Extreme Radioresistance Research Articles

From Ashes to Life - The Indestructible D. radiodurans

Deinococcus radiodurans (D. radiodurans) was accidentally discovered in 1956 when cans of ground meat were exposed to massive doses of ionizing gamma radiation, intended to kill dangerous bacteria. The bacterium can survive doses of radiation, even up to 1,000 times that which is deadly to humans. Among biologists and biophysicists, D. radiodurans is often humorously called “Conan the Bacterium.” This extreme radioresistance of the bacterium has been attributed to its ability to protect the proteome from ROS, which originates from water radiolysis, and also to carry out the effective repair of a large amount of DNA damage.

ITRAQ-based proteomic analysis of Deinococcus radiodurans in response to 12C6+ heavy ion irradiation

BackgroundDeinococcus radiodurans (D. radiodurans) is best known for its extreme resistance to diverse environmental stress factors, including ionizing radiation (IR), ultraviolet (UV) irradiation, oxidative stress, and high temperatures. Robust DNA repair system and antioxidant system have been demonstrated to contribute to extreme resistance in D. radiodurans. However, practically all studies on the mechanism underlying D. radiodurans’s extraordinary resistance relied on the treated strain during the post-treatment recovery lag phase to identify the key elements involved. The direct gene or protein changes of D. radiodurans after stress have not yet been characterized.ResultsIn this study, we performed a proteomics profiling on D. radiodurans right after the heavy ion irradiation treatment, to discover the altered proteins that were quickly responsive to IR in D. radiodurans. Our study found that D. radiodurans shown exceptional resistance to 12C6+ heavy ion irradiation, in contrast to Escherichia coli (E.coli) strains. By using iTRAQ (Isobaric Tags for Relative and Absolute Quantitation)-based quantitative mass spectrometry analysis, the kinetics of proteome changes induced by various dosages of 12C6+ heavy ion irradiation were mapped. The results revealed that 452 proteins were differentially expressed under heavy ion irradiation, with the majority of proteins being upregulated, indicating the upregulation of functional categories of translation, TCA cycle (Tricarboxylic Acid cycle), and antioxidation regulation under heavy ion irradiation.ConclusionsThis study shows how D. radiodurans reacts to exposure to 12C6+ heavy ion irradiation in terms of its overall protein expression profile. Most importantly, comparing the proteome profiling of D. radiodurans directly after heavy ion irradiation with research on the post-irradiation recovery phase would potentially provide a better understanding of mechanisms underlying the extreme radioresistance in D. radiodurans.

DETERMINATION OF THE OCCURENCE OF NUCLEAR ANOMALIES IN DANIO RERIO PERIPHERAL BLOOD ERYTH- ROCYTES DEPENDING ON THE DOSE OF IONIZING RADIATION

Ionizing radiation induces double-stranded breaks in the DNA structure, following by the formation of dicentric chromosomes. With the passage of subsequent postradiation mitoses, dicentric chromosomes are unevenly distributed to the poles of the cell, as a result of which nuclear anomalies of various types are formed, distinguishable at the light-optical level. The aim of this study was to assess the occurrence of nuclear anomalies in the peripheral blood erythrocytes of freshwater fish Danio rerio after exposure to ionizing radiation, as well as to assess the possibility of using these organisms as model animals in radiobiological studies. To determine the frequency of occurrence of nuclear anomalies in peripheral blood erythrocytes of freshwater fish Danio rerio, fish were exposed to X-ray radiation at doses of 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0 Gy. 48 hours after irradiation in fish peripheral blood erythrocytes by light microscopy, four types of nuclear anomalies were identified: micronuclei, nuclear protrusions, nucleoplasmic bridges, dumbbell-shaped nuclei. The frequency of detection of micronuclei and nuclear protrusions was found to be dose-dependent (r = 0.9245, p < 0.05 and r = 0.9062, p < 0.05 respectively), while the appearance of nucleoplasmic bridges and dumbbell-shaped nuclei did not correlate with the dose. More than that, the frequencies of micronuclei and nuclear protrusions detected after irradiation of fish at doses of more than 4 Gy (4.0, 6.0 and 8.0 Gy) significantly differed from the control values. Thus, Danio rerio can be used as a laboratory test system for radiobiological research, for example, to determine the effect of the developed radioprotectors and radiosensitizers, despite the extreme radioresistance of these organisms. It is advisable to use micronuclei or nuclear protrusions as cell markers, given, however, that the identification of these markers will allow detecting radiation exposure only in doses above 4 Gy.

Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA.

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

Natural Transformation in Deinococcus radiodurans: A Genetic Analysis Reveals the Major Roles of DprA, DdrB, RecA, RecF, and RecO Proteins.

Horizontal gene transfer is a major driver of bacterial evolution and adaptation to environmental stresses, occurring notably via transformation of naturally competent organisms. The Deinococcus radiodurans bacterium, characterized by its extreme radioresistance, is also naturally competent. Here, we investigated the role of D. radiodurans players involved in different steps of natural transformation. First, we identified the factors (PilQ, PilD, type IV pilins, PilB, PilT, ComEC-ComEA, and ComF) involved in DNA uptake and DNA translocation across the external and cytoplasmic membranes and showed that the DNA-uptake machinery is similar to that described in the Gram negative bacterium Vibrio cholerae. Then, we studied the involvement of recombination and DNA repair proteins, RecA, RecF, RecO, DprA, and DdrB into the DNA processing steps of D. radiodurans transformation by plasmid and genomic DNA. The transformation frequency of the cells devoid of DprA, a highly conserved protein among competent species, strongly decreased but was not completely abolished whereas it was completely abolished in ΔdprA ΔrecF, ΔdprA ΔrecO, and ΔdprA ΔddrB double mutants. We propose that RecF and RecO, belonging to the recombination mediator complex, and DdrB, a specific deinococcal DNA binding protein, can replace a function played by DprA, or alternatively, act at a different step of recombination with DprA. We also demonstrated that a ΔdprA mutant is as resistant as wild type to various doses of γ-irradiation, suggesting that DprA, and potentially transformation, do not play a major role in D. radiodurans radioresistance.

Single-molecule observation of ATP-independent SSB displacement by RecO in Deinococcus radiodurans.

Deinococcus radiodurans (DR) survives in the presence of hundreds of double-stranded DNA (dsDNA) breaks by efficiently repairing such breaks. RecO, a protein that is essential for the extreme radioresistance of DR, is one of the major recombination mediator proteins in the RecA-loading process in the RecFOR pathway. However, how RecO participates in the RecA-loading process is still unclear. In this work, we investigated the function of drRecO using single-molecule techniques. We found that drRecO competes with the ssDNA-binding protein (drSSB) for binding to the freely exposed ssDNA, and efficiently displaces drSSB from ssDNA without consuming ATP. drRecO replaces drSSB and dissociates it completely from ssDNA even though drSSB binds to ssDNA approximately 300 times more strongly than drRecO does. We suggest that drRecO facilitates the loading of RecA onto drSSB-coated ssDNA by utilizing a small drSSB-free space on ssDNA that is generated by the fast diffusion of drSSB on ssDNA.

Clinical Sequencing Analysis of the Mutation Profiles Associated with Extreme Radioresistance

