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• W2068761995 abstract "Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to γ radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of ionizing irradiation. Results of two-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose γ ray exposure 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turnover, and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, was down-regulated in irradiated R1. Additionally phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knock-out analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways. Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to γ radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of ionizing irradiation. Results of two-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose γ ray exposure 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turnover, and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, was down-regulated in irradiated R1. Additionally phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knock-out analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways. The Gram-positive nonpathogenic bacterium Deinococcus radiodurans is characterized by extreme resistance to ionizing radiation, UV irradiation, desiccation, and a variety of DNA-damaging agents without resulting in lethality or mutagenesis (1Minton K.W. DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans..Mol. Microbiol. 1994; 13: 9-15Crossref PubMed Scopus (250) Google Scholar, 2Battista J.R. Earl A.M. Park M.J. Why is Deinococcus radiodurans so resistant to ionizing radiation?.Trends Microbiol. 1999; 7: 362-365Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). This dramatic capability is ascribed to its outstanding efficiency in reconstructing a functional genome with high fidelity from hundreds of double strand breaks (DSBs) 1The abbreviations used are: DSB, double strand break; PprI, inducer of pleiotropic proteins promoting DNA repair; RecA, recombinase A; PprA, pleiotropic protein promoting DNA repair A; SsB, single strand DNA-binding protein; 2-DE, two-dimensional electrophoresis; COGs, clusters of orthologous groups; Gy, grays; kGy, kilogray(s); 2-D, two-dimensional; NCBI, National Center for Biotechnology Information; PMF, peptide mass fingerprint. generated by DNA-damaging agents (2Battista J.R. Earl A.M. Park M.J. Why is Deinococcus radiodurans so resistant to ionizing radiation?.Trends Microbiol. 1999; 7: 362-365Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 3Lin J. Qi R. Aston C. Jing J. Anantharaman T.S. Mishra B. White O. Daly M.J. Minton K.W. Venter J.C. Schwartz D.C. Whole-genome shotgun optical mapping of Deinococcus radiodurans.Science. 1999; 285: 1558-1562Crossref PubMed Scopus (156) Google Scholar), whereas few other organisms can tolerate DSBs (4Narumi I. Unlocking radiation resistance mechanisms: still a long way to go.Trends Microbiol. 2003; 11: 422-425Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Exponentially growing D. radiodurans is able to withstand 50–100 times more ionizing radiation than Escherichia coli and can survive a 15-kGy acute ionizing radiation dose with no loss of viability. Its ability of continuous growth without any delay when exposed to a maximum of 60 Gy/h γ ray (5Venkateswaran A. McFarlan S.C. Ghosal D. Minton K.W. Vasilenko A. Makarova K. Wackett L.P. Daly M.J. Physiologic determinants of radiation resistance in Deinococcus radiodurans.Appl. Environ. Microbiol. 2000; 66: 2620-2626Crossref PubMed Scopus (116) Google Scholar) has made it one of the most distinguished candidates for bioremediation of radioactive wastes and contaminants (6Brim H. McFarlan S.C. Fredrickson J.K. Minton K.W. Zhai M. Wackett L.P. Daly M.J. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments.Nat. Biotechnol. 2000; 18: 85-90Crossref PubMed Scopus (276) Google Scholar, 7Lange C.C. Wackett L.P. Minton K.W. Daly M.J. Engineering a recombinant Deinococcus radiodurans for organopollutant degradation in radioactive mixed waste environments.Nat. Biotechnol. 1998; 16: 929-933Crossref PubMed Scopus (142) Google Scholar). More than 50 years of research has provided many lines of evidence supporting the benefits of the extreme radioresistance of D. radiodurans from its highly efficient DNA damage repair system and its remarkable antioxidation system (1Minton K.W. DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans..Mol. Microbiol. 