ISDra2 transposition in Deinococcus radiodurans is downregulated by TnpB

Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single‐stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ‐irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB‐mediated inhibition does not involve Deinococcus‐specific factors. TnpB therefore appears to play a regulatory role in ISDra2 transposition.     

Pleiotropic effects of a cold shock protein homolog PprM on the proteome of Deinococcus radiodurans

An extremophile D. radiodurans encodes a non-cold shock inducible cold shock protein homolog DR_0907 (also known as PprM). The DR_0907 ORF was deleted by knockout mutagenesis and the resultant deletion mutant (ΔpprM D. radiodurans) displayed growth defect as well as gamma-radiation sensitivity (D₁₀ values = ΔpprM D. radiodurans: 12.1 kGy versus wild type (WT) D. radiodurans: 14 kGy). 2D gel based comparative proteomics revealed a comparable induction of DNA repair proteins in ΔpprM D. radiodurans and WT D. radiodurans recovering from 5 kGy gamma irradiation (⁶⁰Co gamma source, dose rate: 2 kGy/h), suggesting that pprM does not cause radiation sensitivity through modulation of DdrO-regulated DNA repair genes. However, deletion of pprM did result in repression of several proteins that belonged to vital housekeeping pathways such as metabolism and protein homeostasis that might contribute to slow growth phenotype. These deficiencies intrinsic to ΔpprM D. radiodurans might also contribute to its radiation sensitivity.     

DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans

The bacterium Deinococcus radiodurans can survive extremely high exposure to ionizing radiation. The repair mechanisms involved in this extraordinary ability are still being investigated. ddrB is one gene that is highly up-regulated after irradiation, and it has been proposed to be involved in RecA-independent repair in D. radiodurans. Here we cloned, expressed and characterized ddrB in order to define its roles in the radioresistance of D. radiodurans. DdrB preferentially binds to single-stranded DNA. Moreover, it interacts directly with single-stranded binding protein of D. radiodurans DrSSB, and stimulates single-stranded DNA annealing even in the presence of DrSSB. The post-irradiation DNA repair kinetics of a ddrB/recA double mutant were compared to ddrB and recA single mutants by pulsed-field gel electrophoresis (PFGE). DNA fragment rejoining in the ddrB/recA double mutant is severely compromised, suggesting that DdrB-mediated single-stranded annealing plays a critical role in the RecA-independent DNA repair of D. radiodurans.     

Transposases are responsible for the target specificity of IS1397 and ISKpn1 for two different types of palindromic units (PUs)

Insertion sequences (IS)1397 and ISKpn1, found in Escherichia coli and Klebsiella pneumoniae, respectively, are IS3 family members that insert specifically into short palindromic repeated sequences (palindromic units or PUs). In this paper, we first show that although PUs are naturally absent from extrachromosomal elements, both ISs are able to transpose from the chromosome or from a plasmid into PUs artificially introduced into target plasmids. We also show that ISKpn1 target specificity is restricted to K.pneumoniae Z¹ PU type, whereas IS1397 target specificity is less stringent since the IS targets the three E.coli Y, Z¹ and Z² PU types indifferently. Experiments of transposition of both ISs driven by both transposases demonstrate that the inverted repeats flanking the ISs are not responsible for this target specificity, which is entirely due to the transposase itself. Implications on ISs evolution are presented.     

Physiologic Determinants of Radiation Resistance in Deinococcus radiodurans

Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium Deinococcus radiodurans is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of D. radiodurans and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that D. radiodurans can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of D. radiodurans and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete D. radiodurans genomic sequence support the hypothesis that there are several defects in D. radiodurans global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of D. radiodurans in nutritionally limiting radioactive environments.     