Precision medicine using genetic data of individual tumors is emerging in the field of cancer treatment. However, mutation profiles associated with the response of tumors to radiotherapy have not been fully elucidated. We hypothesized that the patients who exhibited extreme resistance to radiotherapy carry relevant mutation profiles. The patient with uterine cervical cancer that repeatedly locally-recurred after multiple rounds of curative radiotherapy, was selected as analytical subject. Biopsy samples were obtained from treatment-naïve tumor and the tumors that recurred after initial and secondary radiotherapy. Exon sequencing of 409 cancer-related genes was performed using a next-generation sequencer. Prevalence of candidate mutation profiles in clinical malignancy specimens was investigated using cBioportal. Contribution of candidate mutation profiles on radioresistance was examined by (i) meta-analysis of published in vitro radiosensitivity data combined with genomics data of Cancer Cell Line Encyclopedia (CCLE), and (ii) isogenic cell-based experiments using gene knock-in and siRNA techniques. The Differences between two groups were examined by Mann–Whitney test. The sequencing analysis identified activating mutations in PIK3CA (E545K) and KRAS (G12D), and putative biallelic inactivating mutations in SMAD4 (R361C and R261H) as trunk mutation signatures that persisted over the clinical course. Mining of published genomics data revealed that simultaneous mutations of KRAS and SMAD4 have not been reported previously for uterine cervical cancer (n = 0/343); the mutation signature was also rare among various types of malignancies (1.2%, n = 279/22928). Interestingly, this mutation signature was most prevalent in pancreatic cancer (23.7%, n = 195/820 cases) that is known to be clinically radioresistant. These data indicated that simultaneous mutations of KRAS and SMAD4 contributed to extreme radioresistant nature of the tumors analyzed in this study. Mining of CCLE genomics data identified 12 cancer cell lines carrying simultaneous mutations of KRAS and SMAD4. Meta-analysis of 94 publications identified by systemic literature review demonstrated that SF2, surviving fraction after 2 Gy-irradiation as assessed by clonogenic assays, was significantly higher for the KRAS/SMAD4-mutated cell lines compared to that for the background-matched control cell lines (P = 0.010). Experiments using KRASG12D-knock in- and SMAD4-knock down-SW48 cells demonstrated that SF2 for the KRAS/SMAD4-modulated cells was significantly higher than that for the parental cells (P = 0.034). These data indicate that simultaneous mutations in KRAS and SMAD4 are associated with radioresistance. The mutation signature is potentially useful as the biomarker for radioresistance in the practice of precision medicine.

Unraveling the mechanisms of extreme radioresistance in prokaryotes: Lessons from nature.

The last 50 years, a variety of archaea and bacteria able to withstand extremely high doses of ionizing radiation, have been discovered. Several lines of evidence suggest a variety of mechanisms explaining the extreme radioresistance of microorganisms found usually in isolated environments on Earth. These findings are discussed thoroughly in this study. Although none of the strategies discussed here, appear to be universal against ionizing radiation, a general trend was found. There are two cellular mechanisms by which radioresistance is achieved: (a) protection of the proteome and DNA from damage induced by ionizing radiation and (b) recruitment of advanced and highly sophisticated DNA repair mechanisms, in order to reconstruct a fully functional genome. In this review, we critically discuss various protecting (antioxidant enzymes, presence or absence of certain elements, high metal ion or salt concentration etc.) and repair (Homologous Recombination, Single-Strand Annealing, Extended Synthesis-Dependent Strand Annealing) mechanisms that have been proposed to account for the extraordinary abilities of radioresistant organisms and the homologous radioresistance signature genes in these organisms. In addition, and based on structural comparative analysis of major radioresistant organisms, we suggest future directions and how humans could innately improve their resistance to radiation-induced toxicity, based on this knowledge.

Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library.

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.

DdrO is an essential protein that regulates the radiation desiccation response and the apoptotic-like cell death in the radioresistant Deinococcus radiodurans bacterium.

Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus deserti, two genes encoding α-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage.

Protease activity of PprI facilitates DNA damage response: Mn2+-dependence and substrate sequence-specificity of the proteolytic reaction.

The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism.

Interaction of double-stranded DNA with polymerized PprA protein from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of Deinococcus radiodurans. To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs (i.e., super coiled, linear, or nicked circular dsDNA) was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 μM with Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number (280) of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was >1.3 μM. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA. These findings are important for the understanding of the mechanism underlying effective DNA repair involving PprA.

Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation

Purpose: To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells.Materials and methods: Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500–2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit.Results: A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism.Conclusions: Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50–200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

Endogenous nitric oxide production in the radioresistant bacterium deinococcus radiodurans determines lipid composition and is essential for rapid growth recovery after gamma-irradiation

The eubacteria Deinococcus radiodurans (Drad) is the most radioresistant organism known. The extreme radioresistance of Drad primarily owes to its efficiency at mitigating oxidative damage, repairing double-strand DNA breaks, and possibly additional mechanisms that await elucidation. Recently, it was found that a NOS knockout strain of Drad (ΔNOS) is highly-sensitive to UV irradiation, establishing a role for NO in growth recovery after UV exposure. To further test the involvement of Drad NOS in radiation resistance, WT and ΔNOS Drad strains were subjected to extreme doses of gamma-irradiation (3000 Gy, 60 Co source) and the impact of NO on post-irradiation growth recovery, metabolite, gene and protein expression were assessed. Notably, the ΔNOS cell line displayed severely retarded growth following gamma-irradiation, compared to WT cells. NO supplementation with the long-lived NO donor, DETA-NONOate (25 μM), rescued the ΔNOS defect in growth recovery, restoring it to the control level. Clonogenic assays demonstrated that NO selectively facilitates growth recovery, but does not diminish gamma-irradiation cell killing. To discover consequences of endogenously-produced NO on biochemical pathways in Drad, untargeted metabolite profiling studies were performed. NO gene-deletion was associated with profound changes in levels of multiple metabolites, including specific carotenoids, glycerolipids and some additional cell membrane/wall building blocks. Further studies aim to identify biochemical pathways, enzymes and molecular mechanisms by which NO regulates Drad metabolism and thereby attenuates radioresistance. In this effort, untargeted metabolite profiling is providing novel details about NO signaling and may rationalize the enigmatic basis for radiosensitization by NOS inhibitors of some mammalian tumors.

Unusual radioresistance of nitrogen-fixing cultures of Anabaena strains

Nitrogen-fixing cultures of two species of the filamentous, heterocystous cyanobacterium Anabaena, namely Anabaena sp. strain L-31 and Anabaena torulosa were found to be highly tolerant to 60Co gamma radiation. No adverse effect on diazotrophic growth and metabolism were observed up to a dose of 5 kGy. At higher doses, radiation tolerance showed a correspondence with the inherent osmotolerance, with Anabaena L-31 being the more radiation tolerant as well as osmotolerant strain. In Anabaena L-31, exposure to 6 kGy of gamma rays resulted in genome disintegration, but did not reduce viability. Irradiation delayed heterocyst differentiation and nitrogen fixation, and marginally affected diazotrophic growth. All the affected parameters recovered after a short lag, without any discernible postirradiation phenotype. The radiation tolerance of these Gram-negative photoautodiazotrophs is comparable with that of the adiazotrophic photoautotrophic cyanobacterium Chroococcidiopsis or adiazotrophic heterotroph Deinococcus radiodurans. This is the first report of extreme radioresistance in nitrogen-fixing Anabaena cultures.

IrrE, an Exogenous Gene from Deinococcus radiodurans, improves the Growth of and Ethanol Production by a Zymomonas mobilis Strain under Ethanol and Acid Stresses

During ethanol fermentation, bacterial strains may encounter various stresses, such as ethanol and acid shock, which adversely affect cell viability and the production of ethanol. Therefore, ethanologenic strains that tolerate abiotic stresses are highly desirable. Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation, ultraviolet light, and desiccation, and therefore constitute an important pool of extreme resistance genes. The irrE gene encodes a general switch responsible for the extreme radioresistance of D. radiodurans. Here, we present evidence that IrrE acting as a global regulator confers high stress tolerance to a Zymomonas mobilis strain. Expression of the gene protected Z. mobilis cells against ethanol, acid, osmotic, and thermal shock. It also markedly improved cell viability, the expression levels and enzyme activities of pyruvate decarboxylase and alcohol dehydrogenase, and the production of ethanol under both ethanol and acid stress. These data suggest that irrE is a potentially promising gene for improving the abiotic stress tolerance of ethanologenic bacterial strains.

