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Molecular Microbiology (2008) 67(6), 1211–1222 䊏

doi:10.1111/j.1365-2958.2008.06113.x First published online 13 February 2008

DrRRA: a novel response regulator essential for the extreme radioresistance of Deinococcus radiodurans Liangyan Wang,1,2† Guangzhi Xu,1† Huan Chen,3 Ye Zhao,1 Nan Xu,1 Bing Tian1 and Yuejin Hua1* 1 Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, 310029, China. 2 College of Life Sciences, Zhejiang University, 310029, China. 3 James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou 310008, China.

Summary Two-component systems are predominant signal transduction pathways in prokaryotes, and also exist in many archaea as well as some eukaryotes. A typical TCS consists of a histidine kinase and a cognate response regulator. In this study, a novel gene encoding a response regulator (we designate it drRRA) is identified to be essential for the extreme radioresistance of Deinococcus radiodurans. DrRRA null mutant (we designate it MR) is sensitive to gamma-radiation compared with the wild-type strain. Transcriptional assays show that numerous genes are changed in their transcriptional levels in MR at exponential growth phase under normal or gammaradiation condition. Most of them are related to stress response and DNA repair. Antioxidant activity assays exhibit that both superoxide dismutases and catalases are decreased in the mutant, whereas Western blotting assays show that RecA and PprA are also reduced in MR, verifying the microarray and quantitative real-time PCR data. Furthermore, pulsed-field gel electrophoresis assay demonstrates that deletion of drRRA results in the delay of genome restitution. These data support the hypothesis that DrRRA contributes to the extreme radioresistance of D. radiodurans through its regulatory role in multiple pathways such as antioxidation and DNA repair pathways.

Accepted 4 January, 2008. *For correspondence. E-mail yjhua@ zju.edu.cn; Tel. (+86) 57186971703; Fax (+86) 57186971703. †These authors contributed equally to this work.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

Introduction Deinococcus radiodurans, one of the most radioresistant organisms in the world, is characterized by its unusual capability of tolerating extensive DNA damages from diverse sources such as gamma-radiation, ultraviolet, hydrogen peroxide and desiccation (Makarova et al., 2001). The wild-type strain shows no loss of viability when exposed to gamma-radiation at the dosage of 5 kGy during exponential growth phase (Moseley and Evans, 1983). For years it has been an ideal model organism for studying genes involved in maintaining DNA integrity and stability, because of its extraordinary ability to survive heavy irradiation. However, the molecular mechanisms underlying these phenotypes remain unknown. Just like other microorganisms, D. radiodurans might sense and respond to ambient conditions to avoid various damages through signal pathways, the exploration of which might be helpful to reveal the basis of the extreme radioresistance of this bacterium. Recently, a lipoprotein kinase in Escherichia coli has been suggested to play an important role in radiation-induced DNA strand break repair, illuminating the significance of signal transduction in DNA repair (Khairnar et al., 2007). Two-component systems (TCS) are predominant signal transduction pathways in prokaryotes (Bekker et al., 2006; Hwang et al., 2002). Transcriptome analyses of two-component regulatory system mutants of E. coli show that transcriptional levels of numerous genes are significantly altered (Oshima et al., 2002; Zhou et al., 2003). Despite the great interest in mechanisms underlying DNA damage response, little attention has been paid to two-component signal pathways in D. radiodurans. There is a large family of two-component signal pathways in D. radiodurans (Makarova et al., 2001). Some of them might be related to the radioresistance of this special organism. A typical TCS consists of a histidine kinase (HK) and a partner response regulator (RR). The HK containing an invariant histidine residue is auto-phosphorylated in response to external or internal signals. Then, the phosphoryl group is transferred to the aspartate residue on the RR. The RR is generally composed of a conserved receiver domain and a variable effector domain. This particular modular architecture of RRs enables them to function as versatile components that couple a series

1212 L. Wang et al. 䊏

NP_296139_DEIRA YP_605612_DEIGE YP_144628_THETH ZP_01725055_BACSP ZP_01699248_ESCCO

MNEQRILVIEDDHDIANVLRMDLTDAGYVVDHADSAMNGLIKAREDHPDLILLDLGLPDFDGGDVVQRLRKNS-ALP MSAQRILVIEDDLDIANVLRLDLTDAGYAVEHADSAMNGLIRAREDHPDLILLDLGLPDFDGGDVVQRLRKNS-AVP M--KRILLIEDDPEVARLVEAELKEAGFQVDWAKTGMEGLIRHREGKPDLVVLDLGLPDLDGAEVARRIRATD-DTP MS-NRILIIEDEENIARVLQLELQFEGYEAVMAHTGADGLLQYREQQWDLILLDIMLPEMSGIDVLKRIRATESQTP M---KILIVEDEIKTGEYLSKGLTGAGFVVDHADNGLTGYHLAMTAEYDLVILDIMLPDVNGWDIIRMLRTAGKGTP

76 76 74 76 74

N-terminal receiver domain NP_296139_DEIRA YP_605612_DEIGE YP_144628_THETH ZP_01725055_BACSP ZP_01699248_ESCCO

IIVLTARDTVEEKVRLLGLGADDYLIKPFHPDELLARVKVQLRQR-----TSESLSMGDLTLDPQKRLVTYKGEELR IIVLTARDTVDEKVRLLGLGADDYLIKPFHPDELIARVKVQLRQR-----VSESLSMGDLTLDPQKRLVTYKNEELR ILVLTAQDAVDRKVSLLTGGADDYLVKPFHPAELLARIQVQLRHKE----GSEVLAVGQLELYPGKRQVFFREREVR VIMLTAKSEVEDKVKGLDLGANDYVTKPFEIEELLARIRNALRFSQKASPTKVGVSFGQLSINEQTREVIYYGKEIQ VLLLTALGTIEHRVKGLELGADDYLVKPFAFAELLARVRTLLRRGN-TMITESQFKVADLSIDLVSRKVSRAGKRIV

