taken into account in the design of reactors based on site-specific conditions of temperature and light intensity exposure.     

Indicators for photoreactivation and dark repair studies following ultraviolet disinfection

Repair of DNA in bacteria following ultraviolet (UV) disinfection can cause reactivation of inactivated bacteria and negatively impact the efficiency of the UV disinfection process. In this study, various strains of E. coli (wild-type, UV-resistant and antibiotic-resistant strains) were investigated for their ability to perform dark repair and photoreactivation, and compared based on final repair levels after 4 h of incubation, as well as repair rates. Analysis of the results revealed that the repair abilities of different E. coli strains can differ quite significantly. In photoreactivation, the log repair ranged from 10 to 85%, with slightly lower log repair percentages when medium-pressure (MP) UV disinfection was employed. In dark repair, log repair ranged from 13 to 28% following low-pressure (LP) UV disinfection. E. coli strains ATCC 15597 and ATCC 11229 were found to repair the fastest and to the highest levels for photoreactivation and dark repair, respectively. These strains were also confirmed to repair to higher levels when compared to a pathogenic E. coli O157:H7 strain. Hence, these strains could possibly serve as conservative indicators for future repair studies following UV disinfection. In addition, dimer repair by photoreactivation and dark repair was also confirmed on a molecular level using the endonuclease sensitive site (ESS) assay.     

Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log(10) reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm(2) at 20 degrees C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm(2) for a 2-log(10) reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm(2). Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10 degrees C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log(10) reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.     

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.     

Photoreactivation of UV induced cell killing, chromosome aberrations, sister chromatid exchanges, mutations and pyrimidine dimers in Xenopus laevis fibroblasts

Summary Fibroblasts from Xenopus laevis, which possess photoreactivating enzyme were used to study the influence of photoreactivating light on the frequency of pyrimidine dimers in DNA, chromosomal aberrations, sister chromatid exchanges, cell killing and the induction of gene mutations (ouabain-resistance) induced by 254 nm ultraviolet irradiation. The frequency of all biological endpoints studied were reduced following exposure to photoreactivating light parallel to the reduction in the frequencies of pyrimidine dimers (determined as endonuclease sensitive sites). However there was not always an absolute quantitative relationship between the reduction in the frequency of pyrimidine dimers and the reduction in the biological effects. This probably reflects a fast fixation process for the biological effects prior to removal of the dimers by photoreactivation.Abbreviations UV ultraviolet - PR photoreactivating - ESS endonuclease sensitive site - SCE sister chromatid exchanges - BrdUrd 5-brothodeoxyuridine     

The influence of the wavelength of ultraviolet radiation on survival,mutation induction and DNA repair in irradiated chinese hamster cells

Chinese hamster ovary cells were used to compare the cytotoxicity and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with that of near-UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse “Sun Lamp”), of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal number of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation.The numbers of dimers induced were determined by a biochemical method detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers.On the basis of induced dimers, more cells were killed by >310-nm UV than by >290-nm UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5–6 times as many mutants were induced per dimer by >310-nm UV than by >290-nm UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were not caused by differences in repair of dimers: cells with an equal number of dimers induced by either 254-nm or near-UV showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical amount of repair replication.The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance.     

Biofouling control in water by various UVC wavelengths and doses

UV light irradiation is being increasingly applied as a primary process for water disinfection, effectively used for inactivation of suspended (planktonic) cells. In this study, the use of UV irradiation was evaluated as a pretreatment strategy to control biofouling. The objective of this research was to elucidate the relative effectiveness of various targeted UV wavelengths and a polychromatic spectrum on bacterial inactivation and biofilm control. In a model system using Pseudomonas aeruginosa, the inactivation spectra corresponded to the DNA absorption spectra for all wavelengths between 220 and 280 nm, while wavelengths between 254 nm and 270 nm were the most effective for bacterial inactivation. Similar wavelengths of 254-260-270 nm were also more effective for biofilm control in most cases than targeted 239 and 280 nm. In addition, the prevention of biofilm formation by P. aeruginosa with a full polychromatic lamp was UV dose-dependent. It appears that biofilm control is improved when larger UV doses are given, while higher levels of inactivation are obtained when using a full polychromatic MP lamp. However, no significant differences were found between biofilms produced by bacteria that survived UV irradiation and biofilms produced by control bacteria at the same microbial counts. Moreover, the experiments showed that biofilm prevention depends on the post-treatment incubation time and nutrient availability, in addition to targeted wavelengths, UV spectrum and UV dose.     

Effect of flash photoreactivation on Escherichia coli recA induction by ultraviolet light

Excision-deficient Escherichia coli, carrying the gene for the photolyase on a multicopy plasmid, were irradiated with ultraviolet (UV) light then photoreactivated by illumination delivered from a camera flash unit. Such instantaneous illumination monomerizes only cyclobutane pyrimidine dimers already bound by the photolyase. Whereas the lethal effect of UV light and the number of C-to-T transition-type mutations induced by UV irradiation were both significantly reduced by subsequent irradiation with a single flash of light, single-flash photoreactivation did not reverse the induction of the recA gene by UV light. The results indicate, therefore, that non-photoreactivable DNA lesions play a role in recA induction.     

