Effects of carotenoids from Deinococcus radiodurans on protein oxidation

Aims:  To evaluate the antioxidant effect of carotenoids from Deinococcus radiodurans on protein.
Methods and Results:  Deinococcus radiodurans strain R1 (ATCC 13939) and its mutant strain R1ΔcrtB were used for this study. The total carotenoids (R1ex) from D. radiodurans were obtained by extraction with acetone/methanol (7 : 2, by vol), and their antioxidant activity was measured using the DPPH˙ (2,2-diphenyl-1-picrylhydrazyl) system. The protein oxidation level, in vitro and in the cell, was measured using the DNPH (2,4-dinitrophenyl hydrazine) method. The carotenoid extract R1ex scavenged 40·2% DPPH˙ radicals compared to β-carotene (31·7%) at a concentration of 0·5 mg ml⁻¹. The intracellular level of protein oxidation in mutant R1ΔcrtB, which does not contain carotenoid, was 0·0212 mmol mg⁻¹ protein which is significantly greater than that in the wild type (0·0169 mmol mg⁻¹ protein) following the treatment with H₂O₂. The purified major carotenoid product (deinoxanthin) from the wild type showed a greater inhibition of oxidative damage in bovine serum albumin than lycopene or lutein.
Conclusions:  Carotenoids prevent protein oxidation and contribute to the resistance to cell damage in D. radiodurans .
Significance and Impact of the Study:  Our results provide the evidence that carotenoids can protect proteins in D. radiodurans against oxidative stress.     

A new role of Deinococcus radiodurans RecD in antioxidant pathway

In Deinococcus radiodurans, RecBCD holoenzyme is not intact because of the absence of RecB and RecC, but a RecD-like protein does indeed exist. In this work, D. radiodurans recD disruptant was constructed and its possible biological functions were investigated. The results showed that disruption of the recD gene of D. radiodurans resulted in a remarkably increased sensitivity to hydrogen peroxide but had no apparent effect on the resistance to gamma and UV radiation. Furthermore, complementation experiments showed that Escherichia coli RecD, helicase domain or N-terminal domain of D. radiodurans RecD could not individually restore the resistant phenotype to hydrogen peroxide of the recD disruptant, whereas the complete D. radiodurans RecD protein could. Further studies showed that D. radiodurans RecD took part in antioxidant process by stimulating catalase activity and reactive oxygen species scavenging activity in D. radiodurans. These results suggest that D. radiodurans RecD has a new role in the antioxidant pathway.     

Role of RecA in DNA damage repair in Deinococcus radiodurans

It has been shown previously that the RecA protein of Deinococcus radiodurans plays a unique role in the repair of DNA damage in this highly DNA damage-resistant organism. Despite the high level of amino-acid identity, previous work has shown that Escherichia coli RecA does not complement D. radiodurans RecA mutants, further suggesting the uniqueness of D. radiodurans RecA. The work presented here shows that E. coli RecA does in fact provide partial complementation to a D. radiodurans RecA null mutant, suggesting that the RecA protein from D. radiodurans may not be as unique as believed previously.     

耐辐射球菌recQ双突变株的构建及逆境分析

RecQ解螺旋酶是生物有机体在进化中高度保守的SF1超级家族解螺旋酶的一个亚族,它对维持基因组的稳定性有重要的作用。耐辐射球菌野生型菌株R1有两个具有特殊结构的解螺旋酶DR1289和DR2444,运用PCR突变法克隆具有自身groEL启动子、KAT启动子与卡那霉素抗性基因、氯霉素抗性基因融合的DNA片段反向重组到基因组中,首次构建并鉴定了卡那霉素抗性完全突变株ΔDR1289,氯霉素抗性完全突变株ΔDR2444,双突变株ΔrecQ。辐射条件下和H2O2氧化压力下突变株生存率结果表明:ΔDR2444与R1存活率趋势线基本一致,而ΔDR1289和ΔrecQ双突变株较为敏感。根据上述结果推测,DR1289是一个对R1保持极端抗性的必须基因,而DR2444则是极端抗性的非必须基因。     

耐辐射奇球菌dps突变株的构建和蛋白功能初步研究

Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H2O2)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H2O2(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。     

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．     

The key residue for SSB-RecO interaction is dispensable for Deinococcus radiodurans DNA repair in vivo

The RecFOR DNA repair pathway is one of the major RecA-dependent recombinatorial repair pathways in bacteria and plays an important role in double-strand breaks repair. RecO, one of the major recombination mediator proteins in the RecFOR pathway, has been shown to assist RecA loading onto single-stranded binding protein （SSB） coated single-stranded DNA （ssDNA）. However, it has not been characterized whether the protein-protein interaction between RecO and SSB contributes to that process in vivo. Here, we identified the residue arginine-121 of Deinococcus radiodurans RecO （drRecO-R121） as the key residue for RecO-SSB interaction. The substitution of drRecO-R121 with alanine greatly abolished the binding of RecO to SSB but not the binding to RecR. Meanwhile, SSB-coated ssDNA annealing activity was also compromised by the mutation of the residue of drRecO. However, the drRecO-R121A strain showed only modest sensitivity to DNA damaging agents. Taking these data together, arginine-121 of drRecO is the key residue for SSB-RecO interaction, which may not play a vital role in the SSB displacement and RecA loading process of RecFOR DNA repair pathway in vivo.     

