Mol Genet Genomics 62001) 266: 72±78 DOI 10.1007/s004380100520

ORIGINAL PAPER

E. Ge rard á E. Jolivet á D. Prieur á P. Forterre DNA protection mechanisms are not involved in the radioresistance of the hyper thermophilic archaea P yrococcus abyssi and P. furiosus

Received: 1 February 2001 / Accepted: 4 May 2001 / Published online: 20 June 2001 Ó Springer-Verlag 2001

Abstract Hyperthermophilic archaea of the genus environments. They are able to grow at low pH, in high Pyrococcus are resistant to gamma radiation, suggesting salt concentrations or at high temperatures 6Woese et al. that ecient mechanisms for DNA repair exist in these 1990). Pyrococcus abyssi and P. furiosus are two hyper- organisms. To determine whether protective mechanisms thermophilic archaea that are found in geothermal might also be implicated in this radioresistance, we have habitats. They grow optimally at 100°C and 95°C, estimated the linear density of DNA double-stranded respectively 6Fiala and Stetter 1986; Erauso 1993). These breaks caused by gamma irradiation in the genomic extremely high temperatures accelerate the spontaneous DNA of two Pyrococcus species, using Escherichia coli degradation of DNA. The main problem with respect to and the radioresistant bacterium Deinococcus radiodu- DNA stability at high temperatures is thermal degrada- rans as controls. The linear density of double-stranded tion via depurination and subsequent breakage of the breaks was essentially the same in all four microorgan- phosphodiester bonds 6Marguet and Forterre 1994). This isms when irradiation was carried under similar suggests that speci®c mechanisms might exist in hyper- anaerobic conditions, indicating that no speci®c DNA thermophilic archaea that protect and repair DNA. The protection mechanisms exist in Pyrococcus species. thermal degradation of DNA in P. furiosus has been Using one- and two-dimensional gel electrophoresis we compared to that of Escherichia coli by estimating the compared the protein patterns from Pyrococcus abyssi number of DNA backbone breaks after incubation of the and P. furiosus cells that had or had not been exposed to cells at 105°C. P. furiosus DNA was found to be 20 times gamma rays. We did not detect any signi®cant protein more resistant to thermal degradation than E. coli DNA induction following DNA damage in either species. 6Peak et al. 1995). The authors of that study observed that some unidenti®ed proteins remained tightly bound to the Keywords Hyperthermophile á Archaea á DNA. They suggested that the resistance of DNA to Radioresistance á DNA double-stranded breaks thermal degradation could be partly due to proteins that protect the DNA by limiting its contact with water. Fur- thermore, DiRuggiero and collaborators 61998) reported Introduction that P. furiosus could fully repair double-stranded DNA breaks induced in its chromosome by exposure to The phylogenetic domain Archaea includes many or- 2500 Gy of gamma radiation, suggesting that this ganisms that are speci®cally adapted to extreme organism possesses very ecient DNA repair systems. Several years ago, Kopylov and collaborators 61993) reported that two hyperthermophilic archaea, Communicated by R. Devoret Desulfurococcus amyloliticus and Thermococcus stetteri, were radioresistant. They compared the radioresistance E. Ge rard 6&) á P. Forterre Institut de Ge ne tique et Microbiologie, Baà t. 409, of these organisms with those of the model bacterium CNRS, UMR 8621, Universite Paris-Sud, Escherichia coli and the bacterium Deinococcus radio- 91405 Orsay Cedex, France durans, which is the most radioresistant organism E-mail: [email protected] known. At doses that provoke the death of 50% or 90% Tel.: +33-1-69153346 Fax: +33-1-69153423 of the cells, D. amyloliticus and T. stetteri were 12±25 times more radioresistant than E. coli, and only about E. Jolivet á D. Prieur IUEM/UBO, Technopole Brest-Iroise, three times less resistant than D. radiodurans 6Kopylov Place Nicolas Copernic, CNRS, UMR 6539, et al. 1993). P. furiosus is also particularly radioresistant. 