Thioredoxin System from Deinococcus radiodurans

This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-Å resolution. The overall structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K_m (5.7 μM) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K_m, 44.4 μM). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.Deinococcus radiodurans is a gram-positive bacterium capable of withstanding exposure to extreme gamma ray and UV radiation, oxidants, and desiccation (6, 10, 26). The mechanism behind the ability of D. radiodurans to survive exposure to extreme conditions has been a subject of intense research (10, 43). Its ability to survive exposure to extreme conditions has been attributed a number of factors, as follows: a high number of genome copies (8), ring-like nucleoid organization (22), high manganese content (8), and a higher ability to scavenge reactive oxygen species (ROS) (43). However, the mechanism responsible for its extremophilic nature is not clearly understood (25).Efforts to understand the mechanism behind the capability of D. radiodurans to tolerate extreme conditions have focused on understanding its ability to prevent or repair genomic damage, because if unrepaired, genomic damage is lethal to the cell (7). The ability of D. radiodurans to repair genomic damage is likely due to its ability to prevent proteome damage, i.e., its ability to maintain sufficient enzymatic activity for genome repair after irradiation. Therefore, genome repair probably plays a bigger role than prevention of genome damage in making D. radiodurans radiation tolerant (7, 8). Indeed, some experimental evidence suggests that efficient DNA repair is solely responsible for the ability of D. radiodurans to withstand ionizing radiation. D. radiodurans DNA sustains the same amount of genome damage at high radiation doses as other bacteria, but unlike other bacteria, its damage is mended within hours (25). However, some recent evidence suggests that it is likely that prevention of DNA damage (reactive oxygen species [ROS] scavenging) supplements DNA repair to make D. radiodurans ionizing radiation tolerant. It is worth noting that only about 20% of radiation-induced damage to the genome is due to the direct effect of irradiation (the rest is due to radiation-induced ROS) and that cellular extracts of D. radiodurans are more effective in scavenging ROS than Escherichia coli extracts when subjected to oxidative stress (43). Moreover, D. radiodurans has higher basal levels of some antioxidant enzymatic systems (catalase and superoxide dismutase), and disruption of superoxide dismutase (sodA) and catalase (katA) genes results in increased sensitivity of D. radiodurans to ionizing radiation. In addition D. radiodurans catalase is more resistant to inhibition by substrate H₂O₂ than bovine or Aspergillus niger catalase (17). Taken together, these experimental results suggest a significant contribution of antioxidant systems to the ability of D. radiodurans to withstand extreme ionizing radiation.While the contribution of some antioxidant enzymatic systems to the extremophilic nature of D. radiodurans has been extensively studied, the role of the thioredoxin system has not been investigated (40, 43). The thioredoxin system is composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and various cellular targets. The system is found in both prokaryotes and eukaryotes, and homologues of both TrxR and Trx have been isolated from many species. Trx proteins are low-molecular-mass proteins (12 kDa) that possess a highly conserved active site motif, WCGPC (27, 41). TrxR is a homodimeric enzyme and is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Each monomer possesses a flavin adenine dinucleotide (FAD) prosthetic group, a NADPH-binding site, and an active site comprising a redox-active disulfide. There are two distinct forms of this enzyme, as follows: low-molecular-mass TrxR (35 kDa), found in prokaryotes and some eukaryotes, and high-molecular-mass TrxR (55 kDa), found in eukaryotes (41). The two types of TrxR proteins have some differences in structure and mechanism. However, in both cases, reducing equivalents are transferred from NADPH to TrxR, from TrxR to Trx, and finally, from Trx to various cellular proteins (29, 41). Trx targets include proteins which take part in the scavenging of ROS-like thioredoxin-dependent thiol peroxidase (29). The thioredoxin system is thus an important antioxidant enzymatic system.In this study we report the expression, purification, and biochemical characterization of the main components of the D. radiodurans thioredoxin system. In addition, the structural characterization of D. radiodurans TrxR is reported.     

Oxidative Stress Resistance in Deinococcus radiodurans

Summary: Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health.     

Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn²⁺ and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn²⁺-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.     

Differential Radio-Tolerance of Nutrition-Induced Morphotypes of Deinococcus radiodurans R1

Deinococcus radiodurans R1 is a highly radio-tolerant bacterium. Depending on the nutrient availability D. radiodurans R1 exists in three morphologies viz. monococcal, diplococcal and tetracoccal. In this study, we examined whether nutrition-induced morphotypes of D. radiodurans showed similar DNA damage upon gamma radiation exposure. Total DNA damage after radiation exposure was estimated by comparing percent double-strand breaks (DSBs) in genomic DNA. It was found that all three morphotypes exhibited different radiation tolerances which were also dependent on the radiation dose given. Monococcal forms were found to be most radio-tolerant at most of the tested radiation doses. Results showed that these nutrient-starved-condition induced morphotypes show lesser DNA DSBs upon irradiation, hence show higher radio-tolerance.     