离子注入对耐辐射微球菌存活及生物量的影响

以耐辐射异常微球菌（Ｄ．ｒａｄｉｏｄｕｒａｎｓ）为试材,研究了离子注入对其存活和生物干重的影响。结果表明,不同能量和不同种类的离子注入Ｄ．ｒａｄｉｏｄｕｒａｎｓ,其存活均表现为先降后升再降的变化,即存活曲线呈现为相似的“马鞍型”,只是在不同种类的离子注入中,注入能量为２０ｋｅＶ的原子质量较大的Ｎ＋离子比Ｈ＋对其存活和生物干重的影响较大;在不同能量的离子注入中,能量相对较高的３０ｋｅＶ的Ｎ＋离子注入比２０ｋｅＶ的影响大。离子注入生物体产生这种不同于其它电离辐射的存活规律,意味着其对生物体的作用机理显著不同于其它电离辐射。     

辐射过程中耐辐射奇球菌蛋白酶变化的检测与分析

采用明胶和酪蛋白底物酶谱法以及荧光酪蛋白底物对紫外线以及γ射线辐射后恢复期耐辐射奇球菌R1(Deinococcus radiodurans R1,DRR1)的蛋白酶变化进行了检测。结果发现,DRR1存在高活性大分子量组成性表达蛋白酶,与Karlin等［16］提出的DRR1蛋白酶为预测高表达蛋白(PHX)的设想一致。DRR1包含大量分子量大于140kD 的明胶降解酶和分子量大于120kD的酪蛋白降解酶,其中活性最高的174kD明胶酶在经SDS变性处理后仍有较高活性,该蛋白酶在DRR1受紫外线辐射和电离辐射后恢复期的表达模式存在差异,在γ射线电离辐射过程中以及电离辐射后恢复的晚期活性较高。此外,还发现一些蛋白酶特异性由辐射所诱导,表明这些蛋白酶可能参与细胞信号通路中蛋白的顺序降解,也提示DRR1损伤修复过程中细胞内存在一个精确的蛋白酶系统。这些蛋白酶的表达与细胞的营养状态相关。同时对一株由本实验室从北京地区土壤中分离到的杆状耐辐射菌RR533.2的明胶和酪蛋白蛋白酶谱进行了测定,结果发现其蛋白酶谱与DRR1相类似。     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

耐辐射奇球菌铁结合蛋白DRA0258的抗氧化功能

【目的】通过对极端环境耐受的耐辐射奇球菌Deinococcus radiodurans R1全基因组进行序列比对分析,获得具有铁储备蛋白Ferritin类似功能基序的未知功能蛋白DRA0258,采用分子生物学技术对该蛋白的功能和性质进行了验证和分析。【方法】首先对DRA0258进行克隆表达和纯化,并经络合物显色法测定蛋白上铁结合含量;通过三段连接敲除法构建dra0258突变株,检测突变株在双氧水协迫下的生存率、总抗氧化活性及过氧化氢酶活性;利用实时定量PCR检测突变株内抗氧化酶类及铁转运相关性调控蛋白的基因转录水平。【结果】经体内外蛋白铁含量检测证实DRA0258具有一定的铁结合能力;双氧水生存率实验表明dra0258的缺失导致细胞的抗氧化能力显著下降;过氧化氢酶活性、总抗氧化活性检测及抗氧化酶类的基因转录水平检测证实dra0258基因的缺失导致细胞内一些抗氧化基因转录水平下调,细胞的抗氧化应激系统受到损伤,并影响了一些铁调控网络蛋白的基因转录水平。【结论】本研究证实DRA0258是一种铁结合蛋白,该编码基因的缺失影响胞内铁转运系统并使细胞抗氧化能力下调。     

耐辐射奇球菌crtI基因缺失突变株的构建及其功能研究

利用PCR方法和体内同源重组技术,对耐辐射奇球菌(Deinococcus radiodurans)中控制色素合成的关键基因———crtI进行缺失突变,成功获得红色色素缺失突变株M61。对突变株分别进行不同剂量电离辐射(IR)和不同浓度过氧化氢(H2O2)处理,结果表明:与野生型菌株R1相比,突变株M61对电离辐射的抗性降低;对过氧化氢的敏感性明显上升,在高浓度H2O2条件下表现异常敏感。HPLC分析结果显示,crtI基因的完全缺失对色素合成途径产生重要影响,导致番茄红素和其他红色类胡萝卜素的合成被抑制。证明crtI基因是耐辐射奇球菌中控制红色类胡萝卜素合成的一个关键基因。为阐明耐辐射奇球菌中类胡萝卜素参与的抗辐射和抗氧化机制奠定了一定基础,为进一步研究类胡萝卜素在耐辐射奇球菌中的合成途径及功能提供了思路。     

