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Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China
Correspondence
Yuejin Hua
yjhua{at}zju.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-rays, UV radiation, oxidizing agents and desiccation (Cox & Battista, 2005
; Makarova et al., 2001). The remarkable capacity of D. radiodurans to survive damage has been attributed to three mechanisms: prevention, tolerance and repair (White et al., 1999), and especially to its efficient DNA repair mechanisms (Minton, 1994). Although current research is concentrated on the proteins related to DNA repair, and several important genes involved in DNA repair have been identified in D. radiodurans (Earl et al., 2002; Hua et al., 2003; Narumi et al., 2004), the repair pathways employed by this organism remain unclear.
Although the mechanisms underlying the extraordinary resistance of this bacterium are still poorly understood, it was deduced that free radical scavengers are important contributors to the prevention mechanism, and have an important role in preventing DNA damage in D. radiodurans (Ghosal et al., 2005; Tian et al., 2004). D. radiodurans contains superoxide dismutase (SOD), catalase (CAT) and organic hydroperoxide resistance protein (OHRP), which serve as free radical scavengers (Makarova et al., 2001; Meunier-Jamin et al., 2004). Recently, the role of an antioxidant metabolite pyrroloquinoline–quinone (PQQ) from D. radiodurans in Escherichia coli has been reported; PQQ conferred markedly increased protection against reactive oxygen species in E. coli (Khairnar et al., 2003). Moreover, Daly et al. (2004) reported that non-enzymic Mn(II) ions might act as antioxidants and reinforce enzymic antioxidant systems that defend against oxidative stress during recovery.
Carotenoids are well known for their free radical scavenging activities. The protective role of carotenoids against oxidative damage is essential to various organisms including photosynthetic and non-photosynthetic species (Armstrong & Hearst, 1996). The radioresistance of deinococci and sarcinae appears to be associated with the pigmentation of the strains (Mosely, 1983). D. radiodurans R1 has been reported to synthesize a unique carotenoid product that was identified as deinoxanthin (Laurant et al., 1997). However, few investigations concerning the antioxidant effects of this carotenoid have been done to date (Melin et al., 1998), and its biosynthesis pathway remains unclear. Generally, carotenoid biosynthesis starts from the condensation of two molecules of geranylgeranyl diphosphate, which generates phytoene. This step is followed by four steps of desaturation catalysed by CrtI resulting in the synthesis of lycopene, or three-step desaturation catalysed by CrtP resulting in the production of neurosporene (Armstrong et al., 1990; Ehrenshaft & Daub, 1994; Harada et al., 2001). Further downstream reactions, including cyclization of lycopene, addition of keto groups and hydroxylation of cyclized carotene, lead to the formation of different products.
Results from comparative genomic analysis showed that the D. radiodurans genome includes several genes predicted to be involved in carotenoid synthesis (Makarova et al., 2001). Two genes encoding ketolase (CrtO) and cyclase (CrtLm) in D. radiodurans were described recently (Tao & Cheng, 2004; Tao et al., 2004). Further work needs to be done on other related genes to elucidate the deinoxanthin biosynthesis pathway in this bacterium. On the basis of sequence alignment and conserved amino acid analysis, we identified three genes as putative candidates for the phytoene desaturase of D. radiodurans. Among them, DR0810 showed 34 % identity to the phytoene desaturase of Rubobacter xylanophilus. Although DR0093 had 41 % similarity to the carotenoid desaturase of Nocardia farcinica, a recent study has shown that DR0093 acts as a 
-carotene ketolase (Tao & Cheng, 2004). Another candidate, DR0861, exhibited the highest similarity to the phytoene desaturase sequence from the cyanobacterium Gloeobacter violaceus: about 68 % global identity and nearly 80 % similarity. Two highly conserved regions were detected in DR0861 and DR0810 genes: the putative dinucleotide-binding motif (
fold) in the N-terminal region (Fig. 1a) and the ‘bacterial-type phytoene desaturase signature’ at the C terminus (Fig. 1b).