A model for repair of radiation-induced DNA double-strand breaks in the extreme radiophile Deinococcus radiodurans

The bacterium Deinococcus (formerly Micrococcus) radiodurans and other members of the eubacterial family Deinococaceae are extremely resistant to ionizing radiation and many other agents that damage DNA. Stationary phase D. radiodurans exposed to 1.0-1.5 Mrad γ-irradiation sustains >120 DNA double-strand breaks (dsbs) per chromosome; these dsbs are mended over a period of hours with 100% survival and virtually no mutagenesis. This contrasts with nearly all other organisms in which just a few ionizing radiation induced-dsbs per chromosome are lethal. In this article we present an hypothesis that resistance of D. radiodurans to ionizing radiation and its ability to mend radiation-induced dsbs are due to a special form of redundancy wherein chromosomes exist in pairs, linked to each other by thousands of four-stranded (Holliday) junctions. Thus, a dsb is not a lethal event because the identical undamaged duplex is nearby, providing an accurate repair template. As addressed in this article, much of what is known about D. radiodurans suggests that it is particularly suited for this proposed novel form of DNA repair.     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

Preserving Genome Integrity: The DdrA Protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

抗辐射菌Deinococcus radiodurans的多样性

抗辐射菌Deinococcus radiodurans是一种对电离辐射和其他DNA损伤因子具有极强抵抗能力的细菌,是研究DNA损伤与修复的模式生物。综述了国内外在抗辐射菌研究上取得的最新研究成果,从生存环境、对DNA损伤因子的抗性、抗性机理及其损伤修复关联基因等方面报道了抗辐射菌的多样性,并探讨了该细菌高效正确的DNA损伤修复机理的相关研究成果在生命科学、农业、环境修复及医学等领域的应用前景。     

Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters

Insertion specificity of mobile genetic elements is a rather complex aspect of DNA transposition, which, despite much progress towards its elucidation, still remains incompletely understood. We report here the results of a meta-analysis of IS2 target sites from genomic, phage, and plasmid DNA and find that newly acquired IS2 elements are consistently inserted around abrupt DNA compositional shifts, particularly in the form of switch sites of GC skew. The results presented in this study not only corroborate our previous observations that both the insertion sequence (IS) minicircle junction and target region adopt intrinsically bent conformations in IS2, but most interestingly, extend this requirement to other families of IS elements. Using this information, we were able to pinpoint regions with high propensity for transposition and to predict and detect, de novo, a novel IS2 insertion event in the 3′ region of the gfp gene of a reporter plasmid. We also found that during amplification of this plasmid, process parameters such as scale, culture growth phase, and medium composition exacerbate IS2 transposition, leading to contamination levels with potentially detrimental clinical effects. Overall, our findings provide new insights into the role of target DNA structure in the mechanism of transposition of IS elements and extend our understanding of how culture conditions are a relevant factor in the induction of genetic instability.     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.     

Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)₂ structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA ^? bacteria, replicated copies of the introduced (IS30)₂ structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)₂ are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)₂, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)₂ intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)₂ may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)₂. Evolutionary implications of these findings are discussed.     

ISD1, an Insertion Element from the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough: Structure, Transposition, and Distribution

Insertion element ISD1, discovered when its transposition caused the insertional inactivation of an introduced sacB gene, is present in two copies in the genome of Desulfovibrio vulgaris Hildenborough. Southern blot analysis indicated at least two insertion sites in the sacB gene. Cloning and sequencing of a transposed copy of ISD1 indicated a length of 1,200 bp with a pair of 44-bp imperfect inverted repeats at the ends, flanked by a direct repeat of the 4-bp target sequence. AAGG and AATT were found to function as target sequences. ISD1 encodes a transposase from two overlapping open reading frames by programmed translational frameshifting at an A₆G shifty codon motif. Sequence comparison showed that ISD1 belongs to the IS3 family. Isolation and analysis of the chromosomal copies, ISD1-A and ISD1-B, by PCR and sequencing indicated that these are not flanked by direct repeats. ISD1-A is inserted in a region of the chromosome containing the gapdh-pgk genes (encoding glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase). Active transposition to other loci in the genome was demonstrated, offering the potential of a new tool for gene cloning and mutagenesis. ISD1 is the first transposable element described for the sulfate reducers, a large and environmentally important group of bacteria. The distribution of ISD1 in genomes of sulfate-reducing bacteria is limited. A single copy is present in the genome of D. desulfuricans Norway.