29280 Plouzane , France This archaeon can withstand 2000 Gy of gamma 73 radiation without loss of viability, whereas the viability neutrons in vitro. This protein, which is known to of E. coli begins to decrease at doses of 100 Gy 6DiR- protect DNA against thermal denaturation, binds in a uggiero et al. 1997). Thus, radioresistance seems wide- non-speci®c fashion, and induces bending, supercoiling spread in the archaeal domain, since both Euryarchaea and compaction of DNA 6Isabelle et al. 1993). 6Thermococcus, Pyrococcus) and Crenoarchaea 6Desulf- To obtain insight into the mechanisms of DNA urococcus) show this elevated radioresistance. However, protection, we analyzed the radioresistance of P. abyssi such a high level of radioresistance has only been re- and P. furiosus, whose genomes have recently ported for hyperthermophilic archaea. Radioresistance been completely sequenced 6Maeder et al. 1999; is not a common feature of all thermophilic organisms, www.Genoscope.fr). For this, we compared, under as the thermophilic bacteria Thermotoga maritima and similar conditions, the radioresistance of P. abyssi with Thermodesulfobacterium are not radioresistant 6Kopylov that of the bacteria E. coli and D. radiodurans. We then et al. 1993). measured the linear density of double-stranded breaks D. radiodurans is resistant to a wide range of geno- 6DSBs) resulting from gamma irradiation of the genomic toxic agents, suchas gamma radiation, UV radiation DNA of these three organisms and of P. furiosus using and mitomycin C. Ecient repair of DNA damage is in pulsed-®eld agarose gel electrophoresis. The linear den- large part responsible for the resistance to genotoxic sities were similar in all these organisms, indicating that agents 6Battista 1999). As DNA is commonly considered the radioresistance of Pyrococcus species is not due to to be the critical cellular target of radiation, the radio- speci®c protection of DNA but rather to an ecient resistance of Pyrococcus could be related, as in DNA repair system. Under our anaerobic conditions D. radiodurans, to a high capacity for ecient DNA Escherichia coli was more radioresistant than previously repair 6DiRuggiero et al. 1997). However, in contrast to reported 6Kopylov et al. 1993; Shahmohammadi et al. D. radiodurans, hyperthermophilic archaea are not par- 1997). This work thus shows that the conditions of ticularly resistant to UV irradiation 6Kopylov et al. irradiation are crucial when the radioresistances of 1993). Gamma radiation induces a wider variety of di€erent organisms are being compared. In our experi- DNA lesions than UV radiation 6Friedberg et al. 1995). ments, we found no clear evidence for signi®cant Thus, gamma radiation causes lesions in both the bases induction of any particular protein by gamma radiation, and the sugar residues of DNA, and results in the for- using two-dimensional gel electrophoresis of radioac- mation of DNA strand breaks, whereas UV radiation tively labelled proteins. In contrast, proteins synthesised 6254 nm) provokes mainly lesions in the DNA bases in response to gamma irradiation are detectable in E. coli 6Friedberg et al. 1995). Thus, it seems unlikely that 6West and Emmerson 1977) and D. radiodurans 6Hansen hyperthermophilic archaea are able selectively to repair 1980; Tanaka et al. 1996). Therefore, the method we only the lesions due to gamma radiation. During gamma used appears not to be suciently sensitive to identify irradiation direct ionisation of DNA and an indirect proteins induced in response to gamma irradiation in e€ect due to water radiolysis are involved in the