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.     

D.radiodurans CatB基因的克隆及其在大肠杆菌中的表达

通过生物信息学方法从耐辐射奇球菌（Ｄ．ｒａｄｉｏｄｕｒａｎｓ）全基因组居库中查鼠并克隆了编码过氧化氢酶（Ｃａｒｔａｌａｓｅ,Ｃａｔ）的１６１１ｂｐ长ＣａｔＢ基因,将ＣａｔＢ基因连人ｐＫＫ２２３－３表达载体,转化Ｃａｔ酶链陷型大肠杆菌（Ｅ．ｃｏｌｉ ＵＭ２）。转化菌裂解液ＰＡＧＥ酶活性染色分析实物具有Ｃａｔ酶活性,电泳过移位置与ＣａｔＢ位置相符。Ｄ．ｒａｄｉｏｄｕｒａｎｓ ＣａｔＢ基因的表达可使Ｅ．     

Expression of Deinococcus radiodurans PprI enhances the radioresistance of Escherichia coli

PprI, a newly identified gene switch responsible for extreme radioresistance of Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair and protection pathways in response to radiation stress [Biochem. Biophy. Res. Commun. 306 (2003) 354]. To evaluate whether PprI also functions in the radioresistance in other organisms, D. radiodurans PprI protein (Deira-PprI) was expressed in Escherichia coli. The complemented E. coli strain showed an increase of approximately 1.6-fold radioresistance with a high dose of gamma irradiation. Immunoblotting assays showed that the expression of Deira-PprI in E. coli resulted in a significant increase in RecA protein expression following high dose ionizing radiation. The expression of Deira-PprI protein also significantly enhanced the scavenging ability of free radicals by inducing the enzymatic activity of KatG. These results indicate that exogenous expression of Deira-PprI promotes DNA repair and protection pathways and enhances the radioresistance of E. coli.     

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.     

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.     

耐辐射奇球菌基因组DNA表达文库的构建与鉴定

目的:构建耐辐射奇球茵(Dcinoeoccus radiodurans R1)基因组DNA表达文库,为进一步研究耐辐射奇球茵高抗辐射的调控网络奠定基础.方法:提取耐辐射奇球菌基因组DNA,用Sau3AI酶将基因组DNA部分酶切成0.5-5 kb大小的片段,用T4DNA连接酶将部分酶切片段与经BamH I和碱性磷酸酶(CIAP)处理的pGADT7栽体进行连接后电击转化大肠杆菌DH5a.结果:得到重组子数为2.2×104,扩增后的文库滴度为108 cfu/mL.结论:构建了耐辐射奇球菌基因组pGADT7表达文库,为进一步筛选与高抗辐射相关基因产物的互作蛋白奠定了基础.     

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.     

Nucleoid organization in the radioresistant bacterium Deinococcus radiodurans

Processes favoring the exceptional resistance to genotoxic stress of Deinococcus radiodurans are not yet completely characterized. It was postulated that its nucleoid and chromosome(s) organization could participate in the DNA double strand break repair process. Here, we investigated the organization of chromosome 1 by localization of three chromosomal loci including oriC, Ter and a locus located in its left arm. For this purpose, we used a ParB‐parS system to visualize the position of the loci before and after exposure to γ‐rays. By comparing the number of fluorescent foci with the number of copies of the studied loci present in the cells measured by quantitative polymerase chain reaction (qPCR), we demonstrated that the 4–10 copies of chromosome 1 per cell are dispersed within the nucleoid before irradiation, indicating that the chromosome copies are not prealigned. Chromosome segregation is progressive but not co‐ordinated, allowing each locus to be paired with its sister during part of the cell cycle. After irradiation, the nucleoid organization is modified, involving a transient alignment of the loci in the late stage of DNA repair and a delay of segregation of the Ter locus. We discuss how these events can influence DNA double strand break repair.     

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.     

耐辐射球菌研究进展及其应用前景

耐辐射球菌因其对辐射、紫外线、诱变剂等的极高抗性引起了人们的注意。研究发现,其辐射抗性和干燥抗性主要归因于其在逆境条件下保护蛋白质、减少蛋白质被氧化的程度。耐辐射球菌基础理论的研究成果在环境修复、农作物抗旱育种、动植物抗病育种,以及肿瘤治疗等领域都有广阔的应用前景。其中,最有可能运用在对放射性污染的环境修复中。相对于高昂的填埋处置放射性废料的方法,运用耐辐射球菌构建工程菌进行生物修复是一种廉价的方法。耐辐射球菌对干燥的强忍耐性说明其基因组中含有抗干旱基因。寻找耐辐射球菌抗干旱基因,对于转化耐辐射球菌抗干旱基因,创造抗干旱农作物具有潜在的应用价值。     

Function and biochemical characterization of RecJ in Deinococcus radiodurans

The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.     