1994; 13: 9-15Crossref PubMed Scopus (250) Google Scholar, 8Battista J.R. Against all odds: the survival strategies of Deinococcus radiodurans.Annu. Rev. Microbiol. 1997; 51: 203-224Crossref PubMed Scopus (430) Google Scholar, 9Cox M.M. Battista J.R. Deinococcus radiodurans—the consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (531) Google Scholar, 10Daly M.J. Gaidamakova E.K. Matrosova V.Y. Vasilenko A. Zhai M. Venkateswaran A. Hess M. Omelchenko M.V. Kostandarithes H.M. Makarova K.S. Wackett L.P. Fredrickson J.K. Ghosal D. Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance.Science. 2004; 306: 1025-1028Crossref PubMed Scopus (493) Google Scholar, 11Ghosal D. Omelchenko M.V. Gaidamakova E.K. Matrosova V.Y. Vasilenko A. Venkateswaran A. Zhai M. Kostandarithes H.M. Brim H. Makarova K.S. Wackett L.P. Fredrickson J.K. Daly M.J. How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress.FEMS Microbiol. Rev. 2005; 29: 361-375PubMed Google Scholar, 12Makarova K.S. Omelchenko M.V. Gaidamakova E.K. Matrosova V.Y. Vasilenko A. Zhai M. Lapidus A. Copeland A. Kim E. Land M. Mavrommatis K. Pitluck S. Richardson P.M. Detter C. Brettin T. Saunders E. Lai B. Ravel B. Kemner K.M. Wolf Y.I. Sorokin A. Gerasimova A.V. Gelfand M.S. Fredrickson J.K. Koonin E.V. Daly M.J. Deinococcus geothermalis: the pool of extreme radiation resistance genes shrinks.PLoS ONE. 2007; 2: e955Crossref PubMed Scopus (191) Google Scholar, 13Zahradka K. Slade D. Bailone A. Sommer S. Averbeck D. Petranovic M. Lindner A.B. Radman M. Reassembly of shattered chromosomes in Deinococcus radiodurans.Nature. 2006; 443: 569-573Crossref PubMed Scopus (332) Google Scholar, 14Daly M.J. Modulating radiation resistance: insights based on defenses against reactive oxygen species in the radioresistant bacterium Deinococcus radiodurans.Clin. Lab. Med. 2006; 26: 491-504Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 15Chen H. Xu G. Zhao Y. Tian B. Lu H. Yu X. Xu Z. Ying N. Hu S. Hua Y. A novel OxyR sensor and regulator of hydrogen peroxide stress with one cysteine residue in Deinococcus radiodurans.PLoS ONE. 2008; 3e1602Crossref PubMed Scopus (93) Google Scholar). However, the mechanism underlying its radioresistance is still not completely understood (4Narumi I. Unlocking radiation resistance mechanisms: still a long way to go.Trends Microbiol. 2003; 11: 422-425Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Cox M.M. Battista J.R. Deinococcus radiodurans—the consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (531) Google Scholar). Intriguingly this bacterium not only possesses most of the DNA repair genes found in other organisms but also contains many proteins of yet to be determined functions that have been revealed by genome sequencing and comparative genomics (16White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Moffat K.S. Qin H. Jiang L. Pamphile W. Crosby M. Shen M. Vamathevan J.J. Lam P. McDonald L. Utterback T. Zalewski C. Makarova K.S. Aravind L. Daly M.J. Minton K.W. Fleischmann R.D. Ketchum K.A. Nelson K.E. Salzberg S. Smith H.O. Venter J.C. Fraser C.M. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.Science. 1999; 286: 1571-1577Crossref PubMed Scopus (800) Google Scholar, 17Makarova K.S. Aravind L. Wolf Y.I. Tatusov R.L. Minton K.W. Koonin E.V. Daly M.J. Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.Microbiol. Mol. Biol. Rev. 2001; 65: 44-79Crossref PubMed Scopus (578) Google Scholar). These function-unknown proteins may play crucial roles in radioresistance (9Cox M.M. Battista J.R. Deinococcus radiodurans—the consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (531) Google Scholar, 18Tanaka M. Earl A.M. Howell H.A. Park M.J. Eisen J.A. Peterson S.N. Battista J.R. Analysis of Deinococcus radiodurans's transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance.Genetics. 2004; 168: 21-33Crossref PubMed Scopus (227) Google Scholar) as implied by several studies in this bacterium (19Hua Y. Narumi I. Gao G. Tian B. Satoh K. Kitayama S. Shen B. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans.Biochem. Biophys. Res. Commun. 2003; 306: 354-360Crossref PubMed Scopus (146) Google Scholar, 20Narumi I. Satoh K. Cui S. Funayama T. Kitayama S. Watanabe H. PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation.Mol. Microbiol. 