Identification of PprM: a modulator of the PprI-dependent DNA damage response in Deinococcus radiodurans

Deinococcus radiodurans possesses a DNA damage response mechanism that acts via the PprI protein to induce RecA and PprA proteins, both of which are necessary in conferring extreme radioresistance. In an effort to further delineate the nature of the DNA damage response mechanism in D. radiodurans, we set out to identify novel components of the PprI-dependent signal transduction pathway in response to radiation stress. Here we demonstrate the discovery of a novel regulatory protein, PprM (a modulator of the PprI-dependent DNA damage response), which is a homolog of cold shock protein (Csp). Disruption of the pprM gene rendered D. radiodurans significantly sensitive to gamma-rays. PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from D. geothermalis and Thermus thermophilus. Purified PprM is present as a homodimer under physiological conditions, as the case with Escherichia coli CspD. The pprA pprM double-disruptant strain exhibited higher sensitivity than the pprA or pprM single disruptant strains, suggesting that PprM regulates other hitherto unknown protein(s) important for radioresistance besides PprA. This study strongly suggests that PprM is involved in the radiation response mediated by PprI in D. radiodurans.

IrrE, a Global Regulator of Extreme Radiation Resistance in Deinococcus radiodurans, Enhances Salt Tolerance in Escherichia coli and Brassica napus

BackgroundGlobally, about 20% of cultivated land is now affected by salinity. Salt tolerance is a trait of importance to all crops in saline soils. Previous efforts to improve salt tolerance in crop plants have met with only limited success. Bacteria of the genus Deinococcus are known for their ability to survive highly stressful conditions, and therefore possess a unique pool of genes conferring extreme resistance. In Deinococcus radiodurans, the irrE gene encodes a global regulator responsible for extreme radioresistance.Methodology/Principal FindingsUsing plate assays, we showed that IrrE protected E. coli cells against salt shock and other abiotic stresses such as oxidative, osmotic and thermal shocks. Comparative proteomic analysis revealed that IrrE functions as a switch to regulate different sets of proteins such as stress responsive proteins, protein kinases, glycerol-degrading enzymes, detoxification proteins, and growth-related proteins in E. coli. We also used quantitative RT-PCR to investigate expression of nine selected stress-responsive genes in transgenic and wild-type Brassica napus plants. Transgenic B. napus plants expressing the IrrE protein can tolerate 350 mM NaCl, a concentration that inhibits the growth of almost all crop plants.ConclusionsExpression of IrrE, a global regulator for extreme radiation resistance in D. radiodurans, confers significantly enhanced salt tolerance in both E. coli and B. napus. We thus propose that the irrE gene might be used as a potentially promising transgene to improve abiotic stress tolerances in crop plants.

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans.

Crystal Structure of the IrrE Protein, a Central Regulator of DNA Damage Repair in Deinococcaceae

Deinococcaceae are famous for their extreme radioresistance. Transcriptome analysis in Deinococcus radiodurans revealed a group of genes up-regulated in response to desiccation and ionizing radiation. IrrE, a novel protein initially found in D. radiodurans, was shown to be a positive regulator of some of these genes. Deinococcus deserti irrE is able to restore radioresistance in a D. radiodurans Δ irrE mutant. The D. deserti IrrE crystal structure reveals a unique combination of three domains: one zinc peptidase-like domain, one helix-turn-helix motif and one GAF-like domain. Mutant analysis indicates that the first and third domains are critical regions for radiotolerance. In particular, mutants affected in the putative zinc-binding site are as sensitive to gamma and UV irradiation as the Δ irrE bacteria, and radioresistance is strongly decreased with the H217L mutation present in the C-terminal domain. In addition, modeling of IrrE–DNA interaction suggests that the observed IrrE structure may not bind double-stranded DNA through its central helix-turn-helix motif and that IrrE is not a classic transcriptional factor that activates gene expression by its direct binding to DNA. We propose that the putative protease activity of IrrE could be a key element of transcription enhancement and that a more classic transcription factor, possibly an IrrE substrate, would link IrrE to transcription of genes specifically involved in radioresistance.