Photoreactivation Research Articles

Impact of 280 nm UV-C LEDs on the microbial safety and nutritional quality in tender coconut water

The efficiency and efficacy of 280 nm UV-C LEDs for processing Tender coconut water (TCW) have been investigated. TCW inoculated with E. coli, and L. monocytogenes were irradiated using collimated beam apparatus at a dose of 71 mJ/cm2 and 20.3 mJ/cm2 for ≥ 5 log CFU/mL reduction, resulting in the double Weibull model for E. coli with a 4D value of 61 ± 0.83 mJ/cm2 and the Weibull model with a 4D value of 13 ± 0.2 mJ/cm2 for L. monocytogenes. The irradiated samples were exposed to photoreactivation (365 nm) light for up to 120 min. PR (Photoreactivation) was eventually not observed for E. coli, and L. monocytogenes showed no repair. Scanning Electron Microscope results revealed that, unlike the control sample, 280 nm irradiation results in cell lysis, wrinkle, and distortion. UV treatment did not affect the pH, TA, Brix, total phenolics content, mineral content, and sugars of TCW but increased in a* and b* colour value, and ascorbic acid concentration reduced to 18.2% of its original concentration. Sensory evaluation studies reported that control samples scored high for freshness and pleasantness, and attributes like astringency, fermented and cooked aroma have been scored low. Overall, the quality of UV-treated samples has been preferred by panellists.

Ultrafast repair dynamics of CPD photolesion isosteres by DNA photolyase.

UV-B rays from sunlight can cause damage to DNA by inducing photolesions, two major forms being the cyclobutane pyrimidine dimer (CPD) and a less frequently formed 6-4 pyrimidine pyrimidone (64PP) photoproduct. Photolyases (PLs) are flavoenzymes which identify these photolesions and repair them using blue light. CPD PLs utilize 10 ultrafast elementary steps, including 7 electron transfer (ET) steps, to complete the photorepair cycle and modulate the quantum repair yield1. This can be influenced by the interplay between various molecular factors at the active site: amino acid residues, the bound substrate, solvation activity and plasticity/flexibility. Here, we have utilized a combination of steady-state kinetic studies, ultrafast laser spectroscopy and molecular dynamics simulations to approach the role of chemical properties of the substrate and binding configuration. We have measured the binding constants, quantum repair yields and ultrafast reaction rates of each elementary step involved for photorepair of 3 novel CPD isosteres (CPD1 and CPD2: 5’-Thymine<>Toluene-3’ adducts2; CPD3: T<>T dimer with 5’ pyranose sugar instead of furanose) by EcPL. We have found that EcPL can repair all 3 isosteres, despite weaker binding, albeit with lower repair yields. The enzyme utilizes universally conserved pathways to repair the isosteres. The binding configuration and redox properties of the substrate may play a direct role in modulating the reaction rates of intermolecular ET and intramolecular bond splitting steps in the repair photocycle, which in turn determines their overall repair quantum yield.

The crystal structure of Vibrio cholerae (6-4) photolyase reveals interactions with cofactors and a DNA-binding region

Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/β domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors.

Transcriptome Analysis Reveals Candidate Genes Involved in Light-Induced Primordium Differentiation in Pleurotus eryngii.

Pleurotus eryngii, a highly valued edible fungus, is one of the major commercially cultivated mushrooms in China. The development of P. eryngii, especially during the stage of primordium differentiation, is easily affected by light. However, the molecular mechanism underlying the response of primordium differentiation to light remains unknown. In the present study, primordium expression profiles under blue-light stimulation, red-light stimulation, and exposure to darkness were compared using high-throughput sequencing. A total of 16,321 differentially expressed genes (DEGs) were identified from three comparisons. GO enrichment analysis showed that a large number of DEGs were related to light stimulation and amino acid biosynthesis. KEGG analyses demonstrated that the MAPK signaling pathway, oxidative phosphorylation pathway, and RNA transport were most active during primordium differentiation. Furthermore, it was predicted that the blue-light photoreceptor WC-1 and Deoxyribodipyrimidine photolyase PHR play important roles in the primordium differentiation of P. eryngii. Taken together, the results of this study provide a speculative mechanism that light induces primordium differentiation and a foundation for further research on fruiting body development in P. eryngii.