for- Pyrococcus species. Another possibility is that most mation of DNA lesions 6Ward 1998). The hydroxyl proteins involved in Pyrococcus radioresistance are ex- radical intermediates formed during water radiolysis are pressed constitutively. Further investigations using other thought to cause 65% of the cell death 6Ward 1998). methods must be performed to answer this question. This indirect e€ect, due to hydroxyl radicals, does not arise in cells irradiated withUV 6Friedberg et al. 1995). Thus, hyperthermophilic archaea could be speci®cally Materials and methods resistant to the indirect e€ect of gamma irradiation because they possess mechanisms that protect DNA Strains and media against hydroxyl radicals. Indeed both radiosensitising agents and radioprotectants are known. Soluble intra- Pyrococcus abyssi 6Erauso 1993) and P. furiosus 6Fiala and Stetter 1986) were grown in YPC medium 6Yeast Peptone Cystine), which is cellular compounds suchas thiols6Ward 1998) and similar to YPS medium 6Erauso 1993) except that the sulphur is DNA-bound proteins 6Ljungman et al. 1991, Boubrik replaced by cystine. E. coli strain AB1157 and D. radiodurans RI were and Rouviere-Yaniv 1995) are thought to protect the kindly supplied by Adriana Bailone 6Institut Curie, Orsay, France). DNA against the damaging e€ects of ionising radiation. The E. coli strain was cultivated in Luria Broth6LB) medium or LB agar. D. radiodurans was cultivated in enriched 2´TGY 61% tryp- In contrast, oxygen acts as a radiosensitising agent that tone, 0.6% yeast extract, and 0.2% glucose) or on TGY agar. ®xes lesions which could have been repaired in its ab- sence 6Ward 1990; Spotheim-Maurizot 1991). As some proteins may be strongly attached to the DNA of Gamma irradiation of cells P. furiosus 6Peak et al. 1995), DNA protection in hy- The strains were irradiated at the end of the exponential growth perthermophilic archaea could be due to proteins that phase in YPC or in mineral medium 6YPC without yeast extract, limit the accessibility of DNA to hydroxyl radicals. In peptone or cystine) under anaerobic conditions in Hungate tubes. agreement with this hypothesis, Isabelle and collabora- Air was removed applying a vacuum and replaced by saturating the tors 61993) have shown that the protein MC1 of the tubes withN 2. Resazurin was added at 1 mg/l as a redox indicator and Na2S was added at 0.1% to ensure complete anaerobiosis. hyperthermophilic archaeon Methanococcus sp. CHTI55 Cultures were irradiated at a rate of 60 Gy/min using a 137Ce c-ray can protect against the radiolysis of DNA by fast source 6Institut Curie, Orsay, France). The number of viable 74 cells was estimated by plating serial dilutions, under anaerobic radiation dose under aerobic or anaerobic conditions, to conditions for Pyrococcus and aerobic conditions for E. coli and look at the e€ect of the presence of oxygen during D. radiodurans, in the appropriate growth medium. For E. coli and D. radiodurans, the number of viable cells was also estimated after irradiation. Under anaerobic conditions, all the P. abyssi plating of di€erent dilutions onto LB agar or TGY agar. cells survived a dose of up to 2000 Gy 6Fig. 1). At higher doses, viability decreased exponentially. As expected, oxygen increases the radiosensitivity dramatically. The Pulsed-®eld agarose gel electrophoresis 6PFGE) doses of radiation allowing 37% survival 6D37) in min- After irradiation, the cells were washed in arti®cial sea water for eral medium are 2111 Gy under anaerobiosis and Pyrococcus or in 0.9% NaCl for E. coli and D. radiodurans, and 305 Gy under aerobic conditions. The slope of the sur- suspended in 0.125 M EDTA at a density of 5´108 cells/ml. The vival curve is 1.67 times steeper in the presence of oxy- suspensions were then mixed with low-melting-point agarose to gen. In previous reports, oxygen