| The extremely radioresistant bacterium, Deinococcus radiodurans (D. radiodurans), is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet radiation, and desiccation. D. radiodurans is capable of surviving 15,000 grays (Gy) of ionizing radiation, whereas doses below 10 Gy are lethal to all other organisms. Studies on mechanisms of radioresistance of D. radiodurans were thought to be the point to breakthrough the ability of organisms to surviving in extreme environment in recent years.D. radiodurans belongs to non-pathogenic gram-positive micrococcus. The ability of radioresistant of Bacillus spp. attribute to the characteristics of spore structure. Considering the critical role of spore in Bacillus spp., the mechanisms of DNA repair and modulation of proteins ought to be further investigated.Transmission electron microscopy studies showed that a membranous framework demarcates the four compartments of a single D. radiodurans cell, but orifices were evident in the internal membranes of both growing and stationary-state cells. Chromatin was present in all compartments, and it adopted a distinctive toroidal shape. DNA labeling revealed that, whereas DNA toroids are present in all compartments of stationary-state bacteria, the nucleoid in one or two compartments of growing cells is dispersed. The mechanisms of radioresistance included several pathway as follows:1.Homologous recombination at the fragment ends: In this way, the strain utilize recombination sequences as template to manipulate the gap DNA. dsDNA damage was mainly repaired by this way. In D. radiodurans, the RecFOR pathway plays a critical role in DNA damage repair due to a deficiency of RecBCD.2.Non-homologous end-joining (NHEJ): One major mechanism of DSB repair in eukaryotes is non-homologous end-joining (NHEJ), in which the two ends of a broken chromosome are rejoined directlyusing only the base-pairing information of the ends themselves to guide ligation. In the early phase after irradiation, the broken fragments maybe repaired by NHEJ.3.Intra- and interchromosomal single-strand annealing: The single strand DNA could be formed by the specific exonucleace at the dsDNA broken site until the occurrence of complement strand DNA. And then the single strand gap was repaired.4.Synthesis dependent strand annealing (SDSA): Recent research has suggested a compelling model for genome restitution in this species, in which the related but more efficient process called synthesis-dependent singlestrand annealing (SDSA) plays a necessary part. During SDSA, the 3'end of a strand derived from a DNA double-strand break invades the homologous region of a sister duplex. The invading 3′end is used to prime DNA synthesis, unwinding the sister duplex and enlarging the D-loop. The displaced strand in the undamaged complex anneals to the remaining free 3'end created by the double-strand break.5.Extended synthesis-dependent strand annealing (ESDSA): How does ESDSA differ from SDSA? Crucially, it requires at least two genome copies that are broken at different positions. Once overlapping fragments have aligned, a single-round multiplex PCR-like step- a variant of PCR that simultaneously amplifies different target sequences by using multiple primer pairs occurs to produce long single-stranded overhangs that anneal accurately to produce reassembled chromosomal segments. Last, RecA-mediated homologous recombination using long intermediates synthesized by DNA-polymerase-I produces full-length chromosomes.In order to investigate the mechanisms of radioresistance of D. radiodurans, our studies were divided into four parts:1.The function of recF in UV-C resistance was characterized by the construction of the recF mutant D. radiodurans R1 strain and the transformation of D. radiodurans recF into E. coli BL21.2.The critical genes of RecFOR pathway were cloned and expressed in E. coli BL21. The purified proteins were further confirmed by Western-blot.3.To investigate the mechanisms of UVC radiation resistance in D. radiodurans, we produced an enhanced UVC-resistant strain (eD. radiodurans) by selective pressure of direct UVC radiation. We then examined proteome changes between the wild-type strain and eD. radiodurans following UVC irradiation using 2-DE and silver-staining. Genes encoding proteins that may play important roles in UVC radiation resistance were further investigated by RT-PCR.4.To investigate the application of gene