Detection and analysis of UV-induced mutations in the chromosomal DNA of Arabidopsis

Under natural conditions, plants are exposed to solar ultraviolet (UV) radiation, which damages chromosomal DNA. Although plant responses to UV-induced DNA damage have recently been elucidated in detail, revealing a set of DNA repair mechanisms and translesion synthesis (TLS), limited information is currently available on UV-induced mutations in plants. We previously reported the development of a supF-based system for the detection of a broad spectrum of mutations in the chromosomal DNA of Arabidopsis. In the present study, we used this system to investigate UV-induced mutations in plants. The irradiation of supF-transgenic plants with UV-C (500 and 1000 J/m2) significantly increased mutation frequencies (26- and 45-fold, respectively). G:C to A:T transitions (43–67% of base substitutions) dominated in the mutation spectrum and were distributed throughout single, tandem, and multiple base substitutions. Most of these mutations became undetectable with the subsequent illumination of UV-irradiated plants with white light for photoreactivation (PR). These results indicated that not only G:C to A:T single base substitutions, but also tandem and multiple base substitutions were caused by two major UV-induced photoproducts, cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). In contrast, a high proportion of A:T to T:A transversions (56% of base substitutions) was a characteristic feature of the mutation spectrum obtained from photoreactivated plants. These results define the presence of the characteristic feature of UV-induced mutations, and provide insights into DNA repair mechanisms in plants.

The rice germin-like protein OsGLP1 participates in acclimation to UV-B radiation.

Exposure to ultraviolet B radiation (UV-B) stress can have serious effects on the growth and development of plants. Germin-like proteins (GLPs) may be involved in different abiotic and biotic stress responses in different plants, but little is known about the role of GLPs in UV-B stress response and acclimation in plants. In the present study, knockout of GLP 8-14 (OsGLP1) using the CRISPR/Cas9 system resulted in mutant rice (Oryza sativa L.) plants (herein called glp1) that exhibited UV-B-dependent formation of lesion mimic in leaves. Moreover, glp1 grown under solar radiation (including UV-B) showed decreased plant height and increased leaf angle, but we observed no significant differences in phenotypes between wild-type (WT) plants and glp1 grown under artificial light lacking UV-B. Fv/Fm, Y (II) and the expression of many genes, based on RNA-seq analysis, related to photosynthesis were also only reduced in glp1, but not in WT, after transfer from a growth cabinet illuminated with artificial white light lacking UV-B to growth under natural sunlight. The genes-associated with flavonoid metabolism as well as UV resistance locus 8 (OsUVR8), phytochrome interacting factor-like 15-like (OsPIF3), pyridoxal 5'-phosphate synthase subunit PDX1.2 (OsPDX1.2), deoxyribodipyrimidine photolyase (OsPHR), and deoxyribodipyrimidine photolyase family protein-like (OsPHRL) exhibited lower expression levels, while higher expression levels of mitogen-activated protein kinase 5-like (OsMPK3), mitogen-activated protein kinase 13-like (OsMPK13), and transcription factor MYB4-like (OsMYB4) were observed in glp1 than in WT after transfer from a growth cabinet illuminated with artificial white light to growth under natural sunlight. Therefore, mutations in OsGLP1 resulted in rice plants more sensitive to UV-B and reduced expression of some genes for UV-B protection, suggesting that OsGLP1 is involved in acclimation to UV-B radiation.