Photoreactivation of cells and phages injuried by ultraviolet radiation in the ecological long-wave band]

Photoreactivation (PR) was measured after inactivation by far (254 nm), middle (300-315 nm) and near (315-400 nm) UV radiation of Paramecium caudatum and 8 strains of Escherichia coli differing in PR and dark repair capability. PR volume was high and practically the same after irradiation by far and middle UV, but PR was not observed in near UV-inactivated cells of all the strains. It is proposed that pyrimidine dimers are not significant in near UV lethal lesions in cells, as near UV-irradiated phages (T7 and lambdacI 857) are not photoreactivated in undamaged host bacterial cells.     

Photoreactivation of pyrimidine dimers in the DNA of normal and xeroderma pigmentosum cells.

Photoproducts formed in the DNA of human cells irradiated with ultraviolet light (uv) were identified as cyclobuytl pyrimidine dimers by their chromatographic mobility, reversibility to monomers upon short wavelength uv irradiation, and comparison of the kinetics of this monomerization with that of authentic cis-syn thymine-thymine dimers prepared by irradiation of thymine in ice. The level of cellular photoreactivation of these dimers reflects the level of photoreactivating enzyme measured in cell extracts. Action spectra for cellular dimer photoreactivation in the xeroderma pigmentosum line XP12BE agree in range (300 nm to at least 577 nm) and maximum (near 400 nm) with that for photoreactivation by purified human photoreactivating enzyme. Normal human cells can also photoreactivate dimers in their DNA. The action spectrum for the cellular monomerization of dimers is similar to that for photoreactivation by the photoreactivating enzyme in extracts of normal human fibroblasts.     

UV Disinfection of Adenoviruses: Molecular Indications of DNA Damage Efficiency

Adenovirus is a focus of the water treatment community because of its resistance to standard, monochromatic low-pressure (LP) UV irradiation. Recent research has shown that polychromatic, medium-pressure (MP) UV sources are more effective than LP UV for disinfection of adenovirus when viral inactivation is measured using cell culture infectivity assays; however, UV-induced DNA damage may be repaired during cell culture infectivity assays, and this confounds interpretation of these results. Objectives of this work were to study adenoviral response to both LP and MP UV using (i) standard cell culture infectivity assays and (ii) a PCR assay to directly assess damage to the adenoviral genome without introducing the virus into cell culture. LP and MP UV dose response curves were determined for (i) log inactivation of the virus in cell culture and (ii) UV-induced lesions per kilobase of viral DNA as measured by the PCR assay. Results show that LP and MP UV are equally effective at damaging the genome; MP UV is more effective at inactivating adenovirus in cell culture. This work suggests that the higher disinfection efficacy of MP UV cannot be attributed to a difference in DNA damage induction. These results enhance our understanding of the fundamental mechanisms of UV disinfection of viruses—especially double-stranded DNA viruses that infect humans—and improve the ability of the water treatment community to protect public health.     

Effects of UV Radiation on Photolyase and Implications with Regards to Photoreactivation following Low- and Medium-Pressure UV Disinfection

Photolyase activity following exposure to low-pressure (LP) and medium-pressure (MP) UV lamps was evaluated. MP UV irradiation resulted in a greater reduction in photolyase activity than LP UV radiation. The results suggest that oxidation of the flavin adenine dinucleotide in photolyase may have caused the decrease in activity.     

Comparative studies on photoreactivation of ultraviolet light-induced T4 endonuclease susceptible sites and sister-chromatid exchanges in Potorus cells

Photoreactivation (PR) of T4 endonuclease-susceptible sites (ESS) and sister-chromatid exchanges induced by ultraviolet light was investigated in Potorous tridactylis Pt K2 cells, using monochromatic light from a grating monochromator. Both ESS and SCE showed maximum PR at 350 nm and the action spectra of PR essentially overlapped between ESS and SCE at 350, 400 and 450 nm. Exposure to 325-nm light after UV irradiation induced additional ESS and SCE, but reduction of ESS was shown by increasing exposure to 325-nm light, and further induction of SCE was observed by the same treatment. A possible difference in mechanisms between induction of ESS and SCE is suggested at 325 nm, while similar causes for ESS and SCE, presumably pyrimidine dimers, are suggested by UV (254-nm) irradiation.     

Role of DNA damage in the inactivation of Saccharomyces cerevisiae yeasts by 313-nm ultraviolet light

The relative contribution of respiration photoinhibition and DNA damage in the lethal effect induced by 313 nm ultraviolet light (UV) has been investigated in some strains of the yeast Saccharomyces cerevisiae. It has been shown that cells inactivation is essentially due to photo-induced damage to DNA. By photoreactivation experiments it has been found that dimers of the pyrimidine bases are the main lethal photoproducts induced in the DNA by 313 nm ultraviolet light.