engineering in D. radiodurans, the levels of antibiotics in Three Gorge Reservoir of Chongqing region were tested. The MIC values were determined by Kirby-Bauer disc diffusion method and VITEK-II. In addition and the frequency of transformation were analyzed by chemical transformation and electro transformation with pET32a(+).Materials and methods1.recF mutant constructionThe kanamycin resistance gene was obtained from the pET30b(+) plasmid using primers K1 and K2 (containing BamHI and PstI restriction sites) and ligated to groEL promoter which was obtained from the D. radiodurans genome using primers G1 and G2 (containing KpnI and BamHI restriction sites). A 994-bp DNA fragment corresponding to the immediate upstream portion of the dr1089 (recF D. radiodurans) initial codon was amplified using primers MF1 and MF2 (containing EcoRI and KpnI restriction sites), and a 1063-bp DNA fragment corresponding to the immediate downstream region of the dr1089(recF D. radiodurans) stop codon was amplified using primers MF3 and MF4 (containing PstI and HindIII restriction sites). The PCR products were digested with their respective enzymes listed above and then ligated to a groEL promoter-kanamycin cassette. The tripartite ligation products were amplified using L1 and R2, and the resulting fragments were ligated to pUC18 that had been digested with EcoRI and HindIII. The fragments were then transformed into D. radiodurans R1 as follows: 2 ml mid-exponential phase cultures were harvested at room temperature by successive 30 sec. centrifugations at 16,000×g, resuspended in 0.6 ml water at 4oC, and placed on ice; the cells were then centrifuged for 30 sec. at 4oC, washed once more in 0.6 ml water on ice, washed once in 0.3 ml 10% (v/v) glycerol on ice, and resuspended in 50μL 10% glycerol. The recF::GK mutant strain was selected on TGYF (0.5% tryptone, 0.1% glucose, 0.3% yeast extract, and 15 mM fructose) agar containing 10μg/ml kanamycin.2.UV-C irradiation experimentsD. radiodurans, recF-mutant strain, E.coli-recF, and E.coli BL21 cells were grown to early stationary phase and used for determining cell survival rate following UV-C irradiation (254nm). About 108 CFU cells of D. radiodurans, recF-mutant strain and E. coli BL21were used to UV-C treatment. These cells were washed twice with phosphate buffered saline (PBS) containing 0.02% Triton X-100 to ensure the separation of individual CFU and then irradiated in phosphate suspension with a mercury low pressure lamp (Shenxing Company, Jiangyin, Jiangsu, China) at a rate of 1.0J/m2/s with a main emission line of 254 nm. During irradiation, the strain suspension was stirred continuously to ensure homogeneous exposure at room temperature. The spectral irradiance was monitored by use of a UV Light Meter (TAINA, TN-2254). After UV-C radiation at defined fluences, 100μl from the aqueous suspension was taken for further analysis. Survival was determined from appropriate dilutions in PBS as colony forming ability (CFA) after cells were plated in TGY (D. radiodurans and recF-mutant strain) or LB (E. coli BL21and E.coli-recF) and incubated in the dark overnight at 30°C (D. radiodurans and recF-mutant strain) or 37°C (E. coli BL21 and E.coli-recF) for 2 to 4 days.3.Numerical and statistical analysisThe surviving fractions of these cells were determined from the quotient N/N0, with N = colony formers after UV-C irradiation and N0 = colony formers without UV-C irradiation (i.e. the non-treated controls). Inactivation curves were obtained by plotting the logarithm of survival versus the applied UV fluence. The characteristics of the survival curves F10, extrapolation number, and inactivation constant IC, were determined according to Moeller. All experiments were repeated at least three times.4.2-DE.Samples were prepared as described for the comparison of the UVC resistance between D. radiodurans and eD.radiodurans. Approximately 150μg of protein was loaded onto an IPG strip (pH 4-7, 18 cm) and subjected to isoelectric focusing in IPGphor. The treated strips were subjected to 12% SDS-PAGE in the Ettan DALT II System for secondary electrophoresis. The proteins on the 2-DE gels were visualized using silver staining. All 2-DE gels were scanned with Image Scanner. Mass spectra of peptides were obtained using an Ultraflex MALDI- TOF MS.5.Semi-quantitative RT-PCRFor semi-quantitative RT-PCR analysis, 16S rRNA which has relatively constant transcription