Photocatalytic inactivation of airborne bacteria in a polyurethane foam reactor loaded with a hybrid of MXene and anatase TiO2 exposing {0 0 1} facets

A hybrid of TiO2 exposing {001} facets and monolayer Ti3C2Tx nanosheet (MXene) was synthesized, characterized and used as a photocatalyst in this study. The introduction of MXene (3.4 wt%) helped to reduce the recombination of photo-induced electrons and holes, and thus enhanced the photocatalytic activity by 30%. A continuous flow-through reactor loaded with the as-prepared photocatalyst coated onto polyurethane foam was developed to inactivate airborne bacteria. The photocatalytic inactivation efficiency of airborne Escherichia coli (E. coli) achieved 3.4 lg order under ultraviolet (UV) irradiation at 254 (UV254), which was superior to that using UV254-only treatment with 2.5 lg order under the same operating condition (95% relative humidity and retention time of 4.27 s). The effect of humidity and bacteria species on inactivation performance was also investigated. The thick cell membrane could protect bacteria from photocatalytic oxidation while high humidity increased the photocatalytic inactivation efficiency by generating more reactive oxygen species. The phenomena of photo reactivation and dark repair of airborne E. coli using UV254-only treatment was observed. However, no reactivation occurred after UV photocatalytic inactivation, and even a continuous decline under visible light. These results suggested a different inactivation mechanism between UV irradiation and UV photocatalysis that the former inactivated bacteria by damaging their DNA, whereas photocatalysis physically damaged their cell structure.

Adaptative transcriptional response of Dietzia cinnamea P4 strain to sunlight simulator.

Responses to sunlight exposure of the oil-degrading Dietzia cinnamea P4 strain were evaluated by transcriptional levels of SOS genes, photoreactivation and genes involved in tolerance to high levels of reactive oxygen species. The P4 strain was exposed for 1 and 2h and the magnitude of level changes in the mRNA was evaluated by qPCR. The results described the activation of the SOS system, with the decline of the repressor lexA gene levels and the concomitant increase of recA and uvrAD genes levels. The genes that participate in the photoreactivation process were also responsive to sunlight. The phrB gene encoding deoxyribodipyrimidine photo-lyase had its expression increased after 1-h exposure, while the phytAB genes showed a progressive increase over the studied period. The protective genes against reactive oxygen species, catalases, superoxides, peroxidases, and thioredoxins, had their expression rates detected under the conditions validated in this study. These results show a fast and coordinated response of genes from different DNA repair and tolerance mechanisms employed by strain P4, suggesting a complex concerted protective action against environmental stressors.

Theoretical insights into the DNA repair function of Arabidopsis thaliana cryptochrome-DASH.

Following the discovery of cryptochrome-DASH (CRYD) as a new type of blue-light receptor cryptochrome, theoretical and experimental findings on CRYD have been reported. Early studies identified CRYD as highly homologous to the DNA repair enzyme photolyases (PLs), suggesting the involvement of CRYD in DNA repair. However, an experimental study reported that CRYD does not exhibit DNA repair activity in vivo. Successful PL-mediated DNA repair requires: (i) the recognition of UV-induced DNA lesions and (ii) an electron transfer reaction. If either of them is inefficient, the DNA repair activity will be low.To elucidate the functional differences between CRYD and PL, we theoretically investigated the electron transfer reactivity and DNA binding affinity of CRYD and also performed supplementary experiments. The average electronic coupling matrix elements value for Arabidopsis thaliana CRYD (AtCRYD) was estimated to be 5.3 meV, comparable to that of Anacystis nidulans cyclobutane pyrimidine dimer PLs (AnPL) at 4.5 meV, indicating similar electron transfer reactivities. We also confirmed the DNA repair activity of AtCRYD for UV-damaged single-stranded DNA by the experimental analysis. In addition, we investigated the dynamic behavior of AtCRYD and AnPL in complex with double-stranded DNA using molecular dynamics simulations and observed the formation of a transient salt bridge between protein and DNA in AtCRYD, in contrast to AnPL in which it was formed stably. We suggested that the instability of the salt bridge between protein and DNA will lead to reduced DNA binding affinity for AtCRYD.