was shown to increase obtain a ®nal concentration of 0.8% agarose. The 300-ll agarose blocks were then incubated overnight in ESP bu€er 60.5 M EDTA the radiosensitivity by a factor of 2.5±3 6Shenoy et al. pH 9±9.5, 1% lauroyl sarcosine, 1 mg/ml proteinase K) at 37°C. 1975), whereas in our experiment, the D37 value is seven One-®fthof eachagarose block was washedonce withTE bu€er times lower in the presence of oxygen. This observation containing 1 mM phenylmethylsulfonyl ¯uoride and then four can be explained by the fact that oxygen kills P. abyssi, times with TE bu€er. The intact chromosomal DNA contained in the agarose plugs was digested with 20 U of NotI restriction which is a strict anaerobe, even in the absence of ionising enzyme overnight at 37°C. Agarose plugs were analysed on 1% radiation 6Erauso 1993). Furthermore, the Na2S added agarose gels in 0.5´TBE using a CHEF-MAPPER electrophoresis to reduce the medium in the anaerobic cultures could system 6Bio-Rad) at 6.5 V/cm2 for 24 hat 16 °C, witha linear pulse also have a radioprotective e€ect. The survival rate of ramp of 15±70 s and a switching angle of 120°C. The gels were stained withwater containing ethidiumbromide 60.5 lg/ml) for the cells is higher in organic medium than in mineral 30 min and destained for 15 min in water. Gel images were digi- medium at high doses of irradiation. This could be due tised witha Sony CDD video camera and analysed using the to a protective e€ect of the cystine ± which contains GELSCAN software written by Yvan Zivanovic in our laboratory. sulphydryl residues ± in the organic medium. We then compared the survival curve for P. abyssi Estimation of the linear density of double-stranded DNA breaks withthosefor D. radiodurans and E. coli after irradia- introduced by gamma irradiation tion under the same anaerobic conditions 6Fig. 2). The three strains were irradiated under a nitrogen atmo- The linear density of DNA breaks was calculated using the fol- sphere in the presence of the reducing agent Na Sata lowing equation, where Pir is the proportion of each DNA band in 2 concentration of 0.1%. The curves presented here are the irradiated extracts, P0 represents the proportion of the same DNA band in the non-irradiated extract, L is the size of the DNA based on the average survival values from two or three band in bp and D is the dose of irradiation in Grays 6Gy). independent experiments. P. abyssi was markedly less 1 À Pir radioresistant than D. radiodurans since the latter did P0 not exhibit loss of viability, at least up to a dose of L  D irradiation of 3000 Gy 6the highest dose tested in our experiments). [The clear correlation between the number Two-dimensional gel electrophoresis of proteins of DNA double-stranded breaks and the radiation doses 6see below) con®rms that D. radiodurans was actually After irradiation, 2-ml aliquots of exponential-phase P. abyssi and exposed to the radiation.] In contrast, P. abyssi is more P. furiosus cells, at a density of 1.5´106 cells/ml, were grown in the presence of [35S]methionine 680 lCi/ml) in YPC medium. The cells were lysed in 40 ll of bu€er A [9.5 M urea 6Promega), 2% CHAPS 6Sigma), 0.1% DTT 6Boehringer Mannheim), 0.8% ampholytes 3±10 6ESA), and 8 mM PMSF 6Sigma)] by three cycles of freezing and thawing in liquid nitrogen. Linear immobilised pH 3±10 gra- dient strips 613 cm, Pharmacia) were rehydrated in bu€er A con- taining the proteins, mixed with 175 ll of Bu€er B [9 M urea 6Promega), 2% Chaps 6Sigma), 0.23% DTT 6Boehringer Mann- heim), 0.8% ampholytes 3±10 6ESA), 0.04% bromophenol blue, 8 mM phenylmethylsulfonyl ¯uoride 6Sigma)]. The proteins were focused for 5.5±6.5 h at 3500 V. The proteins were then separated on a 12% SDS-PAGE gel 6Laemmli 1970). The autoradiographs of the gels were obtained with a phosphoimager Image Quant 6Molecular Dynamics).