148 148 147 153 150

NP_296139_DEIRA YP_605612_DEIGE YP_144628_THETH ZP_01725055_BACSP ZP_01699248_ESCCO

LSPKEFDILALLIRQPGRVYSRQEIGQEIWQGRLPEGSNVVDVHMANLRAKLRDLDGYGLLRTVRGVATPCAAEAPT LSPKEFDILALLIRQPGRVYSRQEIGQEIWQGRLPEGSNVVDVHMANLRAKLRDLDGYGLLRTVRGVGYALRG---LSPKEFDLLHLLMSRPGRVFPRPEIEERVWGRPLGKDSNVLDVHMANLRAKLREAGAYGYLRTVRGVGYAVRPGRRE LTPREYDLLLYLMKHPKQVLTREQILETVWGFDYYGDTNVVDVYIRYVRQKLEIANVTPIIQTVRGVGYVLKENKSLTSKEFSLLEFFIRHQGEVLPRSLIASQVWDMNFDSDTNAIDVAVKRLRAKIDNDYETKLIQTVRGVGYMLEIPDA-

225 221 224 229 226

C-terminal effector domain Fig. 1. Multiple sequence alignment of five response regulators from several bacteria. DEIRA, Deinococcus radiodurans R1; DEIGE, Deinococcus geothermalis DSM 11300; THETH, Thermus thermophilus HB8; BACSP, Bacillus sp. B14905; ESOCO, Escherichia coli. A well-conserved amino-terminal receiver domain and a variable carboxyl-terminal effector domain are underlined. The shading reflects more than 60% consensus in each column. The more conserved residues were observed, the darker colour shaded.

of cellular behaviours (Gao et al., 2007). In this study, we report an important RR DR2418 (designated DrRRA) that is essential for the radioresistance of D. radiodurans R1.

Results Characterization of DR2418 in D. radiodurans R1 The result of BlastP shows that DR2418 is a homologue of RRs, including two predicted domains: a wellconserved amino-terminal receiver domain and a variable carboxyl-terminal effector domain with DNA-binding signature (Fig. 1). The protein is therefore designated DrRRA. To test whether DrRRA is a RR, we mutated the Asp groups suspected to be involved in the signal transduction and complemented the site-mutated DNA fragments to the dr2418 mutant strain. Just as the dr2418 null mutant strain (see below), the mutation DrRRAD54N results in significant decrease of its radioresistance (Fig. 2). Because Asp-54 within the receiver domain is highly conserved and is the predicted phosphorylation site in nearly all RRs, we propose that DrRRA acts as a RR. To see whether DrRRA possesses DNA-binding activity, the recombinant DrRRA was purified and mixed with FITC-labelled promoter DNA fragments. A band of retarded mobility appeared when it was incubated with dr0997 promoter, indicating that this protein is capable of binding to the specific promoter directly (Fig. 3A and B).

Deletion of drRRA greatly reduces ionizing radioresistance of D. radiodurans R1 In an attempt to identify the role of drRRA in the highly radioresistant bacterium D. radiodurans R1, drRRA null mutant strain (designated MR) was constructed using the deletion mutagenesis technique. The homozygous disruptant was confirmed by PCR and DNA sequencing. The

Fig. 2. Gamma-radiation sensitivity of wild-type strain and its derivatives. Wild-type R1, the mutant complemented with gene drRRA (MRC), the mutant complemented with DrRRAD54N (D54N) and the mutant MR were diluted and treated with various dosages of gamma-radiation respectively. Viable colonies after gamma-irradiation treatment are displayed.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

A response regulator in Deinococcus radiodurans 1213

Marker

A

1

2

3

116 KD 66 KD 45 KD 35 KD DrrrA

25 KD

18 KD B 1

2

3

4

5

6

7

8

Protein-DNA complex

Fig. 3. Purification of the DrRRA protein and its DNA-binding activity. A. SDS-PAGE analysis of DrRRA expression in E. coli BL21 (DE3). Lane ‘Marker’ contains molecular weight markers. Lanes 1 and 2 contain uninduced and induced crude extracts respectively. Lane 3 contains purified DrRRA protein, approximately 25 kD. Note: The actual length of the coding sequence is 666 bp rather than 1122 bp. The result was testified for three times using Pyrobest DNA polymerase (Takara) followed by DNA sequencing. B. Electrophoretic mobility shift assay of DrRRA binding to the FITC-labelled dr0997 promoter specifically. Lanes 1–4, non-specific DNA fragments; Lanes 5–8: the gene dr0997 promoter DNA fragments; Lanes 1 and 5: no DrRRA; Lanes 2 and 6: 1.6 mM DrRRA; Lanes 3 and 7: 2.4 mM DrRRA; Lanes 4 and 8: 3.2 mM DrRRA. Each reaction mixture contained 10 nM of DNA and 50 mg ml-1 of salmon sperm DNA. Protein–DNA complexes were separated in 5% native polyacrylamide gels and analysed by LAS-3000 cooled CCD camera system (Fuji film).

Free DNA

survival curve illustrated in Fig. 4A demonstrates that the complete functional disruption of drRRA in D. radiodurans leads to high sensitivity to ionizing radiation as compared with the wild-type strain. The mutant exhibits a survival rate of 1% following exposure to 2 kGy ionizing radiation, whereas the viability of the wild-type strain is almost unchanged at this dosage. Thus, the sensitivity of the mutant to gamma-radiation is comparable to that of pprI mutant (Hua et al., 2003). Furthermore, the shape of the survival curve for MR is atypical. The viability drops rapidly at low doses. At the dose higher than 2 kGy, the curve flattens. The radioresistance of mutant cells could be fully restored when the null mutant strain was complemented with drRRA. Survival curves following exposure to ultraviolet radiation, hydrogen peroxide and desiccation were also determined. It was found that the mutant is also sensitive to ultraviolet radiation, hydrogen peroxide as well as desiccation (Fig. 4B–D). Deletion of drRRA downregulates the transcriptional levels of numerous genes related to stress response and DNA repair at exponential growth phase To gain insights into the downstream pathways regulated by DrRRA, the transcriptional profile of the disruptant strain was analysed and compared with that of wild-type