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METHODS |
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. D. radiodurans R1 (ATCC 13939) was used as the wild-type strain and for construction of mutants and was grown in TGY medium as described previously (Mosely, 1983). Kanamycin (20 µg ml–1) or chloramphenicol (3 µg ml–1) was added to the medium if required. E. coli strains were cultured in LB broth.
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Construction of D. radiodurans mutants.
Mutants were constructed by double crossover recombination of a kanamycin resistance cassette into the genome. The sequence of D. radiodurans R1, obtained through NCBI (http://www.ncbi.nih.gov/Database), was used to design the primers (Table 2). A 900 bp BamHI fragment upstream and a 900 bp HindIII fragment downstream of the targeted genes were amplified using PCR. The kanamycin resistance cassette containing the groES promoter was obtained from pRADK, a shuttle plasmid modified from pRADZ3 (Gao et al., 2005; Meima & Lidstrom, 2000). The upstream and downstream fragments were digested with BamHI and HindIII respectively, and ligated to the BamHI–HindIII fragment of the kanamycin resistance cassette. The joint product of the three fragments was further amplified by PCR and ligated into the pGEM-T Easy vector to yield pTK (Table 1
). The plasmid was linearized with EcoRV and transformed into D. radiodurans R1 with CaCl2 as described previously (Kitayama et al., 1983). Mutants were selected on TGY plates containing an appropriate antibiotic. The N-terminal motif (amino acids 19–69) deletion mutant was constructed by the same method and designated 
crtIDO. All the recombinants were verified by PCR and sequencing.
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). Another 1489 bp fragment carrying the complete Er. uredovora crtI gene was obtained from the plasmid pACCRT-EIB (Misawa et al., 1995). The fragment was digested with EcoRV and BamHI and ligated into pRADZ3, and the resulting plasmid was named pRADIEu (Table 1).
A plasmid for overexpression of DR0861 in E. coli was also constructed. The intact phytoene desaturase gene homologue was amplified by PCR with engineered NdeI and XhoI restriction sites, then ligated into pET28a to create the expression plasmid pET28I. The fragment was sequenced to confirm the correct sequence and orientation. Plasmid pET28I was transformed into E. coli BL21(DE3) and the resulting strain was named BLI. The E. coli transformant carrying plasmid pACCRT-EB (Misawa et al., 1990) was named BLEB. The strain transformed with pET28I and pACCRT-EB was named BLEBI.
Analysis of carotenoids.
Briefly, carotenoids from D. radiodurans were extracted as follows: exponential-phase cell pellets obtained from 50 ml cell culture were extracted three times with 1 ml acetone/methanol (7 : 2, v/v) in the dark and the supernatant was collected. For the extraction of carotenoids generated in E. coli, cells carrying pACCRT-EB and/or pET28I were incubated in 100 ml LB broth with shaking at 37 °C. After the addition of 0.5 mM IPTG and subsequent incubation for 6 h, cells were harvested by centrifugation at 5100 g for 15 min. Carotenoids in E. coli were extracted by the method described above.
Carotenoids in the pooled extracts were analysed by HPLC using a Waters Alliance 2690 system and a Hypersil C18 column (250x4.6 mm, 5 µm, Alltech). A mixture of acetonitrile/methanol/2-propanol (40 : 50 : 10, by vol.) was used as the mobile phase. The eluted fractions were monitored using a Waters 996 photodiode array detector scanning from 200 to 800 nm. The flow rate was 0.8 ml min–1. Carotenoids were identified by retention time, features of absorption spectra and by comparison with standard compounds or with reported data. The amount of lycopene generated in E. coli transformants was determined from the area under the peak detected at 470 nm using a calibration curve obtained with a lycopene standard (Sigma).
In vitro reaction and enzyme activity assay.