Structural changes within the bifunctional cryptochrome/photolyase CraCRY upon blue light excitation

Cryptochromes (CRYs) are an ubiquitously occurring class of photoreceptors, which are important for regulating the circadian rhythm of animals via a time-delayed transcription-translation feedback loop (TTFL). Due to their protein architecture and common FAD chromophore, they belong to the same superfamily as photolyases (PHLs), an enzyme class that repairs UV-induced DNA lesions upon blue light absorption. Apart from their different functions the only prominent structural difference between CRY and PHL is the highly variable C-terminal extension (CTE) of the former. The nature of the CTE is still unclear and highly speculated. In this study, we show by hydrogen/deuterium exchange and subsequent mass-spectrometric analysis that the CTE of the animal-like cryptochrome from the green algae Chlamydomonas reinhardtii (CraCRY) binds to the surface of the photolyase homology region, which flanks the DNA binding site. We also compared the fully oxidized and fully reduced states of the flavoprotein and designed a tool, so called light chamber, for automated HDX-MS measurements of photoreceptors in defined photostates. We could observe some striking differences between the two photostates and propose a model for light-dependent switching of this bifunctional cryptochrome.

Implications of a Water Molecule for Photoactivation of Plant (6–4) Photolyase

Photolyases (PLs) are flavoproteins able to repair cross-links formed between adjacent pyrimidine bases in DNA in a light-dependent manner via an electron transfer. The catalytically active redox state of the flavin chromophore for the DNA repair is a fully reduced form of flavin adenine dinucleotide (FADH-). PLs and their relative, cryptochromes (CRYs), share a physicochemical process attributable to the light-dependent reduction of the chromophore via an ultrafast successive electron transfer through exclusively conserved three tryptophan side chains. In some (6-4) PLs and animal CRYs, an additional tryptophan participates in this photoactivation process. In a search for the intrinsic difference between the Trp triad and tetrad, a water molecule proximal to the second and third Trp was found in the reported crystal structure of Arabidopsis thaliana (6-4) PL. Here, we investigated the involvement of the water molecule in photoactivation. Molecular dynamics simulations indicated that the water molecule is stably captured in the binding site, while mutation of S412 increased water displacement from the binding site. Photochemical analysis of recombinant proteins revealed that the S412A mutation significantly decelerated the FAD photoreduction as compared to the wild type. The hydrogen-bonding network including the water molecule would play a key role in the stabilization of the FAD-Trp radical pair.

Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair.

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.

What accounts for the different functions in photolyases and cryptochromes: a computational study of proton transfers to FAD.

Photolyases (PL) and cryptochromes (CRY) are light-sensitive flavoproteins, respectively, involved in DNA repair and signal transduction. Their activation is triggered by an electron transfer process, which partially or fully reduces the photo-activated FAD cofactor. The full reduction additionally requires a proton transfer to the isoalloxazine ring. In plant CRY, an efficient proton transfer takes place within several μs, enabled by a conserved aspartate working as a proton donor, whereas in E. coli PL a proton transfer occurs in the 4 s timescale without any obvious proton donor, indicating the presence of a long-range proton transfer pathway. Unexpectedly, the insertion of an aspartate as a proton donor in a suitable position for proton transfer in E. coli PL does not initiate a transfer process similar to plant CRY, but even prevents the formation of a protonated FAD. In the present work, thanks to a combination of classical molecular dynamics and state-of-the-art DFTB3/MM simulations, we identify a proton transfer pathway from bulk to FAD in E. coli PL associated with a free energy profile in agreement with the experimental kinetics data. The free energy profiles of the proton transfer between aspartate and FAD show an inversion of the driving force between plant CRY and E. coli PL mutants. In the latter, the proton transfer from the aspartate is faster than in plant CRY but also thermodynamically disfavoured, in agreement with the experimental data. Our results further illustrate the fine tuning of the electrostatic FAD environment and the adaptability of the FAD pocket to ensure the divergent functions of the members of the PL-CRY family.