strain at exponential growth phase. An array of genes display changes in their expression levels at least twofold statistically, which suggests that these genes are affected by the elimination of drRRA through either direct or indirect mechanisms (Table S1). A majority of downregulated genes are uncharacterized (involved in some unclear regulatory mechanisms), followed by stress response and DNA replication and repair genes. Interestingly, the genes downregulated in MR under non-stress condition overlap with nearly one-third of those genes which are upregulated in the wild-type strain after 2 kGy radiation treatment (Table S2). The overlapping gene set is listed in Table 1. A spectrum of stress response-related genes is suppressed in MR (Table 2). First, many genes related to heat or general stress are downregulated. Meanwhile, three of four putative genes homologous to plant desiccation resistance proteins (DRB0118, DR0105 and DR1172) and nine of 16 DNA damage response genes are repressed. Furthermore, antioxidant proteins directly contributing to the removal of reactive oxygen species (ROS) are also significantly downregulated, including three catalases (KAT: DR1998, DRA0259 and DRA0146), two superoxide dismutases (SOD: DR1279 and DR1546), peroxidase (DRA0145) and thioredoxin (DR0944). Finally, a twentyfold reduction in the transcriptional level of DNA-binding ferritin-like protein dps (DR2263) is observed in MR.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

1214 L. Wang et al. 䊏

A

B

C

D

Fig. 4. Survival curves for D. radiodurans following exposure to gamma-radiation (A), H2O2 (B), UV (C) and desiccation (D). Closed squares, closed circles and closed triangles present wild-type R1, the mutant MR and the mutant complemented with gene drRRA (MRC) respectively. Values are the mean ⫾ standard deviation of four independent experiments.

The transcriptional levels of some genes related to DNA replication and repair are also affected. recA (DR2340) and pprA (DRA0346) are decreased fourfold and 3.3-fold, respectively, in MR. Besides, the transcription abundance of DR2339 (2′-5′ RNA ligase) in MR is 4.6-fold lower than that in wild-type strain. Furthermore, DRB0098, which contains a HD-hydrolase family phosphatase domain and a polynucleotide kinase domain, is repressed 4.4-fold. DRB0100, which is located in the same operon of DRB0098, is repressed 6.3-fold. Additionally, one of DNA gyrase subunits (DR0906) that is induced by the recA-like expression pattern after radiation treatment is also reduced 2.2-fold. Transcriptional profile of MR versus wild-type strain under gamma-radiation is similar to that of under no-stress condition at exponential growth phase To test the role of DrRRA in D. radiodurans under gammaradiation, whole-genome expression profiling of the mutant was performed compared with the wild-type strain after irradiation treatment (2 kGy, Table S3). Changes in transcript abundance were verified by quantitative real-time PCR (Q-RT-PCR; Table S4). A large number of genes are inhibited at least twofold in MR compared with the parent

strain under irradiation stress. For instance, the DRB0098 operon, the desiccation resistance protein (DR1172), the SOD (DR1279 and DR1546) and the DNA-binding ferritinlike protein dps (DR2263) are all downregulated. Notably, 12 genes induced by gamma-radiation (2 kGy) in wild-type strain are muted in the mutant under irradiation stress (Table 1). Furthermore, the Q-RT-PCR data of some important genes such as recA and pprA strongly support the microarray data (Table 1). Therefore, we conclude that these genes are affected by the deletion of DrRrA. Deletion of drRRA decreases the antioxidant ability of D. radiodurans Our microarray data reveal that a number of antioxidant proteins are downregulated in MR strain (Table 2). We therefore tested the scavenging activity of mutant strain against H2O2 treatment. As shown in Fig. 5, the mutant strain exhibits a reduced ROS-scavenging activity under either non-stress or irradiation condition. Even when the concentration of the protein extract of the mutant strain is increased from 1 to 4 mg ml-1, H2O2 scavenging does not exceed that of the wild-type protein extracts. This suggests that deletion of DrrA has a deleterious effect on ROS-scavenging activity of D. radiodurans.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

A response regulator in Deinococcus radiodurans 1215

Table 1. Overlapping genes upregulated in irradiated wild-type R1 strain and downregulated in MR strain under normal condition or gammaradiation stress. Fold change MR/R1

R1(+g)/R1

MR(+g)/R1(+g)

Locus

Annotation

Microarray

Q-RT-PCR

Microarray

Microarray

Q-RT-PCR

DR0003 DR0096 DR0232 DR0613

Predicted protein, DdrC ABC transporter with ATPase domain Predicted protein Uncharacterized protein, similar to ycgI bacillus and yao3 Schizosaccharomyces pombe Uncharacterized protein similar to YGY4_HALSQ DNA gyrase B subunit, GYRB NH2-acetyltransferase Ro-like RNA binding protein Protein of uncharacterized conserved family predominantly in Caenorhabditis elegans, YBIA_ECOLI in E. coli Predicted protein, DdrK Cation-transporting ATPase, authentic frameshift NH2-acetyltransferase family Catalase, CATX Glutaredoxin-like protein NRDH Rio1 family protein kinase UvrB CinA orthologue, CinA MoeB family LigT, 2′-5′ RNA ligase RecA NH2-acetyltransferase, DdrN NH2-acetyltransferase Predicted protein, PprA Homologue of eukaryotic DNA ligase III, DdrP Proline dehydrogenase Pyrroline-5 carboxylate dehydrogenase, ROCA Von-willebrand-factor A domain containing protein Predicted protein