The substrate carotene (phytoene) was extracted from E. coli containing pACCRT-EB by the method described above. The hydrophobic carotene was treated with soybean L--phosphatidylcholine (20 %, w/v) to make a suspension and processed as previously described (Schneider et al., 1997). The recombinant enzyme was produced in E. coli BL21(DE3) with pET28I and purified by a method described previously (Fraser et al., 1992). Enzyme activity was assayed as follows. The reaction mixture (0.5 ml) contained the appropriate substrate (approx. 4 µg), 1.5 µM NADP and 30 µg purified enzyme in 100 mM Tris/HCl buffer (pH 7.9). The reaction was established by adding catalase (20 000 U ml–1), glucose (2 mM) and glucose oxidase (20 U ml–1). Incubation was in the dark at 37 °C for 4 h with shaking at 200 r.p.m. The reaction was terminated by adding methanol (1 ml) containing 6 % (w/v) KOH and heating at 60 °C for 15 min. The products formed were extracted from the incubation mixture with diethyl ether/light petroleum (1 : 9, v/v). The above solvent phase was evaporated to dryness and the residue was redissolved in cool acetone/methanol (7 : 2, v/v). The reaction products were identified by HPLC using a Waters 600 system and a Hypersil ODS2 column (250x4.6 mm, 5 µm, Elite). The other conditions were as described in the preceding section.
Measurement of cell survival rate.
The survival of D. radiodurans exposed to H2O2 and -radiation was determined by methods described previously (Carbonneau et al., 1989; Funayama et al., 1999). For the survival rate of E. coli exposed to -rays, cells grown to an OD600 of about 0.5 were induced with 0.5 mM IPTG for an additional 6 h, and then were subjected to irradiation. The survival rate of E. coli treated with hydrogen peroxide was assayed as described previously (Konola et al., 2000) with some modifications. Cultures of E. coli were induced with 0.5 mM IPTG for 6 h, then 30 % H2O2 solution was added to the cultures to obtain final concentrations of 1, 2, 3 and 4 mM. No H2O2 was added to the control culture. After incubation with shaking at 200 r.p.m. for 15 min, catalase (Sigma) was added in excess (100 µg ml–1) to each culture to inactivate H2O2. The cells were diluted appropriately, spread on TGY agar plates and incubated at 37 °C for 20 h. Survival rate was expressed as the number of colonies obtained from treated samples as a percentage of the number for untreated controls.
Statistical analysis.
For cell survival experiments, experiments were performed four times, with three replicates in each experiment. The values were represented as mean±SD (n=12). Results were assessed by Student’s t-test using Microsoft Excel, and P<0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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crtI) turned out to be colourless. However, the DR0810 mutant was still red in colour and identical to the wild-type strain. Interestingly, the phenotype of the N-terminal motif (amino acids 19–69) deletion mutant (crtIDO) was also colourless like crtI.
Subsequently, the carotenoid compositions of wild-type and mutants were analysed by HPLC. For the wild-type, more than 10 peaks were present in the elution profile of carotenoid extracts as detected at 470 nm (Fig. 2a). Peak 1 was identified as deinoxanthin, based on its retention time and absorption spectral features (Laurant et al., 1997). No peak was detected in the elution profiles of crtI by HPLC at 470 nm (Fig. 2b), indicating that most of the carotenoid synthesis was blocked owing to the loss of DR0861. Moreover, as shown in Fig. 2(e), mutation of DR0861 resulted in the accumulation of phytoene, which is the substrate of phytoene desaturase. These results suggested that DR0861 might encode phytoene desaturase.
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crtI with heterologous or homologous phytoene desaturase genescrtI. The complementary transformant exhibited red pigment when plasmid pRADIDR, containing the intact DR0861 gene, was introduced into the colourless crtI mutant. HPLC analysis revealed that its carotenoid composition was restored (Fig. 2c, f) and similar to that of the wild-type (Fig. 2a, d). Plasmid pRADIEu containing the crtI gene from Er. uredovora was also introduced into crtI and it restored the carotenoid composition to that of the wild-type (data not shown).