A Review of Spectroscopic and Biophysical-Chemical Studies of the Complex of Cyclobutane Pyrimidine Dimer Photolyase and Cryptochrome DASH with Substrate DNA.

Cyclobutane pyrimidine dimer (CPD) photolyase (PL) is a structure-specific DNA repair enzyme that uses blue light to repair CPD on DNA. Cryptochrome (CRY) DASH enzymes use blue light for the repair of CPD lesions on single-stranded (ss) DNA, although some may also repair these lesions on double-stranded (ds) DNA. In addition, CRY DASH may be involved in blue light signaling, similar to cryptochromes. The focus of this review is on spectroscopic and biophysical-chemical experiments of the enzyme-substrate complex that have contributed to a more detailed understanding of all the aspects of the CPD repair mechanism of CPD photolyase and CRY DASH. This will be performed in the backdrop of the available X-ray crystal structures of these enzymes bound to a CPD-like lesion. These structures helped to confirm conclusions that were drawn earlier from spectroscopic and biophysical-chemical experiments, and they have a critical function as a framework to design new experiments and to interpret new experimental data. This review will show the important synergy between X-ray crystallography and spectroscopic/biophysical-chemical investigations that is essential to obtain a sufficiently detailed picture of the overall mechanism of CPD photolyases and CRY DASH proteins.

An Ethenoadenine FAD Analog Accelerates UV Dimer Repair by DNA Photolyase.

Reduced anionic flavin adenine dinucleotide (FADH- ) is the critical cofactor in DNA photolyase (PL) for the repair of cyclobutane pyrimidine dimers (CPD) in UV-damaged DNA. The initial step involves photoinduced electron transfer from *FADH- to the CPD. The adenine (Ade) moiety is nearly stacked with the flavin ring, an unusual conformation compared to other FAD-dependent proteins. The role of this proximity has not been unequivocally elucidated. Some studies suggest that Ade is a radical intermediate, but others conclude that Ade modulates the electron transfer rate constant (kET ) through superexchange. No study has succeeded in removing or modifying this Ade to test these hypotheses. Here, FAD analogs containing either an ethano- or etheno-bridged Ade between the AN1 and AN6 atoms (e-FAD and ε-FAD, respectively) were used to reconstitute apo-PL, giving e-PL and ε-PL respectively. The reconstitution yield of e-PL was very poor, suggesting that the hydrophobicity of the ethano group prevented its uptake, while ε-PL showed 50% reconstitution yield. The substrate binding constants for ε-PL and rPL were identical. ε-PL showed a 15% higher steady-state repair yield compared to FAD-reconstituted photolyase (rPL). The acceleration of repair in ε-PL is discussed in terms of an ε-Ade radical intermediate vs superexchange mechanism.

Methyl-CpG binding domain protein acts to regulate the repair of cyclobutane pyrimidine dimers on rice DNA.

UVB radiation causes cyclobutane pyrimidine dimers (CPDs) to form on the DNA of living organisms. This study found that overexpression of the silicon absorbance gene Lsi1 reduced the accumulation of CPDs in rice, which profited from the reactivation by photolyase. The transcript abundance of deoxyribodipyrimidine photolyase (Os10g0167600) was generally correlated with the silicon content of the rice, and the up-regulation of Os10g0167600 was found to be highest in the UVB-treated Lsi1-overexpressed (Lsi1-OX) rice. A trans-acting factor, methyl-CpG binding domain protein (OsMeCP), was found to interact with the cis-element of Os10g0167600. The nucleic location of OsMeCP effectively enabled the transcriptional regulation. Compared with the WT, the level of OsMeCP was lower in the Lsi1-OX rice but higher in the Lsi1-RNAi line. Rice cultured in a high silicate-concentration solution also exhibited less OsMeCP abundance. Overexpression of OsMeCP led to lower Os10g0167600 transcript levels and a higher CPD content than in the WT, but the reverse was true in the OsMeCP-RNAi line. These findings indicate that OsMeCP acts as a negative regulator of silicon, and can mediate the repression of the transcription from Os10g0167600, which inhibits the photoreactivation of the photolyase involved in the repair of CPDs.

