Recombinant D. radiodurans cells for bioremediation of heavy metals from acidic/neutral aqueous wastes

The stability and superior metal bioremediation ability of genetically engineered Deinococcus radiodurans cells, expressing a non-specific acid phosphatase, PhoN in high radiation environment has already been established. The lyophilized recombinant DrPhoN cells retained PhoN activity and uranium precipitation ability. Such cells also displayed an extended shelf life of 6 months during storage at room temperature and showed surface associated precipitation of uranium as well as other metals like cadmium. Lyophilized cells, immobilized in polyacrylamide gels could be used for uranium bioprecipitation in a flow through system resulting in 70% removal from 1mM input uranium solution and a loading of 1 g uranium/g dry weight cells. Compared with a batch process which achieved a loading of 5.7 g uranium/g biomass, the efficiency of the column process was low due to clogging of the column by the precipitate.     

Contact-free cold atmospheric plasma treatment of Deinococcus radiodurans

In this study we investigated the sensitivity of Deinococcus radiodurans to contact-free cold atmospheric plasma treatment as part of a project to establish new efficient procedures for disinfection of inanimate surfaces. The Gram-positive D.?radiodurans is one of the most resistant microorganisms worldwide. Stationary phases of D.?radiodurans were exposed to cold atmospheric plasma for different time intervals or to ultraviolet?C (UVC) radiation at dose rates of 0.001-0.0656?J?cm(-2), respectively. A methicillin-resistant Staphylococcus aureus strain (MRSA) served as control for Gram-positive bacteria. The surface microdischarge plasma technology was used for generation of cold atmospheric plasma. A plasma discharge was ignited using ambient air. Surprisingly, D.?radiodurans was sensitive to the cold atmospheric plasma treatment in the same range as the MRSA strain. Survival of both bacteria decreased with increasing plasma exposure times up to 6 log(10) cycles (>99.999?%) within 20?s of plasma treatment. In contrast, UVC radiation of both bacteria demonstrated that D.?radiodurans was more resistant to UVC treatment than MRSA. Cold atmospheric plasma seems to be a promising tool for industrial and clinical purposes where time-saving is a critical point to achieve efficient disinfection of inanimate surfaces and where protection from corrosive materials is needed.     

Deinococcus radiodurans strain R1 contains N6-methyladenine in its genome

Methylation of DNA is known to be involved in DNA repair mechanisms in bacteria. Deinococcus radiodurans strain R1 on exposure to high radiation undergoes significant DNA damage, which is repaired without mutations. However, the presence of modified nucleotides has not been reported in its genome. We report here the detection of N6-methyladenine in the genome of D. radiodurans strain R1 using immunochemical techniques. This N6-methyladenine is not a part of GATC restriction-modification system. D. radiodurans cell extract also exhibited a DNA adenine methyltransferase activity which was reduced in the early post-irradiation recovery phase.     

Engineering of Deinococcus radiodurans R1 for Bioprecipitation of Uranium from Dilute Nuclear Waste

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an ~27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of ⁶⁰Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste.     

Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30

Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.     

Protein recycling is a major component of post-irradiation recovery in Deinococcus radiodurans strain R1

Exposure to 6kGy dose of (60)Co gamma-rays resulted in immediate growth arrest, followed by complete recovery of Deinococcus radiodurans strain R1 cells. Selective degradation and resynthesis of several predicted highly expressed proteins (including major chaperones, key TCA cycle enzymes, and few stress proteins) and several hypothetical proteins marked the lag period, preceding resumption of growth. A major exercise in protein recycling appears to be an integral component of post-irradiation recovery in D. radiodurans and complements the extensive DNA repair, characteristic of this extremely radioresistant bacterium.     

Functional analysis of γ-carotene ketolase involved in the carotenoid biosynthesis of Deinococcus radiodurans

Deinococcus radiodurans strain R1 synthesizes a unique ketocarotenoid product named deinoxanthin. The detailed steps involved in the biosynthesis of deinoxanthin remain unresolved. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans . Analysis of the carotenoids in the resultant mutant R1ΔcrtO demonstrated that dr0093 encodes γ-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position of γ-carotene derivatives to form ketolated carotenoids. The mutant R1ΔcrtO became more sensitive to H₂O₂ treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans . Carotenoid extracts from mutant R1ΔcrtO exhibited lower 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.     

Shuttle plasmids constructed by the transformation of an Escherichia coli cloning vector into two Deinococcus radiodurans plasmids

An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.     

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.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

The LexA Protein from Deinococcus radiodurans Is Not Involved in RecA Induction following γ Irradiation

The involvement of LexA in induction of RecA was investigated in Deinococcus radiodurans. As in the wild-type strain, an increase in RecA protein synthesis following gamma irradiation was detected in a lexA disruptant, indicating that LexA is not involved in the induction of RecA in D. radiodurans.     

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination

Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed.     

Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments

We have developed a radiation resistant bacterium for the treatment of mixed radioactive wastes containing ionic mercury. The high cost of remediating radioactive waste sites from nuclear weapons production has stimulated the development of bioremediation strategies using Deinococcus radiodurans, the most radiation resistant organism known. As a frequent constituent of these sites is the highly toxic ionic mercury (Hg) (II), we have generated several D. radiodurans strains expressing the cloned Hg (II) resistance gene (merA) from Escherichia coli strain BL308. We designed four different expression vectors for this purpose, and compared the relative advantages of each. The strains were shown to grow in the presence of both radiation and ionic mercury at concentrations well above those found in radioactive waste sites, and to effectively reduce Hg (II) to the less toxic volatile elemental mercury. We also demonstrated that different gene clusters could be used to engineer D. radiodurans for treatment of mixed radioactive wastes by developing a strain to detoxify both mercury and toluene. These expression systems could provide models to guide future D. radiodurans engineering efforts aimed at integrating several remediation functions into a single host.     

Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors

The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.     

Mutation in recR gene of Deinococcus radiodurans and possible involvement of its product in the repair of DNA interstrand cross-links

We previously reported that some Deinococcus radiodurans mutants are sensitive to DNA interstrand cross-linking agents but resistant to UV and gamma-rays. We isolated DNA fragments from a D. radiodurans genomic library which complemented the mitomycin C sensitivity of one of these mutants. One 3.2kb-long fragment contains an open reading frame of approximately 700bp and the deduced amino acid sequence is very homologous to other prokaryotic RecR proteins. This open reading frame in the mitomycin C-sensitive mutant strain contains a frame shift mutation at its carboxyl terminal region. These data suggest that RecR protein plays an important role in the resistance to interstrand cross-links in this bacterium.     

Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.     

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.     

Antibiotic Sensitivity of Micrococcus radiodurans

A wild-type strain of Micrococcus radiodurans and its nonpigmented mutant W(1) were tested for sensitivity to 10 antibiotics selected from the standpoint of their mechanism of action. Representatives of groups of antibiotics inhibiting deoxyribonucleic acid (DNA) synthesis, DNA-dependent ribonucleic acid synthesis, protein synthesis, and cell wall synthesis were selected. M. radiodurans and its mutant exhibited full susceptibility to all antibiotics tested (mitomycin C, actinomycin D, chloramphenicol, dihydrostreptomycin, erythromycin, neomycin, kanamycin, benzylpenicillin, bacitracin, and vancomycin), the degree of susceptibility being of the same order as that of a standard strain of Staphylococcus aureus 209 P, with the exception of dihydrostreptomycin.     

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。