Carotenoid composition in E. coli expressing DR0861
DR0861 was expressed in E. coli strain BL21. The protein extracted from cell homogenates was separated by 12 % (w/v) SDS-PAGE as shown in Fig. 3. The expected protein expressed in E. coli had an apparent molecular mass of 60 kDa (lane 3–7), and was absent in the control strain (lane 2). Even when induced at different temperatures (25–37 °C), the recombinant protein was less than 10 % (w/w) of the total cellular protein. This may be due to the low solubility of membrane-associated proteins in the cells or to biased codon usage (Fraser et al., 1992; Matsumura et al., 1997).
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) and 15,15'-cis-phytoene was the only carotenoid as detected at 280 nm (Fig. 4c). When plasmid pET28I was introduced into E. coli with pACCRT-EB, the cell suspension showed red pigmentation after induction by IPTG. Lycopene accumulated as the major carotenoid and phytoene was scarcely detectable (Fig. 4b, d). This result indicated that DR0861 acts as phytoene desaturase (CrtI), which catalyses lycopene biosynthesis from phytoene.
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. The substrate phytoene was converted into lycopene as the end product of the desaturation reaction, and the peak typical of all-trans-lycopene (peak 1 in Fig. 5b) was found, with absorbance maximum at 470 nm, while 15,15'-cis-phytoene (peak 2 in Fig. 5a) had its absorbance maximum at 280 nm in the elution profile. From the in vitro and in vivo enzyme activity assays, we can conclude that lycopene in D. radiodurans is formed from phytoene through a desaturation reaction catalysed by DR0861 protein.
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crtI-rays. The wild-type strain exhibited a great resistance to irradiation with a D10 dose of 8554 Gy (Fig. 6), whereas the colourless mutant crtI showed a significant decrease of radioresistance at doses higher than 2000 Gy (P<0.05) and its D10 was 6803 Gy. This result indicated that loss of pigment affects the radioresistance of D. radiodurans. The survival rate of the crtIDO mutant was not significantly different from that of the crtI null mutant crtI (P>0.05). Moreover, when complemented with pRADIDR, the ionizing radiation resistance of the mutants was completely restored. From these results, we deduced that carotenoids contribute to radioresistance and provide some protection in D. radiodurans. However, overexpression of the crtI gene in the wild-type hardly improved the extent of resistance (Fig. 6), presumably because the production of carotenoids is controlled by many carotenoid synthesis enzymes, precursor supply and product storage in the bacteria.
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). A greater decrease in resistance was observed for the colourless strain compared to the wild-type (P<0.01) when H2O2 concentration exceeded 20 mM,. Overexpression strain RIO showed no increase in its survival rate when exposed to H2O2, compared with that of the wild-type under the same growth conditions (Fig. 7c).
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). To examine its protective effect against oxidative damage in E. coli, we expressed the crtI gene from D. radiodurans in E. coli containing pACCRT-EB and determined the amount of lycopene generated in the transformants, and investigated the survival rate of E. coli transformants exposed to different doses of H2O2 and -rays.
Table 3 shows the production of lycopene in various strains. Transformant BLEB with pACCRT-EB showed a slight resistance to oxidative damage (Fig. 8a), due to the antioxidant effect of phytoene synthesized in vivo (Table 3). Without induction by IPTG, a small quantity of lycopene (52.40±3.15 µg g–1) was produced in transformant BLEBI (no IPTG). Compared to the control (E. coli host strain), the lycopene accumulated in E. coli BLEB gave significant protection against H2O2 (P<0.001). After induction by IPTG, the cells produced a large quantity of lycopene (221.14±17.37 µg g–1) (Table 3). Overproduction of lycopene in the E. coli transformant had no influence on its survival in the absence of environmental stresses. However, a pro-oxidant phenomenon was observed in the E. coli after the transformant was exposed to H2O2. The cells overproducing lycopene became more sensitive to H2O2 than the wild-type, and lost most of their viability even when exposed to 1 mM H2O2 (Fig. 8a).