levels in bacteria, was chosen as an internal control. cDNA from each sample was amplified with primers for 16S rRNA, and the cDNA samples were normalized by differential dilutions according to the quantity of 16S rRNA product. Selected genes were amplified from normalized cDNA samples with specific primers. The PCR products were analyzed on a 1.2% agarose gel and visualized with ethidium bromide staining.6.Antibiotics analytical methods Samples from 4 hospitals, 1 nursery, 1 slaughter house, 1 wastewater treatment plant and source water samples of Chongqing region of Three Gorge Reservoir were analyzed for macrolide, lincosamide, trimethoprim, fluorouinolone, sulfonamide and tetracycline antibiotics by on-line solid-phase extraction and liquid chromatography- tandem mass spectrometry.7.Antibiotic resistance profies of D. radioduransAntibiotic susceptibility tests (AST) were performed with Kirby-Bauer disc diffusion method recommended by CLSI (2009) and VITEK-II.8.The optimality condition of electroporationTo study the optimality condition of electrporation, different levels of pRADZ3 were transformed into D. radiodurans through electroporation and chemical transformation.Results and Discussion1.A DNA fragment containing the D. radiodurans groEL gene promoter and a kanamycin resistance gene fusion was cloned by PCR amplification and used to displace the recF gene in the wild-type strain R1. The method to construct a mutant strain were established and we could performed further investigation on other function genes by this way.2.recF is needed for the replication of D. radiodurans. Furthermore, as a part of the RecFOR pathway in D. radiodurans, disruption of recF could dramatically decrease the UV-C resistance of D.radiodurans and recF could increase the UV-C resistance of E.coli BL21 cells.3.These purified proteins of RecFOR pathway were got by clone and expression in E. coli BL21 by pET32a(+) vector. We could study the interaction of these proteins in vitro. Furthermore, we could get the antibody of these proteins by animal immune and detect the expression levels of the proteins by Western-blot.4.We generated an extremely UVC-radioresistant strain of D. radiodurans (eD. radiodurans) by directed evolution using UVC radiation. We looked for proteins differentially expressed between D. radiodurans and eD. radiodurans following UVC- irradiation using two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver-staining. The eD. radiodurans strain showed 1.64-fold more UVC resistance than wild-type strains. Furthermore, the expression levels of 25 protein spots showed significant changes under UVC radiation stress. Of these spots, 14 (including 10 up-regulated and 4 down-regulated proteins) were identified with peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ ionizationtime of flight mass spectrometry (MALDI- TOF MS) after tryptic in-gel digestion. These proteins are involved in various cellular functions, including 1) transcription, 2) photosynthetic reactions, 3) nucleotide/peptide binding, 4) PRM (protein turnover), 5) protein against oxidative and desiccation stress, 6) ion transport, 7) carbohydrate metabolism, and 8)proteolysis enzymes. Maf protein, v-type ATP synthase, amino acetyltransferase, TerD, DR1172 (LEA76 family desiccation resistance protein) and DR0392 were detected in eD. radiodurans but not in D. radiodurans following UVC radiation. Most of these proteins have not previously been reported to be relevant to UVC resistance. eD. radiodurans also had higher levels of Yellow-related protein, carbohydrate kinase, serine protease and DR0972. Maf protein, Yellow-related protein, TerD and a LEA76 family desiccation resistance protein were found to be involved in UVC resistance in D.radiodurans. Genes encoding proteins that may play important roles in UVC radiation resistance were further investigated by RT-PCR. These data provide an in-depth analysis of the response of the D. radiodurans proteome to UVC resistance, and they reveal protein alterations that are associated with UVC resistance. TerD may play an important protective role against oxidation and desiccation to ensure the repair of general stress-induced damage. LEA76 family desiccation resistance protein ) in eD.radiodurans following UVC radiation indicates a potential relationship between UV radiation resistance and desiccation resistance. Considering the role of yellow locus in Drosophila