-2.84 -4.34 -2.35 -6.31

– – – –

10.04 2.45 2.54 2.15

-3.04 -2.08 -2.58 -6.11

– – – –

-2.30



8.96

-1.60



-2.23 -2.31 -4.54 -3.16

– – -9.10 –

5.87 2.20 2.68 4.07

-1.53 -1.52 -2.00 1.20

– – -5.40 –

-3.61 -8.51

– –

3.37 5.72

1.42 -1.10

– –

-2.54 -2.43 -5.75 -3.80 -3.03 -4.32 -4.61 -4.00 -6.95 -3.38 -3.32 -6.26

– – – – – – – -4.00 – – -3.37 -2.20

2.14 2.11 2.62 3.09 3.41 3.18 11.30 3.80 3.32 2.40 5.73 2.33

-1.50 1.20 -2.77 1.43 -1.80 -1.20 -1.27 -1.30 -2.90 -1.28 -1.32 -2.39

– – – – – – – -5.07 – – -3.43 -3.05

-1.14 -1.64

– –

2.00 2.03

-3.45 -3.90

– –

-2.00



2.13

-2.17



-1.90



2.74

-2.46



DR0905 DR0906 DR1057 DR1262 DR1263

DR1264 DR1440 DR1978 DR1998 DR2085 DR2209 DR2275 DR2338 DR2339 DR2340 DR2441 DRA0019 DRA0346 DRB0100 DR0814 DR0813 DR2391 DR0234

Function annotation is based on KEGG (http://www.genome.jp/kegg/). Q-RT-PCR, quantitative real-time PCR; –, the data undone.

To analyse the enzymatic activities of KAT and SOD, PAGE gel activity-staining assays were used. It was observed that deficiency of drRRA causes significant decrease of KAT and SOD activities in the disrupted strain (Fig. 6A). KatE (upper), KatA (lower) and SOD are downregulated to 0.38-, 0.78- and 0.60-fold, respectively, in non-stressed MR. Although the activities of KAT and SOD in MR are induced by irradiation, their activities could not reach the levels of wild-type strain at the same radiation dosage. Total KAT and SOD activities were also determined from total protein extracts of the mutant and wild-type cells. The results are in consistent with both the gel activity staining results and the microarray data (Fig. 6B). These results suggest that deletion of drRRA attenuates the protective effect of antioxidant proteins.

Deletion of drRRA leads to a reduction of RecA and PprA It is indicated above that the transcriptional levels of recA and pprA are reduced upon deletion of drRRA in D. radiodurans R1. In order to investigate the effect of drRRA disruption on the expression of RecA and PprA in vivo, Western blotting analysis was used. As shown in Fig. 7, the expression levels of RecA and PprA in the mutant strain exhibit a reduction relative to its parent under both normal and irradiation stress conditions. This implies that DrRRA affects the expression of RecA and PprA negatively, consonant with the microarray assay results. However, the expression levels of RecA and PprA are also increased to a certain degree in mutant strain after gamma-radiation treatment, implying there are addi-

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

1216 L. Wang et al. 䊏

Table 2. Stress response-related genes suppressed in MR compared with wild-type R1. Stress type

Gene name

Locus

Annotation

Heat, general

grpE dnaK DnaJ Lon htrA ftsh yebL

DR0128 DR0129 DR0126 DR1974 DR1599 DRA0290 DR2523

thiJ

DR0491

uspA dps yehZ

DR2363 DR2263 DRA0135

pspA katE katE Peroxidase sodA sodC

DR1473 DR1998 DRA0259 DRA0145 DR1279 DR1546 DR0944 DR1849 DR2085

Hsp20 molecular chaperonin Hsp70 molecular chaperonin Hsp70 chaperonin cofactor ATP-dependent lon serine protease Serine protease, with regulatory PDZ domain ATP-dependent Zn protease Zn-binding (lipo)protein of the ABC type Zn transport system (surface adhesin A) Protease I, related to general stress protein 18, ThiJ superfamily protein universal stress protein, nucleotide-binding starvation inducible DNA-binding protein Proline/glycine betaine ABC-type transport, periplasmic binding subunit Phage shock protein A, controls membrane integrity Catalase Catalase with C-terminal domain similar to FABB domain Present only in plant Polyporaceae spp. Superoxide dismutase (Mn), SODM Copper/zinc-superoxide dismutase, SODC Thioredoxin Peptide methionine sulphoxide reductase Glutaredoxin-like protein NRDH

General

Starvation Osmotic Phage Oxidative

Oxidative/ detoxication Detoxication Desiccation

DNA damage

msrA grxA

DR1187 DRB0118

ddrC ddrE

DR0105 DR1172 DR0003 DR0194

ddrG ddrI ddrJ

DR0227 DR0997 DR1263

ddrK ddrM ddrN ddrP cinA

DR1264 DR1440 DR2441 DRB0100 DR2338

Fold change

Conserved membrane protein YGDQ Desiccation-related protein from Craterostigma plantagineum; found to date only in plants LEA76 family desiccation resistance protein LEA76/LEA29-like desiccation resistance protein Predicted protein Predicted HTPX superfamily Metalloprotease, Bacillus yugP orthologue Predicted protein HTH transcription factor, CAP family Protein of uncharacterized conserved family predominantly in C. elegans, YBIA_ECOLI in E. coli Predicted protein Cation-transporting ATPase, authentic frameshift NH2-acetyltransferase Homologue of eukaryotic DNA ligase III Competence damage protein, mitomycininduced

tional mechanisms of their regulation under radiation stress.

Deletion of drRRA delays the restitution of the genome following gamma-radiation From the assays above, we were aware that disruption of drRRA might affect DNA repair process in D. radiodurans. To further testify whether DNA strand break repair is influenced by deletion of drRRA, we performed the pulsedfield gel electrophoresis (PFGE) assay. As illustrated in Fig. 8, an intact genome is formed within 6 h after gamma-radiation treatment in wild-type cells, whereas the MR genome is not restituted until 18 h following irradiation. The result demonstrates that deletion of drRRA retards the process of DNA repair in D. radiodurans.