Low-steady-state metabolism induced by elevated CO2 increases resilience to UV radiation in the unicellular green-algae Dunaliella tertiolecta

Global change factors derived from anthropogenic activities such as increased CO2 and ultraviolet radiation (UVR) have direct impacts on the physiological responses of organisms in both terrestrial and aquatic ecosystems. We assessed in this work the mechanistic, physiological and molecular response of the unicellular chlorophyte Dunaliella tertiolecta exposed to combinatorial present (390 ppmv, LC) and future CO2 levels predicted for the year 2100 (900 ppmv, HC) and different UVR doses. Growth rates, cell esterase activity, reactive oxygen species accumulation (ROS), and 14C fixation decreased under HC compared to LC. The deleterious effects of UVR were attenuated in HC, except for decreased photosynthetic capacities. Transcriptome analysis showed different expression patterns depending on UVR and CO2 levels. PsbA, LhcII and eCA gene expression was downregulated at HC compared to LC. However, photolyase (PL) gene expression was upregulated at HC with respect LC in UVR. This suggests that HC (as opposed to LC) promoted a “low-metabolic steady state”, decreasing UVR exposure-activated repair by means of protein replacement. Only PL photoreactivation was active under HC. Our data add evidence on D. tertiolecta possibly becoming more resilient to UVR exposure under future CO2 regimes, and maybe other marine unicellular chlorophytes, warranting serious considerations on the consequent ecological implications.

Photochemistry of Wild-Type and N378D Mutant E. coli DNA Photolyase with Oxidized FAD Cofactor Studied by Transient Absorption Spectroscopy.

DNA photolyases (PLs) and evolutionarily related cryptochrome (CRY) blue-light receptors form a widespread superfamily of flavoproteins involved in DNA photorepair and signaling functions. They share a flavin adenine dinucleotide (FAD) cofactor and an electron-transfer (ET) chain composed typically of three tryptophan residues that connect the flavin to the protein surface. Four redox states of FAD are relevant for the various functions of PLs and CRYs: fully reduced FADH(-) (required for DNA photorepair), fully oxidized FADox (blue-light-absorbing dark state of CRYs), and the two semireduced radical states FAD(.-) and FADH(.) formed in ET reactions. The PL of Escherichia coli (EcPL) has been studied for a long time and is often used as a reference system; however, EcPL containing FADox has so far not been investigated on all relevant timescales. Herein, a detailed transient absorption study of EcPL on timescales from nanoseconds to seconds after excitation of FADox is presented. Wild-type EcPL and its N378D mutant, in which the asparagine facing the N5 of the FAD isoalloxazine is replaced by aspartic acid, known to protonate FAD(.-) (formed by ET from the tryptophan chain) in plant CRYs in about 1.5 μs, are characterized. Surprisingly, the mutant protein does not show this protonation. Instead, FAD(.-) is converted in 3.3 μs into a state with spectral features that are different from both FADH(.) and FAD(.-) . Such a conversion does not occur in wild-type EcPL. The chemical nature and formation mechanism of the atypical FAD radical in N378D mutant EcPL are discussed.

