Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.     

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.     

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.     

Expression of recA in Deinococcus radiodurans.

Deinococcus (formerly Micrococcus) radiodurans is remarkable for its extraordinary resistance to ionizing and UV irradiation and many other agents that damage DNA. This organism can repair > 100 double-strand breaks per chromosome induced by ionizing radiation without lethality or mutagenesis. We have previously observed that expression of D. radiodurans recA in Escherichia coli appears lethal. We now find that the RecA protein of D. radiodurans is ot detectable in D. radiodurans except in the setting of DNA damage and that termination of its synthesis is associated with the onset of deinococcal growth. The synthesis of Shigella flexneri RecA (protein sequence identical to that of E. coli RecA) in recA-defective D. radiodurans is described. Despite a large accumulation of the S. flexneri RecA in D. radiodurans, there is no complementation of any D. radiodurans recA phenotype, including DNA damage sensitivity, inhibition of natural transformation, or inability to support a plasmid that requires RecA for replication. To ensure that the cloned S. flexneri recA gene was not inactivated, it was rescued from D. radiodurans and was shown to function normally in E. coli. We conclude that neither D. radiodurans nor S. flexneri RecA is functional in the other species, nor are the kinetics of induction and suppression similar to each other, indicating a difference between these two proteins in their modes of action.     

Salt-mediated multicell formation in Deinococcus radiodurans.

The highly radiation-resistant tetracoccal bacterium Deinococcus radiodurans exhibited a reversible multi-cell-form transition which depended on the NaCl concentration in the medium. In response to 0.8% NaCl addition into the medium, the pair/tetrad (designated 2/4) cells in a young culture grew and divided but did not separate and became 8-, 16-, and 32-cell units successively. In exponential growth phase, the cells divided in a 16/32 pattern. Potassium ions were equally effective as Na+ in mediating this multicell-formation effect; Mg2+, Li+, and Ca2+ also worked but produced less multiplicity. This effect appears to be species specific. This-section micrographs revealed that in a 16/32-cell unit, eight 2/4 cells were encased in an orderly manner within a large peripheral wall, showing five cycles of septation. Our results suggest the presence of a salt-sensitive mechanism for controlling cell separation in D. radiodurans.     

Deinococcus radiodurans - the consummate survivor

Relatively little is known about the biochemical basis of the capacity of Deinococcus radiodurans to endure the genetic insult that results from exposure to ionizing radiation and can include hundreds of DNA double-strand breaks. However, recent reports indicate that this species compensates for extensive DNA damage through adaptations that allow cells to avoid the potentially detrimental effects of DNA strand breaks. It seems that D. radiodurans uses mechanisms that limit DNA degradation and that restrict the diffusion of DNA fragments that are produced following irradiation, to preserve genetic integrity. These mechanisms also increase the efficiency of the DNA-repair proteins.     

An RNA ligase from Deinococcus radiodurans

Although DNA repair pathways have been the focus of much attention, there is an emerging appreciation that distinct pathways exist to maintain or manipulate RNA structure in response to breakage events. Here we identify an RNA ligase (DraRnl) from the radiation-resistant bacterium Deinococcus radiodurans. DraRnl seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA: DNA hybrid, but it cannot seal 3'-OH/5'-PO4 DNA nicks. The specificity of DraRnl arises from a requirement for RNA on the 3'-OH side of the nick. DraRnl is a 342-amino acid monomeric protein with a distinctive structure composed of a C-terminal adenylyltransferase domain linked to an N-terminal module that resembles the OB-fold of phenylalanyl-tRNA synthetases. RNA sealing activity was abolished by mutation of the predicted lysine adenylylation site (Lys-165) in the C-terminal domain and was reduced by an order of magnitude by deletion of the N-terminal OB module. Our findings highlight the existence of an RNA repair capacity in bacteria and support the hypothesis that contemporary DNA ligases, RNA ligases, and RNA capping enzymes evolved by the fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.     

Heat shock and cold shock in Deinococcus radiodurans

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.     

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.     

Glucosyl diglyceride lipid structures in Deinococcus radiodurans.

The structures of two lipids from the radiation-resistant bacterium Deinococcus radiodurans are reported here: 1,2-diacyl-3-alpha-glucopyranosyl-glycerol and 3-O-[6'-O-(1",2"-diacyl- 3"-phosphoglycerol)-alpha-glucopyranosyl]-1,2-diacylglycerol. These lipids are strikingly different from previously characterized polar lipids from this organism, in that they are not unique to the genus Deinococcus and indeed have counterparts in both gram-negative and gram-positive bacteria. Moreover, as examples of glucose-containing lipids, they further illustrate the diversity of carbohydrate-containing lipids in D. radiodurans, from which lipids containing galactose and N-acetylglucosamine have already been structurally characterized.     