P-value

-2.59 -2.17 -2.48 -2.49 -2.16 -2.07 -2.21

3.73 3.50 8.41 1.00 1.25 1.00 1.72

-2.38

9.00 E-04

-2.58 -20.39 -8.09

5.14 E-05 2.74 E-06 2.91 E-06

-5.03 -2.43 -2.70 -2.54 -4.43 -2.83 -8.39 -2.12 -5.75

7.20 E-03 1.27 E-02 1.11 E-02 1.00 E-04 1.29 E-06 4.00 E-04 3.47 E-07 1.60 E-04 4.15 E-05

-2.85 -2.53

3.18 E-05 7.00 E-04

-10.92 -5.60 -2.84 -3.05

9.18 2.92 9.37 6.43

E-05 E-05 E-05 E-04 E-02 E-04 E-02

E-05 E-05 E-05 E-06

-2.02 -4.51 -3.16

7.41 E-03 1.84 E-05 4.20 E-05

-3.61 -8.51 -6.95 -6.26 -4.32

1.53 1.15 1.81 1.56 1.00

E-05 E-04 E-04 E-04 E-04

Discussion We have shown that DR2418 (DrRRA) is a DNA-binding RR. Its null mutant strain (MR) exhibits a remarkably reduced survival after gamma-irradiation treatment, demonstrating that it is a key element involved in the extraordinary radioresistance of D. radiodurans. Numerous genes involved in stress response are strongly suppressed in MR. Specially, three desiccation resistance genes and nine DNA damage response (ddr) genes encoding proteins induced in response to desiccation and ionizing radiation (Makarova et al., 2001; Tanaka et al., 2004) are downregulated. A close correlation between desiccation and gamma-radiation resistance in D. radiodurans has been reported (Mattimore and Battista, 1996; Battista et al., 2001). Consequently, the mutant strain is sensitive to both ionizing radiation and desiccation.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

A response regulator in Deinococcus radiodurans 1217

Fig. 5. ROS-scavenging activities of wild type and MR. The reaction mixture contained 20 ml of 0.01% H2O2, 200 ml of 50 mM PBS, and 280 ml of luminol. Closed squares, closed circles, open squares and open circles present wild-type R1 under non-stress condition, the mutant MR under non-stress condition, wild-type R1 under irradiation stress and the mutant MR under irradiation stress respectively. All the experiments were performed three times and values were mean ⫾ standard deviation.

Notably, antioxidative metabolism, which has been widely regarded as a process critical to the extreme radioresistance of D. radiodurans (Ghosal et al., 2005), is influenced by DrRRA. The ROS-scavenging activity of MR is obviously reduced under either normal or irradiation stress condition, revealing the positive effect of DrRRA on antioxidant process of this bacterium. Particularly, KAT and SOD are the major ROS-scavenging proteins that prevent DNA damage caused by hydrogen peroxide and superoxide anion radicals. Both KAT and SOD mutant strains are sensitive to ionizing radiation (Mirsa and Fridovich, 1976; Markillie et al., 1999). As three KAT and two SOD are repressed obviously in their transcriptional and expression levels, the decrease of radioresistance of MR is inevitable. Furthermore, various cellular components can be further damaged by hydroxyl radicals in the presence of elevated ROS and intracellular iron ions. It is shown that DNA-binding ferritin-like protein Dps (DR2263) can sequester free irons and spatially bind DNA in nonspecific manner (Zhao et al., 2002). As DR2263 is drastically downregulated in MR under gamma-radiation, the damage effect of ROS on cells would be exacerbated. Distinctively, DNA replication and repair processes are directly or indirectly linked with DrRRA. RecA and PprA are two essential DNA repair proteins in D. radiodurans. RecA plays a critical role in homologous recombination and recombinational DNA repair (Bianco et al., 1998; Roca and Cox, 1997), and is essential for the regulation of the SOS repair system in E. coli (Anderson and Kowalczykowski, 1998). Following extensive DNA damage, it is transiently induced to higher levels in D. radiodurans

(Kim and Cox, 2002; Kim et al., 2002). PprA is unique to D. radiodurans and predicted to be a RecA-independent DNA repair-related protein (Narumi et al., 2004). Both RecA-defective and PprA-defective D. radiodurans mutants display a remarkable reduction in their ability to recover from acute DNA damage (Tanaka et al., 2004; Gutman et al., 1994; Narumi et al., 1999). The expression levels of RecA and PprA are both reduced in MR, suggesting that DrRRA influences them positively. In addition, DR2339 is possibly involved in the DNA repair process by radiation (Arn and Abelson, 1996). DRB0098 resembles a similarly configured human protein that plays an important role in DNA double-strand breaks (DSB) repair (Makarova et al., 2001). DRB0100 is likely an ATP-dependent DNA ligase that specifically promote non-homologous endjoining (NHEJ) in eukaryotes and exhibits a recA-like activation pattern after acute ionizing radiation (Liu et al., 2003). DR0906 is a DNA gyrase subunit induced by DNA DSB (Tanaka et al., 2004). As they are all downregulated,

A

R1



R1

MR

MR

–γ

–γ



Kat E Kat A SOD

B

Fig. 6. Enzyme activities of KAT and SOD in wild-type and mutant strains. A. PAGE gel of enzymatic activity. The relative intensities of the bands were scanned with a Gel Imager System (Bio-Rad Laboratories, Hercules, CA). Each lane contained 20 mg of protein. B. Total enzyme activities. Assays were performed three times and values were presented as mean value ⫾ standard deviation. Minus g (-g) and plus g (+g) indicate non-stress and post gamma irradiation conditions respectively.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

1218 L. Wang et al. 䊏

R1

R1

MR

MR



–γ

–γ



the DNA repair ability of the mutant strain could be weakened and become sensitive to gamma-radiation. Furthermore, the mutant strain displays a delay in the restitution of an intact genome compared with wild-type strain after gamma radiation treatment, which emphasizes the impact of drRRA disruption on DNA repair process and radioresistance. However, it is mentionable that the retardation of genome reconstitution in the mutant strain might result from multiple influences such as antioxidation protection and DNA repair attenuation, because the reduced ROS-scavenging ability might damage the proteins required in the DNA repair process (Daly et al., 2007). Nevertheless, the dominant pathway in the process is unclear yet. It has been reported that gamma-radiation can induce plentiful ROS and cause a majority of DNA damage including DSB, whereas UV radiation mainly causes the lesions of DNA base (Friedberg et al., 1995). Probably, DrRRA affects multiple pathways such as antioxidation protection and DSB repair. In the present paper, we have proved that DrRRA, a member of two-component signal transduction pathway, functions as an essential component in the extraordinary resistance of D. radiodurans. This provides a new insight into the understanding of the DNA repair and protection mechanism of this peculiar bacterium. We hypothesize

RecA PprA GroEL

R1 C

0

1.5

3

6

12

18

λ-ladder

Fig. 7. Western blot assays of intracellular levels of RecA and PprA in wild-type and mutant strains. Minus g (-g) and plus g (+g) indicate non-stress and post gamma irradiation conditions respectively. Samples of cell extracts equivalent to 100 mg of protein were loaded in each lane. Anti-GroEL antibody was used as the loading control.