Results

Survival curves for P. abyssi, E. coli and D. radiodurans after irradiation withgamma rays

We ®rst measured survival rates for P. abyssi after Fig. 1 Survival curves for P. abyssi irradiated withgamma rays exposure to gamma radiation, as a function of the emitted by 137Ce under various conditions 75 Gamma-ray irradiation does not cause major changes in protein patterns in Pyrococcus

We tried to ®nd induced proteins that might be involved in radioresistance in Pyrococcus species by analysing one- and two-dimensional gel pro®les of total protein extracts from P. abyssi and P. furiosus after irradiation of the cultures with 180±3000 Gy of gamma radiation. In the ®rst set of experiments, the proteins were stained with silver to look at the steady-state pattern of whole protein extracts 6not shown). We failed to detect any signi®cant radiation-dependent changes in these pat- Fig. 2 Survival curves for P. abyssi, D. radiodurans and E. coli terns. To examine the patterns of newly synthesised after exposure to gamma radiation from 137Ce proteins after irradiation, proteins were labelled with [35S]methionine during growth for 10±105 min, and ex- tracted at di€erent times after irradiation 6from 30 min radioresistant than E. coli. The survival curve for E. coli to 6 h). The protein patterns remained essentially similar decreases exponentially at 100 Gy, the lowest dose withand withoutirradiation withdoses of 180±720 Gy tested. The D37 values for P. abyssi and E. coli are 6shown in Fig. 4 for 720 Gy). At higher doses, the 2436 Gy and 614 Gy, respectively. The slope of the ex- amount of newly synthesized proteins in irradiated cells ponential curve for P. abyssi, calculated from the values decreased in some experiments but the protein patterns shown in Fig. 1, is however 1.6 times steeper than that were conserved. Some proteins 6indicated by arrows) constructed from the E. coli data. The level of radiore- seem to be induced or to migrate di€erently after irra- sistance of E. coli is higher than usually reported in the diation in the experiment shown in Fig. 4. However, literature for cultures irradiated in the absence of oxy- these di€erences in protein patterns were not reproduc- gen. In the experiments reported by Kopylov et al. ible. This result shows that there are no striking 61993), 50% survival is obtained at a dose of 100 Gy, modi®cations in protein patterns 6relative induction or whereas a radiation dose of 417 Gy is necessary to re- repression) in P. furiosus or P. abyssi in response to duce survival by this amount under our conditions. The gamma-ray irradiation. di€erence could be due to the high level of reduction of the culture medium obtained in our experiments by addition of Na2S. Discussion

It was previously reported that several hyperthermo- Number of DNA breaks induced philic archaea are more radioresistant than E. coli by gamma irradiation but less resistant than D. radiodurans 6Kopylov et al. 1993; DiRuggiero et al. 1997). We con®rmed this To compare the linear density of DNA breaks between observation here by comparing the e€ect of gamma the two Pyrococcus species, E. coli and D. radiodurans, rays on the two latter organisms with that on two we analyzed their genomic DNAs after irradiation us- Pyrococcus species, P. abyssi and P. furiosus, under the ing PFGE. The genomic DNA isolated from irradiated same conditions. cells was digested with NotI before loading on the gel in An attractive hypothesis was that the radioresistance order to quantify the disappearance of bands with of Pyrococcus was due to some adaptation to high discrete sizes. For eachorganism, induced DSBs in the temperatures. The mechanisms that protect the DNA of DNA were detected at doses of radiation as low as 500 P. furiosus against thermal degradation 6Peak et al. or 1000 Gy 6Fig. 3). As expected, the bands corre- 1995) could also protect DNA against the hydroxyl sponding to the longer DNA fragments disappeared radicals produced during irradiation. We have shown ®rst, since there is a higher probability of ®nding a DSB here, however, that the number of DSB caused by in suchfragments. Thelinear densities of DSBs are gamma radiation in genomic DNA is similar in the two shown in Table 1. The results are averages of the values Pyrococcus species, E. coli and D. radiodurans. Thus, the obtained for eachDNA fragment at eachdose and, strong radioresistance of Pyrococcus is not related to except in the case of P. furiosus, two independent ex- speci®c protection of DNA. It appears therefore that the periments per species. In agreement withtheimpression mechanism that protects the DNA against thermal de- gained by visual inspection of the gel pro®les, values gradation at 100°C does not prevent the formation of obtained for all four species were very similar, varying DNA breaks during irradiation. from 0.72´10±9 DSB/Gy/bp for P. furiosus to 1.09´10±9 The stronger radioresistance of Pyrococcus compared DSB/Gy/bp for D. radiodurans. These results indicate to E. coli should be partly related to the size of the the absence of speci®c mechanisms that prevent the chromosomal DNA 61.8 Mb for Pyrococcus abyssi, formation of DSBs in the two Pyrococcus species. 4.6 Mb for Escherichia coli) since fewer DNA lesions are 76