2004; 54: 278-285Crossref PubMed Scopus (132) Google Scholar, 21Harris D.R. Tanaka M. Saveliev S.V. Jolivet E. Earl A.M. Cox M.M. Battista J.R. Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1.PLoS Biol. 2004; 2: e304Crossref PubMed Scopus (95) Google Scholar, 22Earl A.M. Mohundro M.M. Mian I.S. Battista J.R. The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression.J. Bacteriol. 2002; 184: 6216-6224Crossref PubMed Scopus (147) Google Scholar, 23Wang L. Xu G. Chen H. Zhao Y. Xu N. Tian B. Hua Y. DrRRA: a novel response regulator essential for the extreme radioresistance of Deinococcus radiodurans.Mol. Microbiol. 2008; 67: 1211-1222Crossref PubMed Scopus (73) Google Scholar). Several years ago, our group and that of John Battista (19Hua Y. Narumi I. Gao G. Tian B. Satoh K. Kitayama S. Shen B. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans.Biochem. Biophys. Res. Commun. 2003; 306: 354-360Crossref PubMed Scopus (146) Google Scholar, 22Earl A.M. Mohundro M.M. Mian I.S. Battista J.R. The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression.J. Bacteriol. 2002; 184: 6216-6224Crossref PubMed Scopus (147) Google Scholar) identified and validated a novel protein, PprI (also named IrrE), essential in D. radiodurans radioresistance. It strongly enhanced catalase activities and promoted the expression of RecA and PprA (19Hua Y. Narumi I. Gao G. Tian B. Satoh K. Kitayama S. Shen B. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans.Biochem. Biophys. Res. Commun. 2003; 306: 354-360Crossref PubMed Scopus (146) Google Scholar). Expression of the novel gene, driven by the promoter of D. radiodurans groEL (DR0607), also significantly enhanced the resistance of E. coli to γ irradiation. We found that the expression of PprI in E. coli also increased the expression of RecA and improved catalase activity (24Gao G. Tian B. Liu L. Sheng D. Shen B. Hua Y. Expression of Deinococcus radiodurans PprI enhances the radioresistance of Escherichia coli.DNA Repair (Amst.). 2003; 2: 1419-1427Crossref PubMed Scopus (48) Google Scholar). Both microarray and Western blotting analysis demonstrated that ionizing radiation did not increase PprI transcription or translation levels (19Hua Y. Narumi I. Gao G. Tian B. Satoh K. Kitayama S. Shen B. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans.Biochem. Biophys. Res. Commun. 2003; 306: 354-360Crossref PubMed Scopus (146) Google Scholar, 25Liu Y. Zhou J. Omelchenko M.V. Beliaev A.S. Venkateswaran A. Stair J. Wu L. Thompson D.K. Xu D. Rogozin I.B. Gaidamakova E.K. Zhai M. Makarova K.S. Koonin E.V. Daly M.J. Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4191-4196Crossref PubMed Scopus (300) Google Scholar, 27Chen H. Xu Z.J. Tian B. Chen W.W. Hu S.N. Hua Y.J. Transcriptional profile in response to ionizing radiation at low dose in Deinococcus radiodurans.Prog. Nat. Sci. 2007; 17: 529-536Crossref Scopus (13) Google Scholar). To our knowledge, no homologous protein has been identified through database searches in other organisms except in the closely related Deinococcus geothermalis, which is also an exceptionally radioresistant bacterium. Sequence analysis revealed that PprI contained a functional domain (DUF955) consisting of neutral zinc metallopeptidases and a lacI-type helix-turn-helix that led us, in our previous study, to propose it as a transcriptional regulator (19Hua Y. Narumi I. Gao G. Tian B. Satoh K. Kitayama S. Shen B. PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans.Biochem. Biophys. Res. Commun. 2003; 306: 354-360Crossref PubMed Scopus (146) Google Scholar). However, the precise molecular mechanism by which PprI contributes to radioresistance remains unclear. Identification of the PprI regulatory network should provide insight into the role of PprI in the defense of γ radiation insults. In the current study, we conducted a systematic proteome-wide analysis to identify proteins and pathways regulated by PprI in bacterial cells recovering from radiation damage. Beyond the previous focus on proteomic changes of D. radiodurans in response to irradiation (28Sheng D. Zheng Z. Tian B. Shen B. Hua Y. LexA analog (dra0074) is a regulatory protein that is irrelevant to recA induction.J. Biochem. 2004; 136: 787-793Crossref PubMed Scopus (29) Google Scholar, 32Tanaka A. Hirano H. Kikuchi M. Kitayama S. Watanabe H. Changes in cellular proteins of Deinococcus radiodurans following gamma-irradiation.Radiat. Environ. Biophys. 