MR C

0

1.5

3

6

12

18

Fig. 8. Genome recovery of MR compared with wild-type R1 following gamma-irradiation. MR (lanes 9–15) shows a delay in intact genomic DNA restoration after gammairradiation compared with wild-type strain (lanes 1–7) over an 18 h time-course after irradiation. Lanes C present the samples prior to irradiation. l-DNA ladder was indicated.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

A response regulator in Deinococcus radiodurans 1219

that DrRRA is a RR that might control or influence the expression of numerous genes related to DNA protection and repair such as kat, sod, recA and pprA. Once DrRRA is disrupted, cells become vulnerable to damage. It is interesting that our previous work has shown that activated PprI regulates expression and activation of a variety of DNA repair and protection genes, including recA, pprA and kat in response to gamma-radiation in D. radiodurans. The pprI (also called irrE) disruptant strain is also very sensitive to ionizing radiation (Earl et al., 2002; Hua et al., 2003). Indeed, we have found that the double mutant (DdrRRADpprI ) is more sensitive to gamma-radiation (data not shown). Therefore, it is necessary to elucidate the relationship between these two genes. Another imperative question is to identify the sensor that activates DrRRA. Directly adjacent to drRRA on the D. radiodurans chromosome is a putative sensor kinase gene dr2419, but disruption of this gene shows less radiosensitive than MR (data not shown). Probably, DrRRA is regulated by more than one HK. Also, it is interesting that DrRRA could specially bind to the promoter DNA fragment of DNA damage response gene dr0997 (ddrI ), while the relative expression of this gene was significantly reduced in the mutant compared with the wild-type strain under either normal condition or gamma-radiation stress (Table S5). Additionally, one-third of genes in the genome are unknown or unique to D. radiodurans. Some of them are remarkably changed in the transcriptional levels in the mutant. Speculately, these unknown genes might play a new role through DrRRA in radioresistance. The detailed regulation mechanism of DrRRA is to be carried out in our further work.

Experimental procedures Bacterial strains and growth conditions Deinococcus radiodurans R1 strain was used as wild type. All D. radiodurans strains used were cultured at 30°C in TGY medium (0.5% Bacto tryptone, 0.1% glucose, 0.3% Bacto yeast extract) with aeration or on TGY plates supplemented with 1.5% Bacto agar. Antibiotics were added at an appropriate concentration to cause selection for transfectants.

Protein purification Escherichia coli BL21 (DE3) cells carrying plasmid pET-28adrrrA were grown to an OD600 of 0.5 at 37°C and induced with 1 mM IPTG at 37°C for 4–5 h. Cells were harvested and lysed in 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 10 mM imidazole containing 1 mM PMSF by ultrasonic. Lysates were cleared by centrifugation at 13 000 r.p.m. for 40 min at 4°C. The supernatant was then subjected to affinity chromatography on Ni2+-NTA agarose (Qiagen). After washing, proteins were eluted with 300 mM imidazole and dialysed overnight. Elutions were examined by SDS-PAGE and image analyses. Proteins were estimated to be > 95% purity.

Site-directed mutagenesis To mutate Asp of DrRRA to asparagine, Quick Change Sitedirected Mutagenesis Kit (Stratagene) was used. Following verification by DNA sequencing, the fragment containing the mutated sequence was cloned into shuttle vector and transformed into D. radiodurans.

Electrophoretic mobility shift assay Promoter DNA probes for gel mobility shift assays were amplified by PCR from D. radiodurans and labelled with FITC at the 3′ end. Binding reaction mixtures contained 0–3.2 mM of purified DrRRA protein and 10 nM of FITC-labelled promoter PCR fragments. Binding reactions were performed in 20 ml of binding reaction buffer (50 mM Tris-HCl pH 8.0), 100 mM KCl, 10 mM MgCl2, 3 mM dithiothreitol, 50 mg ml-1 of salmon sperm DNA) for 30 min at room temperature, and the reaction mixtures were loaded onto 5% non-denaturing polyacrylamide gels. The labelled DNA was detected by LAS3000 cooled CCD camera system (Fuji film).

Disruption and complementation of DrRRA Disruption of DrRRA was performed using a deletion mutagenesis technique as described previously (Gao et al., 2005). Briefly, a ~1000 bp DNA fragment immediately upstream and downstream of the gene was amplified from the genome of D. radiodurans R1 strain. These two PCR products were digested with BamHI and HindIII, respectively, and ligated with the kanamycin-resistance DNA fragment from pRADK pretreated with the same enzymes. The construct was then transformed into the competent cells of D. radiodurans R1 at exponential growth phase, and mutant colonies were selected on TGY plates containing 30 mg ml-1 kanamycin. The null mutant strain was confirmed by genomic PCR and DNA sequencing. Complemented plasmid was constructed as described previously and transformed into the null mutant (Gao et al., 2005).