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-rays. Significant differences in survival rate between the colourless E. coli host strain and red-pigment transformant strains were observed. As shown in Fig. 8b, E. coli BLEBI without IPTG induction exhibited much greater radioresistance to the lethal effects of ionizing radiation than the control, E. coli BL21(DE3) (P<0.01). The D10 dose of the former was 175 Gy whereas that of the latter was 136 Gy. However, when lycopene was overproduced after induction by IPTG (Table 3), the survival rate of E. coli BLEBI exposed to -rays decreased significantly (D10=47.8 Gy), compared to that of the control (P<0.05). This negative effect of overproduction of lycopene on cell survival is similar to the pro-oxidant effect of lycopene in the H2O2 treatment experiment. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and this extraordinary ability has been mostly attributed to its efficient DNA repair (Makarova et al., 2001). Although some novel genes involved in DNA repair in D. radiodurans have been identified recently (Earl et al., 2002; Hua et al., 2003; Narumi et al., 2004), the mechanisms underlying its extraordinary resistance have remained unclear. Furthermore, it was discovered that free radical scavengers had an important role in the protective mechanism of D. radiodurans (Daly et al., 2004). The enzymic antioxidants including CAT and SOD act as important free radical quenchers and their mutants became more sensitive to ionizing radiation than the wild-type (Markillie et al., 1999). Expression of PQQ synthase from D. radiodurans increased the radioprotective and oxidative stress protective capabilities of E. coli (Misra et al., 2004). These findings suggest that enzymes and antioxidants contribute to the cellular defence system in D. radiodurans. Carotenoids are strong antioxidants that scavenge reactive oxygen species including singlet oxygen and hydroxyl radicals (Glaeser & Klug, 2005). The red-pigmented D. radiodurans synthesizes a unique carotenoid product that was identified as deinoxanthin. However, the deinoxanthin biosynthesis pathway and its functions in D. radiodurans have not been elucidated.
We determined that DR0861 encodes the bacterial-type phytoene desaturase (CrtI) in D. radiodurans on the basis of gene mutation, function complementation, heterologous expression and in vitro enzyme activity assays. From the sequence alignments and comparison analysis, DR0861 has the highest identity to the phytoene desaturase of G. violaceus, which was identified as the first oxygenic photosynthetic organism using a bacterial-type phytoene desaturase (CrtI) to convert phytoene into lycopene, rather than a cyanobacterial or plant-type phytoene desaturase (CrtP/Pds) (Steiger et al., 2005; Tsuchiya et al., 2005). The dinucleotide-binding motif and signature domains found in some phytoene desaturases of bacteria and fungi (Armstrong et al., 1989; Bartley et al., 1990; Linden et al., 1994; Ruiz-Hidalgo et al., 1997) are also present in the sequence of DR0861 (Fig. 1). These observations indicate that phytoene desaturases including DR0861 existing in bacteria are conserved and may come from a common ancestor. Phylogenetic analysis based on CrtI sequences provided strong support for this hypothesis (data not shown).
We found that DR0861 gene knockout and dinucleotide-binding domain deletion mutations inhibited lycopene synthesis and resulted in phytoene accumulation (Fig. 2b, e). Although the DR0810 gene, another putative phytoene desaturase gene annotated in the database, has more than 30 % identity with the phytoene desaturase of Rubobacter xylanophilus, our results showed that DR0810 was not involved in lycopene synthesis on the basis of gene knockout and HPLC analysis. Complementation of the knockout mutant with the crtI gene from D. radiodurans or Er. uredovora restored carotenoid synthesis in D. radiodurans completely (Fig. 2c), indicating that the inactive crtI gene was responsible for the loss of pigmentation in the crtI mutant.