1996; 35: 95-99Crossref PubMed Scopus (29) Google Scholar), we systematically examined the changes at the proteome level in the wild type strain (R1), compared with the pprI-deleted mutant strain (YR1), in response to 1 kGy of ionizing radiation using two-dimensional electrophoresis (2-DE) and MALDI-TOF MS or MALDI-TOF/TOF MS to identify components of the pprI-mediated responsive pathways for bacterial cell survival. The majority of proteins identified as being regulated by PprI were involved in transcription, translation, DNA replication and repair, signal transduction, protein turnover and chaperoning, energy production and conversion, and metabolism. We found that upon radiation PprI turned on various pathways to funnel the cellular efforts into efficient repair and recovery of its intact genome for cellular survival. The D. radiodurans R1 strain and E. coli strain JM109 were available in our laboratory. D. radiodurans cultures were grown at 32 °C in TGY broth (0.5% Bacto tryptone, 0.1% glucose, 0.3% Bacto yeast extract) with aeration or on TGY plates supplemented with 1.5% agar, whereas E. coli was grown at 37 °C in LB broth (1.0% Bacto tryptone, 0.5% Bacto yeast extract, 1.0% NaCl) or on LB plates solidified with 1.5% agar. D. radiodurans cells were transformed using the modified CaCl2 technique as described previously (33Meima R. Rothfuss H.M. Gewin L. Lidstrom M.E. Promoter cloning in the radioresistant bacterium Deinococcus radiodurans.J. Bacteriol. 2001; 183: 3169-3175Crossref PubMed Scopus (66) Google Scholar). The construction of the PprI function-deficient mutant strain YR1 was described previously (34Gao G. Lu H. Huang L. Hua Y. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans.Chin. Sci. Bull. 2005; 50: 311-316Crossref Google Scholar). Briefly a DNA fragment from the plasmid pRADK (34Gao G. Lu H. Huang L. Hua Y. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans.Chin. Sci. Bull. 2005; 50: 311-316Crossref Google Scholar) containing the kanamycin resistance gene driven by the groEL (DR0607) promoter was reversely inserted into the pprI (DR0167) gene in the D. radiodurans genome. To validate the functional involvement of the identified gene products in radioresistance, we deleted individual genes and tested the radiation resistance capacity of the mutants. Mutants of DR1473, DR2317, DRA0018, and DRA0283 were constructed using the same protocol (34Gao G. Lu H. Huang L. Hua Y. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans.Chin. Sci. Bull. 2005; 50: 311-316Crossref Google Scholar) and designated M1473, M2317, MA0018, and MA0283, respectively (Table I). Primers used for the construction of these mutants are listed in supplemental Table S1. Survival curves under γ irradiation of four mutants, including M1473, M2317, MA0018, and MA0283, were carried out using a published protocol (34Gao G. Lu H. Huang L. Hua Y. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans.Chin. Sci. Bull. 2005; 50: 311-316Crossref Google Scholar).Table IStrains and plasmidsStrains and plasmidsCharacteristicsSourcePlasmids pRADKShuttle plasmids between E. coli and D. radiodurans, AmpR, KanR, ChlRGao et al. (34Gao G. Lu H. Huang L. Hua Y. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans.Chin. Sci. Bull. 2005; 50: 311-316Crossref Google Scholar) pRADA0283mycpRADK containing c-Myc-tagged dra0283This study pRAD1343mycpRADK containing c-Myc-tagged dr1343This studyStrains E. coliDH5αSupE44, ΔlacU169, hsdR17, recA1 endA1, gryA96, thi-1, relA1Lab stock D. radioduransR1Wild type strain (ATCC 13939)Lab stockYR1As R1 but pprI(DR0167)-deletedGao et al. (34Gao G. Lu H. Huang L. Hua Y. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans.Chin. Sci. Bull. 2005; 50: 311-316Crossref Google Scholar)MA0018As R1 but DRA0018-deletedThis studyMA0283As R1 but DRA0283-deletedThis studyM1473As R1 but DR1473-deletedThis studyM2317As R1 but DR2317-deletedThis studyR1pRADKR1 containing pRADKThis studyDRA0283mycR1 containing pRADA0283mycThis studyDR1343mycR1 containing pRAD1343mycThis study Open table in a new tab To find an appropriate time point to obtain bacterial samples for proteomics analysis, we investigated cell growth and genome restitution during postirradiation recovery. Pulse field gel electrophoresis of D. radiodurans R1 and YR1 strains was conducted as described previously (35Mattimore V. Battista J.R. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.J. Bacteriol. 1996; 178: 633-637Crossref PubMed Google Scholar). Strains were grown to A600 = 0.3 and harvested. Cells were washed once with 0.9% NaCl, resuspended in MgSO4 (10 mm), acutely irradiated (1 kGy) at room temperature, and incubated in fresh media, and A600 values were measured at several time points. Concomitantly samples (5 ml) were prepared as DNA-agarose plugs and sequentially treated with lysozyme, proteinase K, and restriction enzyme NotI, and plugs were then sent for pulsed field gel electrophoresis (22 h at 14 °C) using the CHEF-MAPPER electrophoresis system (Bio-Rad). The main electrophoresis parameters were set as 6 V/cm, 40-s linear pulse, and a switching angle of 120° (−60° to +60°). The R1 and YR1 strains were transferred to fresh TGY medium and grown (32 °C) with continuous shaking until early stationary phase (A600 = 0.8). Cells were harvested by centrifugation, washed twice with PBS (pH 7.4), and resuspended in MgSO4 (10 mm). Suspensions were halved: one-half was acutely irradiated on ice with 1 kGy 60Co γ rays; the other was used as the non-irradiated control. After treatment, cells were collected by centrifugation, resuspended in fresh TGY medium, incubated (32 °C for 60 min) with continuous shaking, then washed twice with PBS, and pelleted. The cell pellet was snap frozen in liquid nitrogen and stored (−80 °C). Deep frozen cells were resuspended in lysis buffer (9 m urea, 4% (w/v) CHAPS, 65 mm DTT, 2% IPG buffer pH 3–10 linear, 1 mm PMSF, 40 mm Tris base) and lysed with a Biospec Minibeadbeater (Bartlesville, OK). Lysates were immediately placed on ice to inhibit proteolysis. Cell debris were removed by centrifugation (30,000 × g for 30 min), and the clear supernatant was stored (−80 °C) in aliquots until analysis. Protein concentrations were measured using the Bio-Rad protein assay reagent. 2-DE was performed according to the manufacturer’s instructions (Amersham Biosciences). Briefly each protein sample in the lysis buffer was diluted to 500 μl with rehydration solution (9 m urea, 2% CHAPS, 30 mm DTT, 0.5% IPG buffer pH 4–10 linear, 0.002% bromphenol blue). Immobiline DryStrip gels (pH 4–7, 24 cm; Amersham Biosciences) were rehydrated with 500 μl of mixture solution in 24-cm strip holders and electrofocused with an Ettan IPGphor Isoelectric Focusing System (Amersham Biosciences). The focusing protocol was performed as follows: 50 μA/strip at 20 °C, 30 V for 12 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V for 7 h. After isoelectric focusing, strips were equilibrated (30 min) with gentle shaking in SDS equilibration buffer (6 m urea, 30% glycerol, 2% SDS, 1% DTT, 2.5% iodoacetamide, 50 mm Tris-HCl buffer, pH 8.8, 0.002% bromphenol blue) and then separated by SDS-PAGE (12.5%). The second dimension SDS electrophoresis was performed using a Hoefer SE 600 unit (Amersham Biosciences). Four gels were run simultaneously: non-irradiated R1 and YR1 and radiation-treated R1 and YR1 (n = 4/sample). All resolved protein spots in 2-D gels were visualized by silver staining using a Silver Staining kit (Amersham Biosciences). Other standard chemicals required were purchased from Sigma. Stained gels were scanned on an ImageScanner (Amersham Biosciences), and images were analyzed with ImageMaster 2D Elite software supplied by the manufacturer. A D. radiodurans wild type R1 spot file served as the experiment reference pattern. On average, over 1000 spots were detected on each 2-D gel image. Among these, ∼950 of the most reproducible spots were included in the data analysis. Protein spots separated on 2-D gels were quantitated in terms of their relative volume (spot volume/total spot volume). For comparison, the value of each spot was divided by that of each corresponding spot volume from the untreated R1 strain. Statistical significance of the differences in expression profiles of D. radiodurans with or without the γ irradiation was evaluated by t test with significance set at p < 0.05; all statistical calculations utilized Microsoft Excel software. Only those spots with a 2-fold or greater change in expression levels were considered significant and selected for spot picking, trypsin digestion, and mass spectrometry analysis to identify their protein content. Approximately 200 μg of protein from each sample (non-irradiated R1 and YR1 and irradiated R1 and