Survival curves of D. radiodurans R1 To perform the survival rate assays, D. radiodurans cells were grown in TGY broth to an OD600 of 1.0. For gammaradiation treatment, the cell suspension was diluted in phosphate buffer and irradiated at room temperature for 1 h with 60 Co gamma-rays at several different doses (from 0.8 to 8 kGy), which were adjusted by changing the distance of samples from the gamma-ray source. Control cells were incubated in the absence of gamma-radiation at the same temperature. After the treatment, the cells were plated on TGY plates and incubated at 30°C for 3 days prior to enumeration of colonies. For ultraviolet treatment, the cells were plated on TGY plates at an appropriate concentration, and then exposed to different doses of UV radiation at 254 nm. For H2O2 treatment, the cultures were treated with different concentrations of hydrogen peroxide for 30 min before plating on TGY plates. Desiccation assay was carried out as previously described (Mattimore and Battista, 1996) with some modifications. Briefly, 10 ml of cell suspension was

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1220 L. Wang et al. 䊏

placed inside a sealed desiccator over silica gel-self indicator at 25°C. Relative humidity within the desiccator was measured as less than 5% with a hygrometer. Samples were revived and plated on TGY agar at various intervals.

Scavenging activity (% ) (CL control − CL 0 ) − (CL sample − CL 0 ) = × 100% (CL control − CL 0 ) Where CLcontrol is the luminosity of control, CL0 is the luminosity of background and Clsample is the luminosity of test samples.

Whole-genome array analysis RNA isolation, probe preparation, microarray hybridization and data analysis were performed as described previously (Chen et al., 2007), with some amendnants. For RNA preparation, the mutant and wild-type stain were grown in TGY to an OD600 of 0.25. Total RNA was extracted using TRIZOL Reagent (Invitrogen) following liquid nitrogen pulverization. RNA samples were treated with RNase free DNase I (Promega) and purified by phenol-chloroform extraction. RNA quality and quantity were evaluated by UV absorbance at 260 and 280 nm. Hybridization probes and microarray hybridization were prepared as described previously. The microarrays were scanned using a confocal laser microscope of the GenePix 4000B. Hybridization signals were quantified by GenePix pro 5.1. Normalization and statistical analysis were carried out in the R computing environment using the linear models of Microarray data package. Prior to channel normalization, microarray outputs were filtered to remove spots of poor signal quality by excluding those data points with a mean intensity less than two standard deviations above background in both channels. Within Limma, global LOESS normalization was carried out for each microarray (Chen et al., 2007; Yang et al., 2002).

Quantitative real-time PCR Quantitative real-time PCR was performed as previously described (Chen et al., 2007) with the RNA prepared in microarray assay. Briefly, first-strand cDNA synthesis was carried out in 20 ml of reactions containing 1 mg of DNase I-treated total RNA and 3 mg of random hexamers. The Quant SYBR Green PCR Kit (TIANGEN) was introduced following the manufacturer’s instructions. Seven genes were chosen for assay by quantitative real-time PCR, for which DR0089 was used as a normalization factor.

Antioxidant activity assay The ROS-scavenging activities of wild-type and mutant strains were assayed by chemiluminescence (Murr et al., 1996; Tian et al., 2004). Briefly, D. radiodurans cells (OD600 = 1.0, treated with gamma-radiation or not) were suspended in phosphate buffer and disrupted on ice with an ultrasonicator. The debris was removed by centrifugation. Protein concentrations of the supernatants were determined by the Bradford method using BSA as standards. The reaction mixture contained 20 ml of 0.01% H2O2, 200 ml of PBS and 280 ml of luminol (0.1 mM). Luminosity was monitored in a BPCL Model Ultra Weak Chemiluminescence Analyzer. The samples were measured in triplicate and averaged. Scavenging activity (%) was calculated using the following equation:

Activity assays for catalases and superoxide dismutase Total proteins were prepared as described above. PAGE gel activity-staining and total enzymatic activity measurements of KAT and SOD in the cellular extracts were performed as previously described respectively (Sheng et al., 2005; Tian et al., 2007).

Western blot analysis Cell extracts were prepared as described above. The supernatants were separated on SDS-PAGE and detected with DrRecA and DrPprA antibodies (rabbit IgG, laboratory stock). As a control, D. radiodurans GroEL was detected by E. coli GroEL antiserum (Sigma).

Pulsed-field gel electrophoresis Sample preparation and PFGE analyse were performed as described previously (Harris et al., 2004). Briefly, bacteria (OD600 = 0.3) were exposed to gamma-irradiation in phosphate buffer (pH 7.4) and then incubated in TGY broth. Aliquots of viable cells at various times after irradiation treatment were taken to prepare DNA agarose plugs which were digested with lysozyme, proteinase K and NotI (Takara), successively. After digestion, the plugs were subjected to pulsed field gel electrophoresis for 22 h at 14°C using a CHEF-MAPPER electrophoresis system (Bio-Rad) with the following conditions: 6 V cm-1, linear pulse of 40 s and a switching angle of 120° (-60° to +60°).

Acknowledgements This work is supported by a grant from the National Basic Research Program (2004CB19604), a grant from the National Hi-Tech Development Program (2007AA021305), a grant for Distinguished Young Scientist (30425038) and a key project from the National Natural Science Foundation (30330020) to Y.J.H. Microarray scanning and real-time PCR were carried out at Center of Analysis and Measurement of Zhejiang University, China. We thank Professor Junjie Fu, Xiaojun Zhao and Zhiming Sun (Radiation Center of Zhejiang University) for their help in radiation treatment. We also thank Dr Steve Bates for his critical reading of the manuscript. The article is contributed to the 50th anniversary of Institute of NuclearAgricultural Sciences, Zhejiang University.

References Anderson, D.G., and Kowalczykowski, S.C. (1998) Reconstitution of an SOS response pathway: derepression of transcription in response to DNA breaks. Cell 95: 975–979.