Reconstitution of the Deinococcus radiodurans aposuperoxide dismutase

Deinococcus radiodurans, a radiation-resistant aerobe, synthesized a 43,000 Mr dimeric superoxide dismutase. The holoenzyme, sp act 3300 U/mg, contained 1.5 g-atoms Mn, 0.6 g-atom Fe, and 0.1 g-atom Zn per mole dimer. Apoprotein, prepared by dialysis of the holoenzyme in denaturant plus chelator and then renatured in chelex-treated Tris chloride buffer, rapidly regained superoxide dismuting activity upon incubation in 1 mM MnCl2. Reconstitution was dependent on Mn concentration and pH. The Mn-reconstituted protein, sp act 3560 U/mg, contained 1.7 g-atoms Mn per mole dimer. The holoenzyme and Mn-reconstituted apoprotein migrated with the same patterns in 10% acrylamide gels and focused to the same pattern upon isoelectric focusing. Fluorescence emission maxima of the holoenzyme, Mn-reconstituted apoprotein, and the renaturated apoprotein were 329 +/- 1 nm but differed from the denatured apoprotein (352 nm). Apoprotein bound 1.7 g-atoms Zn and from 3-7 g-atoms Fe per mole dimer on incubation with 1 mM ZnSO4 and Fe(NH4)2(SO4)2, respectively. Although neither Zn nor Fe restored superoxide dismuting activity, the ferrous and the zinc salt inhibited reconstitution of the apoprotein with manganese. Metal addition to renatured aposuperoxide dismutase offers a novel approach to reconstitution of procaryote superoxide dismutases.     

A novel serralysin metalloprotease from Deinococcus radiodurans

A hypothetical protein (DR2310) from the radiation resistant organism Deinococcus radiodurans harbors highly conserved Zn(+2)-binding (HEXXH) domain and Met-turn (SVMSY), characteristic of the serralysin family of secreted metalloproteases from Gram negative bacteria. Deletion mutagenesis of DR2310 confirmed that the ORF is expressed in Deinococcus radiodurans as a secreted protease of 85 kDa. Biochemical analysis revealed DR2310 to be a Ca(+2) and Zn(+2)-requiring metalloprotease. Unique features such as a long N-terminus, replacement of the highly conserved C-terminal glycine rich Ca(+2)-binding repeats with a single N-terminal aspartate rich eukaryotic thrombospondin type-3 Ca(+2)-binding repeat and absence of C-terminal secretion signals make it a novel member of serralysin family. This is the first report of a functional serralysin family metalloprotease from a Gram positive organism.     

Promoter cloning in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the -10 and -35 regions of these promoters resembled the sigma(70) consensus sequence of E. coli.     

耐辐射奇球菌代谢产物中的化学成分

通过甲醇提取、硅胶柱色谱、重结晶分离纯化,从耐辐射奇球菌(Deinococcus radiodurans)代谢产物中分离得到五个化合物,根据光谱数据鉴定为:腺嘌呤(1),胸腺嘧啶(2),尿嘧啶(3),腺苷(4)和L-丙氨酸(5)。所有成分均为首次从该细菌的代谢产物中得到。     

Sensitivity of Deinococcus radiodurans to near-ultraviolet radiation

Although Dienococcus radiodurans is notoriously resistant to far-ultraviolet radiation (FUV; 254 nm), it is highly sensitive to near-ultraviolet radiation (NUV; 300-400 nm), thus demonstrating that the mechanisms of damage (and/or recovery) by the two types of irradiation are different. This observed difference between FUV and NUV effects in D. radiodurans agrees with previous studies with Escherichia coli. Near-ultraviolet radiation produces DNA damage which is presumed to be single-strand breaks (SSB) in the DNA of D. radiodurans. Unique lesions, such as DNA-protein crosslinks could not be demonstrated in this study. Cells that were pre-irradiated with a small dose of NUV were subsequently protected against inactivating doses of NUV. The data presented are consistent with induced DNA repair following NUV damage in D. radiodurans; this is in contrast to FUV damage where DNA repair is constitutive but not induced.     

耐辐射异常球菌抗辐射机理的研究新进展

报道了自1956年Anderson发现耐辐射异常球菌(Deinococcusradiodurans)以来,国外在其生理生化和遗传学特性、特殊的细胞膜结构、各种诱变因素所致的DNA损伤与其高效的修复机制和生物化学、分子生物学应用于该菌的研究新进展。对该菌的研究在辐射生物学与医学上具有特殊的意义,因此,我国的辐射生物学、微生物学和医学研究人员应尽快开展这方面的研究。     

Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans.

Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of either locus results in a partial loss of resistance to ionizing radiation. The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant. In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation. The irrB and irrI mutations also reduce D. radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds. Subclones of each gene have been isolated, and the loci have been mapped relative to each other. The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes.     

Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.