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 ecient 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 ecient 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. Ecient 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 ecient resistance of Pyrococcus could be related, as in DNA repair system. Under our anaerobic conditions D. radiodurans, to a high capacity for ecient 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 dierent 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 suciently sensitive to identify irradiation direct ionisation of DNA and an indirect proteins induced in response to gamma irradiation in eect 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 eect, 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 eect 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 eects 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