To investigate the protective role of carotenoids in D. radiodurans, we measured the cell survival rates of the colourless mutants and wild-type strain exposed to ionizing radiation and oxygen stress. The colourless mutants became significantly more sensitive to -rays and H2O2 than the wild-type. The sensitivity of colourless mutants was not as marked as that of DNA repair-defective strains. The resistance of the mutants could be recovered when the pigment was restored after the intact crtI gene from D. radiodurans or Er. uredovora was used for complementation. These results demonstrated that carotenoids provide some protection against radiation and oxygen damage in D. radiodurans. The relatively modest effect of carotenoids on cell survival may be due to the fact that the carotenoids are not involved in the DNA repair process directly, but participate in the protective mechanism of D. radiodurans as ROS scavengers. The carotenoids can quench harmful ROS generated from water radiolysis to prevent oxidative damage to proteins, including DNA repair proteins, and membrane lipid peroxidation. It has been reported that carotenoids, including lycopene, can efficiently inhibit membrane lipid peroxidation (Stahl et al., 1998). Pyrroloquinoline quinone, another antioxidant metabolite in this bacterium, has also shown obvious protective effects on the cell (Khairnar et al., 2003). It is not clear whether there is a synergetic effect between this compound and the carotenoids. Further research needs to be done to elucidate the protection mechanism of carotenoids and the relationships of these antioxidant metabolites in D. radiodurans.
Furthermore, our results showed that overexpression of the crtI gene in D. radiodurans did not result in a notable increase in the carotenoid content and the cells showed no markedly enhanced resistance to -rays and H2O2 (Fig. 6, 7). This was probably due to carotenoid synthesis being controlled by many carotenoid synthesis enzymes, precursor supply and product storage in the bacteria.
To confirm the function of the DR0861 gene, it was expressed in E. coli containing pACCRT-EB. The transformant became red-pigmented and accumulated lycopene (Fig. 4b). Desaturation activity of DR0861 protein was further tested by in vitro enzyme activity assays. The substrate 15,15'-cis-phytoene was converted into all-trans-lycopene as the end product of the desaturation reaction (Fig. 5). These results indicated that DR0861 had desaturase activity, which was responsible for the conversion of phytoene to lycopene through four-step, not three-step, desaturation in D. radiodurans. We also determined the amount of lycopene synthesized in E. coli transformants, and assessed its protective effects on E. coli. Without IPTG induction, the production of lycopene in E. coli transformants was less than that in IPTG-induced transformants, and led to a significant enhancement of resistance to H2O2 and -rays, suggesting that carotenoid synthesis can supply E. coli cells with some protection. However, after induction with IPTG, the superfluous production of lycopene had a pro-oxidant effect when the cells were exposed to H2O2 or -rays, which caused a significant decrease in cell survival rate (Fig. 8). On the other hand, the survival capacity of E. coli containing only pACCRT-EB or pET28I receiving the same treatment with IPTG was not affected. Pro-oxidant actions of lycopene have also been reported in other biological systems (Lowe et al., 1999; Yeh & Hu, 2000). The antioxidant activity of some carotenoids may shift into pro-oxidant activity, depending on their concentrations as well as on the biological environment including oxygen tension (Palozza, 1998). Therefore, the pro-oxidant effect of lycopene in this study may result from its high levels in the E. coli transformants and the oxidative stress from the treatment with H2O2 and -rays.
In this study, we identified a typical crtI-type phytoene desaturase gene (DR0861) involved in the deinoxanthin biosynthesis pathway of D. radiodurans and investigated the protective effects of carotenoids against radiation and oxidative damage in D. radiodurans and E. coli. Further investigation of the remaining unidentified carotenogenic enzyme genes and the function of the corresponding carotenoid metabolites would facilitate elucidation of carotenoid biosynthesis pathways and actions of carotenoid metabolites in D. radiodurans.
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ACKNOWLEDGEMENTS |
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Edited by: J. Green
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Received 27 September 2006;
revised 24 January 2007;
accepted 29 January 2007.
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), 
), 
), RIO (
) and KIDR (
). Values are the mean±SD of four independent experiments (n=12).
, 19.61 mM H2O2; 
, BL21 carrying pET-28I (with IPTG induction);