Fig. 3 Pulsed-®eld gel electrophoresis of genomic DNA from P. abyssi, P. furiosus, D. radiodurans and E. coli. The strains were irradiated with0±3000 Gy gamma radiation. Total DNA was digested by NotI and analyzed by pulsed-®eld gel electrophoresis. On the gel at the top left, the lengths of the chromosomes of Saccharomyces cerevisiae are indicated in comparison witha NotI digest of genomic DNA of P. abyssi

Table 1 Linear density of DNA double-stranded breaks produced calculated that about 2 and 4 DSBs were created per by gamma irradiation in Pyrococcus furiosus, P. abyssi, Deinococ- chromosome for E. coli and P. abyssi, respectively, at the cus radiodurans and Escherichia coli D37 dose. However, this comparison probably underes- Species Linear density of DSB/Gy/bpa timates the di€erence in radioresistance between E. coli and Pyrococcus species. In contrast to E. coli, the two ±9 Pyrococcus furiosus 0.72 6‹0.24) ´10 Pyrococcus species possess systems that fully counteract Pyrococcus abyssi 0.84 6‹0.23) ´ 10±9 Deinococcus radiodurans 1.09 6‹0.40) ´10±9 the deleterious e€ects of radiation at dosages of up to Escherichia coli 0.82 6‹0.35) ´ 10±9 2000 Gy. D. radiodurans shows 100% survival when exposed to a dose of radiation equivalent to 3000 Gy, a The linear density of double-stranded DNA breaks was calculated which induces the formation of nearly 10 DSBs in its as described in Materials and methods chromosome. The capacity to repair DSB is thus higher in D. radiodurans than in E. coli or Pyrococcus. accumulated in Pyrococcus for a given dose of irradia- As in E. coli and D. radiodurans, DSBs may be tion. Based on the numbers of DSBs observed following repaired by homologous recombination in Pyrococcus irradiation and the size of the chromosomal DNA, we species. P. abyssi and P. furiosus contain several copies 77

Fig. 4 Comparison of two-dimensional gel pro®les of newly synthesized proteins from P. furiosus and P. abyssi before and after gamma ray irradiation. After irradiation with 720 Gy of gamma rays or no irradiation 6C) the cells were grown in the presence of [35S]methionine. Total protein extracts were analyzed on two-dimension- al gels. The arrows indicate proteins that seem to be induced in this experiment

of their chromosomes during the log phase and the this hypothesis, it has already been observed that some stationary phase of growth 6Bernander, personal com- proteins involved in DNA repair are constitutively ex- munication). Furthermore, the P. abyssi genome encodes pressed in Pyrococcus species. The two RecA/Rad51 several proteins similar to boththeRecA and Rad51 homologues present in P. furiosus, RadA and RadB, proteins, which are implicated in homologous recombi- are not induced by gamma rays or UV irradiation nation in bacteria and eukarya, respectively 6our un- 6Komori et al. 2000). Since P. abyssi and P. furiosus are published observations). We did not ®nd any signi®cant hyperthermophilic organisms, they probably need an protein induction after gamma irradiation. One possi- ecient DNA repair system which is continuously bility is that the proteins involved in the response to expressed in order to maintain the integrity of their gamma irradiation are inducible and that our method is genetic information. However, this situation may be not sensitive enough to detect them. Indeed, the two- more general in Archaea, since pretreatment of the two dimensional gels of proteins allowed us to observe be- archaean species Halobacterium halobium and Sulfolo- tween 200 and 300 individual proteins. This represents bus solfataricus withlow doses of hydrogen peroxide or only a tenthof all theproteins of P. abyssi and P. furiosus. N-methyl-N-nitrosoguanidine does not increase their A more sensitive method, such as microarray technology, survival when exposed to higher doses. This suggests should thus be used to look further for genes and pro- that no adaptive response to DNA damage exists in teins that are induced in response to gamma rays. An- these archaea 6Praul and Taylor 1997). The develop- other possibility is that most proteins involved in the ment of ecient genetic tools is now required to iden- response to gamma irradiation are constitutively tify the mechanisms involved in DNA radioresistance in expressed in P. abyssi and P. furiosus. In agreement with hyperthermophilic archaea. 78

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