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Arn, E.A., and Abelson, J.N. (1996) The 2′-5′ RNA ligase of Escherichia coli. Purification, cloning, and genomic disruption. J Biol Chem 271: 31145–31153. Battista, J.R., Park, M.J., and McLemore, A.E. (2001) Inactivation of two homologues of proteins presumed to be involved in the desiccation tolerance of plants sensitizes Deinococcus radiodurans R1 to desiccation. Cryobiology 43: 133–139. Bekker, M., Teixeira de Mattos, M.J., and Hellingwerf, K.J. (2006) The role of two-component regulation systems in the physiology of the bacterial cell. Sci Prog 89: 213–242. Bianco, P.R., Tracy, R.B., and Kowalczykowski, S.C. (1998) DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci 3: D570–603. Chen, H., Xu, Z.J., Tian, B., Chen, W.W., Hu, S.N., and Hua, Y.J. (2007) Transcriptional profile in response to ionizing radiation at low dose in Deinococcus radiodurans. Prog Nat Sci 17: 529–536. Daly, M.J., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Zhai, M., Leapman, R.D., et al. (2007) Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS Biol 5: e92. Earl, A.M., Mohundro, M.M., Mian, I.S., and Battista, J.R. (2002) The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression. J Bacteriol 184: 6216– 6224. Friedberg, E.C., Walker, G.C., and Siede, W. (1995) DNA Repair and Muta-genesis. Washington, DC: ASM Press, 14–31. Gao, G.J., Lu, H.M., Huang, L.F., and Hua, Y.J. (2005) Construction of DNA damage response gene pprI functiondeficient and function-complementary mutants in Deinococcus radiodurans. Chinese Sci Bull 50: 232–237. Gao, R., Mack, T.R., and Stock, A.M. (2007) Bacterial response regulators: versatile regulatory strategies from common domains. Trends Biochem Sci 32: 225–234. Ghosal, D., Omelchenko, M.V., Gaidamakova, E.K., Matrosova, V.Y., Vasilenko, A., Venkateswaran, A., et al. (2005) How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress. FEMS Microbiol Rev 29: 361–375. Gutman, P.D., Carroll, J.D., Masters, C.I., and Minton, K.W. (1994) Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans. Gene 141: 31–37. Harris, D.R., Tanaka, M., Saveliev, S.V., Jolivet, E., Earl, A.M., Cox, M.M., and Battista, J.R. (2004) Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1. PLoS Biol 2: e304. Hua, Y., Narumi, I., Gao, G., Tian, B., Satoh, K., Kitayama, S., and Shen, B. (2003) PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem Biophys Res Commun 306: 354–360. Hwang, I., Chen, H.C., and Sheen, J. (2002) Two-component signal transduction pathways in Arabidopsis. Plant Physiol 129: 500–515. Khairnar, N.P., Kamble, V.A., Mangoli, S.H., Apte, S.K., and Misra, H.S. (2007) Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli. Mol Microbiol 65: 294– 304.

Kim, J.I., and Cox, M.M. (2002) The RecA proteins of Deinococcus radiodurans and Escherichia coli promote DNA strand exchange via inverse pathways. Proc Natl Acad Sci USA 99: 7917–7921. Kim, J.I., Sharma, A.K., Abbott, S.N., Wood, E.A., Dwyer, D.W., Jambura, A., et al. (2002) RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization. J Bacteriol 184: 1649–1660. Liu, Y., Zhou, J., Omelchenko, M.V., Beliaev, A.S., Venkateswaran, A., Stair, J., et al. (2003) Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation. Proc Natl Acad Sci USA 100: 4191–4196. Makarova, K.S., Aravind, L., Wolf, Y.I., Tatusov, R.L., Minton, K.W., Koonin, E.V., and Daly, M.J. (2001) Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev 65: 44–79. Markillie, L.M., Varnum, S.M., Hradecky, P., and Wong, K.K. (1999) Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants. J Bacteriol 181: 666–669. Mattimore, V., and Battista, J.R. (1996) Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. J Bacteriol 178: 633–637. Mirsa, H.P., and Fridovich, I. (1976) Superoxide dismutase and the oxygen enhancement of radiation lethality. Arch Biochem Biophys 176: 577–581. Moseley, B.E., and Evans, D.M. (1983) Isolation and properties of strains of Micrococcus (Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways. J Gen Microbiol 129: 2437–2445. Murr, C., Baier-Bitterlich, G., Fuchs, D., Werner, E.R., Esterbauer, H., Pfleiderer, W., and Wachter, H. (1996) Effects of neopterin-derivatives on H2O2-induced luminol chemiluminescence: mechanistic aspects. Free Radic Biol Med 21: 449–456. Narumi, I., Satoh, K., Kikuchi, M., Funayama, T., Kitayama, S., Yanagisawa, T., et al. (1999) Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30. Mutat Res 435: 233–243. Narumi, I., Satoh, K., Cui, S., Funayama, T., Kitayama, S., and Watanabe, H. (2004) PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation. Mol Microbiol 54: 278–285. Oshima, T., Aiba, H., Masuda, Y., Kanaya, S., Sugiura, M., Wanner, B.L., et al. (2002) Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12. Mol Microbiol 46: 281–291. Roca, A.I., and Cox, M.M. (1997) RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol 56: 129–223. Sheng, D., Gao, G., Tian, B., Xu, Z., Zheng, Z., and Hua, Y. (2005) RecX is involved in antioxidant mechanisms of the radioresistant bacterium Deinococcus radiodurans. FEMS Microbiol Lett 244: 251–257. Tanaka, M., Earl, A.M., Howell, H.A., Park, M.J., Eisen, J.A.,

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1211–1222

1222 L. Wang et al. 䊏

Peterson, S.N., and Battista, J.R. (2004) Analysis of Deinococcus radiodurans’s transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance. Genetics 168: 21–33. Tian, B., Wu, Y., Sheng, D., Zheng, Z., Gao, G., and Hua, Y. (2004) Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans. Luminescence 19: 78–84. Tian, B., Xu, Z., Sun, Z., Lin, J., and Hua, Y. (2007) Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses. Biochim Biophys Acta 1770: 902–911. Yang, Y.H., Dudoit, S., Luu, P., Lin, D.M., Peng, V., Ngai, J., and Speed, T.P. (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15. Zhao, G., Ceci, P., Ilari, A., Giangiacomo, L., Laue, T., Chiancone, E., and Chasteen, N. (2002) ron and hydrogen per-

oxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli. J Biol Chem 277: 27689–27696. Zhou, L., Lei, X.H., Bochner, B.R., and Wanner, B.L. (2003) Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two-component systems. J Bacteriol 185: 4956–4972.

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