and the up-regulation of Yellow-related protein in D. radiodurans, this protein could function in the regeneration process, or it maybe involved in regulation of carotenoid expression. In D. radiodurans, maf may have critical roles in various cellular processes, including stress signaling, regulation of transcription and cell proliferation.5.The concentration of antibiotics in water environment of Chongqing was far below the antibiotic resistance of D.radiodurans. pRADZ3 could be transformed between E. coli and D. radiodurans. The optimality condition of electroporation was 2.2 kV, 25μF, 200 ?,2μg/ml vector. Point of innovation1.Metholds1.1 Methold of homologous recombinant was applied to investigate the construction of gene mutant strain. pHMR186 vector was used to construct the mutant D. radiodurans. Nucleotide fragment which contained the homologous arm was transformed into receipt strain directly to construct gene mutant strain. In this study, homologous arm was lingaged into pUC18 and the positive strain could be got on the antibiotic resistant plate. The positive strains were further confirmed by PCR.1.2 Until now, plate process was usually used to test the UV resistant ability of strain, but agar surfaces have pits and fissures at the microscopic level. Bacteria inside these microcrevices will be exposed to less, or even no, UV and, hence, appear to be resistant. If one bacterium in a thousand was shielded in this manner, it could limit the linear portion of the survival curve to the -3 log level after which tailing would begin. To avoid these surface effects, many researchers irradiated the bacteria in dilute solutions that were vigorously stirred.2.ResultsThe critical role of recF in UV resistance of D. radiodurans was the first time to reported. recF could induced growth defect of D. radiodurans. Maf protein, Yellow-related protein, TerD and a LEA76 family desiccation resistance protein were found to be involved in UVC resistance in D.radiodurans. this was the first time to report the antibiotics levels in Three Gorge Reservoir of Chongqing region and the antibiotic resistance profiles of D. radiodurans.3.ApplicationThe transformation condition of pRADZ3 was optimized and the antibiotics levels in aquatic environment was far from inhibition of D. radiodurans surviving.Shortcoming1.If we used shuttle vector which comprised recF gene to complement the deficient of recF in recF mutant strain, it would be more convincing than current results to elucidate the role of recF in UV resistance. 2.RecF, RecO, RecA expressed by pET32a(+) belongs to inclusion body. To get the active proteins, the incubation time, incubation temperature and concentration of IPTG ought ot optimized. If the process described above has negative effect on inclusion body, the expression vector should be changed.3.The category of antibiotics selected in water environment wasn't coincidence with antibiotics selected in antibiotic resistance profiles restricted by detection condition.4.We haven't cloned functional gene into D. radiodurans and this research ought to be finished in the future.Project in the future1.The specific mechanism ought to be investigate considering the critical role of RecFOR pathway in UV resistance of D. radiodurans. Several matter which include ionizing radiation, UV radiation and oxidation damage could incure single-strand break and double-strand break. Which damage status could this repair pathway favored to? Which terminal did this repair action started?2.If we could testify the critical role in UV resistance of DR1172, we could deduce the origin of radiation resistance of D.radiodurans.3.Energy metabolism played an important role in replication, surviving and apoptosis. In cells treated with g radiation, the levels of cyclic AMP (cAMP) and ATP increased rapidly by differentially regulating adenylyl cyclase (AC) and cAMP phosphodiesterase. In this study, energy metabolism related proteins exhibit high expression after UV treatment. Further study may included: 1) The relationship between energy metabolism and phosphorylation of proteins; 2) Is the recovery of proteins following radiation related to energy metabolism? If we could verify the relationship between energy metabolism and phosphorylation of proteins, we could explore a newly method or drug to prevente patient from radiation damage after radiotherapy.
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