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。     

非生物胁迫下耐辐射异常球菌drB0118基因功能分析

【目的】鉴定和确定被预测为编码干燥相关蛋白的耐辐射异常球菌(Deinococcus radiodurans) drB0118基因功能,探讨该基因对盐、渗透和氧化胁迫抗性的作用。【方法】构建drB0118基因缺失突变株(ΔB0118),通过氯化钠、D-山梨糖醇和过氧化氢等胁迫冲击实验及氧化胁迫条件下qRT-PCR分析,研究drB0118突变对非生物胁迫反应及氧化胁迫相关基因表达的影响。【结果】drB0118突变导致菌株对NaCl和D-sorbitol胁迫的抗性降低;对氧化胁迫(H2O2)敏感;qRT-PCR分析显示,drB0118突变引起氧化胁迫抗性基因pod和oxyR分别下调4倍和10倍。【结论】D. radiodurans中drB0118参与了盐、渗透和氧化等多种非生物胁迫反应。     

High‐coverage proteomics reveals methionine auxotrophy in Deinococcus radiodurans

Deinococcus radiodurans is a robust bacterium best known for its capacity to resist to radiation. In this study, the SDS‐PAGE coupled with high‐precision LC‐MS/MS was used to study the D. radiodurans proteome. A total of 1951 proteins were identified which covers 63.18% protein‐coding genes. Comparison of the identified proteins to the key enzymes in amino acid biosyntheses from KEGG database showed the methionine biosynthesis module is incomplete while other amino acid biosynthesis modules are complete, which indicated methionine auxotrophy in D. radiodurans. The subsequent amino acid‐auxotrophic screening has verified methionine instead of other amino acids is essential for the growth of D. radiodurans. With molecular evolutionary genetic analysis, we found the divergence in methionine biosynthesis during the evolution of the common ancestor of bacteria. We also found D. radiodurans lost the power of synthesizing methionine because of the missing metA and metX in two types of methionine biosyntheses. For the first time, this study used high‐coverage proteome analysis to identify D. radiodurans amino acid auxotrophy, which provides the important reference for the development of quantitative proteomics analysis using stable isotope labeling in metabolomics of D. radiodurans and in‐depth analysis of the molecular mechanism of radiation resistance.     

Radiation resistance of Deinococcus radiodurans R1 with respect to growth phase

Deinococcus species exhibit an extraordinary ability to withstand ionizing radiation (IR). Most of the studies on radiation resistance have been carried out with exponential phase cells. The studies on radiation resistance of Deinococcus radiodurans R1 with respect to different phases of growth showed that late stationary phase cells of D. radiodurans R1 were fourfold more sensitive to IR and heat as compared with exponential or early stationary phase cells. The increased sensitivity of D. radiodurans R1 to IR in the late stationary phase was not due to a decrease in the intracellular Mn/Fe ratio or an increase in the level of oxidative protein damage. The resistance to IR was restored when late stationary phase cells were incubated for 15 min in fresh medium before irradiation, indicating that replenishment of exhausted nutrients restored the metabolic capability of the cells to repair DNA damage. These observations suggest that stress tolerance mechanisms in D. radiodurans R1 differ from established paradigms.     

RecX is involved in antioxidant mechanisms of the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans shows remarkable resistance to reactive oxygen species (ROS), generated by irradiation. Disruption of recX (dr1310) in D. radiodurans using targeted mutagenesis method enhanced its ROS scavenging activity, and recX overexpression in this bacterium repressed its antioxidant activity significantly. Further analyses on catalase and superoxide dismutase, two important antioxidant proteins in cells, showed that RecX could repress the induction of antioxidant enzymes, revealing that it negatively regulates the ROS scavenging activity in D. radiodurans.     

抗辐射菌中DNA损伤修复主要基因群的研究进展

抗辐射红色球菌对电离辐射具有很高的放射线抵抗性,该菌具有惊人的DNA的二条链切断的修复能力,由辐射等引起的切断损伤DNA在几至十几小时内能高效正确地进行完全修复。在对切断的双链DNA进行修复时,除了大肠杆菌等生物在切断的双链DNA修复时出现的蛋白质以外,还有该菌所特有的修复蛋白质也参与修复。本文对该菌所特有的DNA二条链的切断损伤修复的主要基因及其相互作用进行了简要介绍。     

耐辐射奇球菌超氧化物歧化酶基因的克隆与序列分析

By using a 453 bp length gene fragment of superoxide dismutase(SOD)as a probe,which was firstly amplified from Deinococcus radiodurans genomic DNA by PCR with degenerate oligonucleotide primers corresponding to the conservative regions of known SODs,a putative SOD gene was identified from the database of D.radiodurans whole genome.Its 636 bp length open reading frame and 5′ and 3′ flanking sequence was determined.The conventional E.coli ribosomal and RNA polymerase binding sites were found upstream from SOD encoding region and an inverted repeat sequence downstream of the termination codon.The deduced 211 amino acid sequence of the structural gene showed a high similarity to other manganese and iron containing SODs in normally conserve regions.