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.     

DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans and other members of the same genus share extraordinary resistance to the lethal and mutagenic effects of ionizing and u.v. radiation and to many other agents that damage DNA. While it is known that this resistance is due to exceedingly efficient DNA repair, the molecular mechanisms responsible remain poorly understood. Following very high exposures to u.v. irradiation (e.g. 500 Jm⁻², which is non-lethal to D. radiodurans), this organism carries out extremely efficient excision repair accomplished by two separate nucleotide excision repair pathways acting simultaneously. One pathway requires the uvrA gene and appears similar to the UvrABC excinuclease pathway defined in Escherichia coli. The other excision repair pathway is specific for u.v. dimeric photoproducts, but is not mediated by a pyrimidine dimer DNA glycosylase. Instead, it is initiated by a second bona fide endonuclease that may recognize both pyrimidine dimers and pyrimidine-(6–4)pyrimidones. After high doses of ionizing-radiation (e.g. 1.5Mrad), D. radiodurans can mend >100 double-strand breaks (dsb) per chromosome without lethality or mutagenesis. Both dsb mending and survival are recA-dependent, indicating that efficient dsb mending proceeds via homologous recombination. D. radiodurans contains multiple chromosomes per cell, and it is proposed that dsb mending requires extensive recombination amongst these chromosomes, a novel phenomenon in bacteria. Thus, D. radiodurans may serve as an easily accessible model system for the double-strand-break-initiated interchromosomal recombination that occurs in eukaryotic cells during mitosis and meiosis.     

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。     

Analysis of the recJ gene and protein from Deinococcus radiodurans

The mechanism by which double-strand DNA breaks are repaired in the radiation-resistant bacterium Deinococcus radiodurans is not well understood. This organism lacks the RecBCD helicase/nuclease, which processes broken DNA ends in other bacteria. The RecF pathway is an alternative pathway for recombination and DNA repair in E. coli, when RecBCD is absent due to mutation, and D. radiodurans may rely on enzymes of this pathway for double-strand break repair. The RecJ exonuclease is thought to process broken DNA ends for the RecF pathway. We attempted to delete the recJ gene from D. radiodurans, using homologous recombination to replace the gene with a streptomycin-resistance cassette. We were unable to obtain a complete deletion mutant, in which the gene is deleted from all of the chromosome copies in this polyploid organism. Quantitative real-time PCR shows that the heterozygous mutants have a recJ gene copy that is ca. 10–30% that of the wild-type. Mutants with reduced recJ gene copy grow slowly and are more sensitive than wild-type to UV irradiation, gamma irradiation, and hydrogen peroxide. The mutants are as resistant as wild-type to methyl-methanesulfonate. The D. radiodurans RecJ protein was expressed in E. coli and purified under denaturing conditions. The re-folded protein has nuclease activity on single-stranded DNA with specificity similar to that of E. coli RecJ exonuclease.     

耐辐射球菌pprI在大肠杆菌中表达增强细胞抗氧化能力的研究

将耐辐射球菌(Deinococcus radiodurans)与DNA修复有关的开关基因—pprI通过穿梭质粒pRADZ3导入大肠杆菌TG1中,使其在正常培养条件下(不需诱导剂)表达PprI蛋白,并通过Western blot证实该基因在TG1中可稳定表达。与转化了空白质粒pRADZ3 TG1对照,观察了改造后的两种大肠杆菌在有H2O2氧化压力下的存活率和大肠杆菌中两种过氧化氢酶（KatE, KatG）的活性表达差异。结果表明,无论在指数生长期还是稳定生长期,能表达PprI蛋白的大肠杆菌比对照的存活率要高出10％左右;非变性电泳结果表明,耐辐射球菌pprI 在大肠杆菌中的表达使得KatE活性在指数生长期与稳定生长期分别增加1.5～2倍和2.5～3倍。证明耐辐射球菌pprI 在大肠杆菌中的表达能够增强细胞抗氧化能力。