Extremophile DNA Photolyases: DNA Repair under Extreme Conditions

Adaptation to extreme environments is a seminal characteristic of life on Earth. Nowhere is this property more strongly evident than in single-celled organisms. Bacteria, archaea, and eukaryotes all have representatives that thrive in cold or hot environments. Many of these extremophile enzymes share significant amino acid sequence homology in spite of very different optimal growth temperatures (TG). Based on a simple amino acid sequence analysis it is unclear what adaptive changes are required to tailor homologous enzymes to function in extreme environments.A case in point is DNA photolyase (PL), a monomeric flavoprotein that binds UV-damaged DNA and repairs it by blue light activated picosecond electron transfer from a conserved flavin adenine dinucleotide cofactor (FADH¯) to the tightly bound cyclobutylpyrimidine dimer DNA lesion. These enzymes also include a second light-harvesting cofactor. Here we present a comparative analysis of three recombinant CPD photolyases, hyperthermophilic Sulfolobus solfataricus (TG=353K, rSsPL), mesophilic E. coli (TG=310K, rEcPL), and psychrophilic Colwellia psychrerythraea (TG=281K, rCpPL).All PLs utilize FADH¯ bound in a highly conserved site for repair. In addition, each PL demonstrates different properties for its 2nd cofactor. We used a variety of biochemical, physical, and molecular biological tools to compare these extremophile proteins with regard to repair yield, cofactor reduction potential and excited state properties. The difference in the stability of the redox state of the purified protein suggests different structural adaptations of each PL to their respective thermal environments. Denaturation studies reveal that rSsPL has a very stable structure, whereas rCpPL is extremely sensitive to its thermal and aqueous environments. A comparison of cofactor absorption and emission spectra reveal significant differences in how the flavin cofactor is bound in the binding pocket, in spite of significant amino acid sequence homology for this cavity across all proteins.

Disinfection of biologically treated wastewater and prevention of biofouling by UV/electrolysis hybrid technology: Influence factors and limits for domestic wastewater reuse

Reuse of wastewater contributes significantly to an efficient and sustainable water usage. However, due to the presence of a multitude of pathogens (e.g. bacteria, viruses, worms, protozoa) in secondary effluents, disinfection procedures are indispensable. In decentralized wastewater treatment, UV irradiation represents one of the most common disinfection methods in addition to membrane processes and to a certain extent electrochemical procedures. However, the usage of UV disinfected secondary effluents for domestic (sanitary) or irrigation purposes bears a potential health risk due to the possible photo and dark repair of reversibly damaged bacteria.Against this background, the application of the UV/electrolysis hybrid technology for disinfection and prevention of bacterial reactivation in biologically treated wastewater was investigated in view of relevant influence factors and operating limits. Furthermore, the influence of electrochemically generated total oxidants on the formation of biofilms on quartz glass surfaces was examined, since its preventive avoidance contributes to an enhanced operational safety of the hybrid reactor.It was found that reactivation of bacteria in UV irradiated, biologically treated wastewater can be prevented by electrochemically produced total oxidants. In this regard, the influence of the initial concentration of the microbiological indicator organism Escherichia coli (E. coli) (9.3*102–2.2*105 per 100 mL) and the influence of total suspended solids (TSS) in the range of 11–75 mg L−1 was examined. The concentration of total oxidants necessary for prevention of bacterial regrowth increases linearly with the initial E. coli and TSS concentration. At an initial concentration of 933 E. coli per 100 mL, a total oxidants concentration of 0.4 mg L−1 is necessary to avoid photo reactivation (at 4200 Lux), whereas 0.67 mg L−1 is required if the E. coli concentration is enhanced by 2.4 log levels (cTSS = constant = 13 mg L−1). The prevention of dark repair is ensured with 25–50% lower concentration of total oxidants. An increase of the TSS concentration from 11 mg L−1 to 75 mg L−1 leads to a triplication of the need of total oxidants from 0.6 mg L−1 to 1.8 mg L−1 (3*105E. coli per 100 mL).The energy consumption of the hybrid reactor varies from 0.17 kWh m−3 to 0.94 kWh m−3 depending on the TSS concentration (11–75 mg L−1).Furthermore, biofilm formation on quartz glass surfaces, of which the sleeves of UV lamps consist, can be suppressed by electrochemically produced total oxidants at a concentration of at least 1 mg L−1 which ensures high operational safety of the hybrid reactor combined with large maintenance intervals.