YR1) was separated by isoelectric focusing. After separation by a second SDS-PAGE, proteins were detected by Coomassie Blue R-250 staining (15 h). Protein spots demonstrating different expression patterns compared with controls (induction rate ≥2) were excised from gels and processed for mass spectrometric analysis. Excised spots were reduced at room temperate with tris(2-carboxyethyl)phosphine (Pierce), alkylated with iodoacetamide (Sigma), and digested (20 h) in situ with trypsin (Sigma). Peptides were extracted by addition of a 50% acetonitrile, 5% TFA solution, and extracted solutions were concentrated to 4 μl in a lyophilizer (VirTis, Gardiner, NY). Peptides were treated with ZipTips (Millipore, Bedford, MA) before application to the sample plate in cases where the signal to noise ratio on MALDI-TOF spectra was not ideal. A protein-free gel piece was similarly processed and used as the control to identify autoproteolysis products derived from trypsin. For most of the 2-D gel protein samples (supplemental Table S3), we determined the identity of protein spots in the Research Center for Proteome Analysis, Chinese Academy of Sciences, Shanghai, China. The digested sample was mixed with an equal volume of cyano-4-hydroxycinnamic acid (10 mg/ml; Sigma) saturated with 50% acetonitrile in 0.05% TFA and analyzed by MALDI-TOF MS using an AutoFlex TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). The working mode was set with positive ion reflection mode, an accelerating voltage of 20 kV, and 150-ns delayed extraction time. The spectrum masses ranging from 700 to 3500 Da were acquired with laser shots at 200/spectrum. A Peptide Mixture-1 kit (Bruker Daltonics) was used for external calibration. The matrix and autolytic peaks of trypsin were used for internal calibration. Monoisotopic mass was analyzed with FlexAnalysis 2.0 (Bruker Daltonics) and automatically collected with a signal to noise ratio >4 and a peak quality index >30. The known contaminant ions (human keratin and tryptic autodigest peptides) were excluded. For interpretation of the mass spectra, monoisotopic peptide masses were input into Mascot 2.0 (Matrix Science) for analysis with BioTools 2.1 (Bruker Daltonics). The rest of the protein samples (supplemental Table S3) was analyzed with a Voyager DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) in the Zhejiang University Mass Spectrometry Facility (Hangzhou, China) using a procedure as described previously (30Zhang C. Wei J. Zheng Z. Ying N. Sheng D. Hua Y. Proteomic analysis of Deinococcus radiodurans recovering from gamma-irradiation.Proteomics. 2005; 5: 138-143Crossref PubMed Scopus (75) Google Scholar). Briefly the digested samples were spotted onto a MADLI target with an equal volume of cyano-4-hydroxycinnamic acid (10 mg/ml; Sigma) saturated with 50% acetonitrile in 0.05% TFA. The data were acquired on a Voyager DE STR MALDI-TOF mass spectrometer. The instrument settings were reflector mode with 160-ns delay extraction time, positive polarity, and 20-kV accelerating voltage. The spectrum masses were usually acquired with laser shots at 200/spectrum and ranged from 1000 to 4000 Da. External calibration was performed using a Peptide Mass Standard kit (Perspective Biosystems, Framingham, MA). The matrix and the autolytic peaks of trypsin were used as internal standards for mass calibration. The acquired data were processed with base-line correction, noise removal (5%), and peak deisotoping using Data Explorer 4.0 (Applied Biosystems) following exclusion of known contaminant ions (human keratin and tryptic autodigest peptides). The processed data were input into Mascot 2.0 (Matrix Science) for protein identity searching. Many of processed spectra from the AutoFlex MALDI-TOF/TOF mass spectrometer were searched against the NCBI nonredundant protein database (updated on May 26, 2005), which contained 2,471,633 sequences. The search was restricted to “Bacteria (Eubacteria)” as taxonomy, which contained 944,772 sequences. The remaining spectra were searched against the NCBI nonredundant protein database (updated on August 5, 2005), which contained 2,739,666 sequences. The search was restricted to “Other Bacteria” as taxonomy, which contained 171,641 sequences. The spectra from the Voyager DE STR mass spectrometer were searched against the N" @default.
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