Extremophiles (2006) DOI 10.1007/s00792-006-0002-9 ORIGINAL PAPER Ernest Williams Æ Todd M. Lowe Æ Jeffrey Savas Jocelyne DiRuggiero Microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus exposed to gamma irradiation Received: 21 March 2006 / Accepted: 16 May 2006 Ó Springer-Verlag 2006 Abstract The remarkable survival of the hyperthermo- Keywords Archaea Æ Hyperthermophile Æ philic archaeon Pyrococcus furiosus to ionizing radiation DNA repair Æ Ionizing radiation Æ was previously demonstrated. Using a time course study Oxidative stress Æ Transcriptional analysis and whole-genome microarray analyses of mRNA transcript levels, the genes and regulatory pathways in- volved in the repair of lesions produced by ionizing Introduction irradiation (oxidative damage and DNA strand breaks) in P. furiosus were investigated. Data analyses showed Pyrococcus furiosus is an hyperthermophilic archaeon that radA, encoding the archaeal homolog of the RecA/ that grows optimally at 100°C under anaerobic condi- Rad51 recombinase, was moderately up regulated by tions (Fiala and Stetter 1986). Its genome has been irradiation and that a putative DNA-repair gene cluster sequenced along with two other Pyrococcus species, was specifically induced by exposure to ionizing radia- P. abyssi and P. horikoshii, providing a basis for com- tion. This novel repair system appears to be unique to parative genome analyses (Maeder et al. 1999). We thermophilic archaea and bacteria and is suspected to be demonstrated that exposure of P. furiosus cells to ion- involved in translesion synthesis. Genes that encode for izing radiation—a substitute to heat in inducing DNA a putative Dps-like iron-chelating protein and two strand breaks (Peak et al. 1995)—at 2,500 Grays (Gy) membrane-bound oxidoreductases were differentially resulted in chromosome fragmentation (DiRuggiero expressed following gamma irradiation, potentially in et al. 1997). Similar results were obtained with P. abyssi response to oxidative stress. Surprisingly, the many (Gerard et al. 2001) and within 2–4 h of incubation at systems involved in oxygen detoxification and redox 95°C the chromosomes of both Pyrococcus species were homeostasis appeared to be constitutively expressed. reassembled, producing actively growing cells and Finally, we identified several transcriptional regulators demonstrating highly effective DNA repair systems and protein kinases highly regulated in response to (DiRuggiero et al. 1997). Chromosome reassembly fol- gamma irradiation. lowing fragmentation by ionizing radiation was also reported for the halophilic archaeon Halobacterium sa- linarium strain NRC-1 (Kottemann et al. 2005) and the Electronic Supplementary Material Supplementary material is bacterium Deinococcus radiodurans (Cox and Battista available to authorised users in the online version of this article at 2005) suggesting that adaptation to environmental http://dx.doi.org/10.1007/s00792-006-0002-9. conditions such as high temperature and desiccation may result in enhanced radiation resistance (DiRuggiero Communicated by K. Horikoshi et al. 1997; Kottemann et al. 2005; Cox and Battista E. Williams Æ J. DiRuggiero (&) 2005). Department of Cell Biology and Molecular Genetics, More than 80% of the deleterious effects of gamma University of Maryland, 3221 H.J. Patterson Hall, radiation are the result of hydroxyl radical production College Park, MD 20742, USA E-mail: [email protected] via radiolysis of water, imparting severe oxidative stress Tel.: +1-301-4054598 to all the cell’s components (Hutchinson 1985). Lesions Fax: +1-301-3149081 to the DNA include bases modifications, DNA back- bone breakage and cross-linking of proteins to DNA T. M. Lowe Æ J. Savas Department of Biomolecular Engineering, (Hutchinson 1985; Riley 1994). Clustered lesions are UCSC RNA Center, University of California, Santa Cruz, converted into double-strand breaks when two opposing 1156 High Street, Santa Cruz, CA 95064, USA strand breaks are formed or during attempted base excision repair (Dianov et al. 2001). Therefore, DNA thanesulfonate (EMS) by adding 0.001% of H2O2 or repair systems and cellular defense mechanisms against 0.1 M EMS (final concentrations) to cultures grown to oxidative stress are key components of radiation resis- 5 · 106 cells/ml (Watrin and Prieur 1996; Praul and tance. Taylor 1997). Cultures were incubated at room tem- Maintenance of genome stability is critical to the perature for 30 min under anaerobic conditions. Sodium survival of organisms and in particular to those organ- thiosulfate was added to the EMS treated cultures to a isms living at high temperature. Surprisingly, our final concentration of 200 mM to inactivate EMS. Cells understanding of the pathways and proteins involved in from both treatments were washed twice, resuspended in DNA repair in hyperthermophiles, and in archaea in reduced culture medium and incubated at 90°C for general, is still very limited (Kelman and White 2005). 40 min. Controls were treated in the same way without Comparative genome analyses have shown that archaeal addition of H2O2 or EMS, or without gamma irradia- DNA repair proteins are more closely related to their tion. eukaryal than to their bacterial counterparts and that many proteins, and sometimes entire pathways, are missing in some or in all the archaea (DiRuggiero and RNA extraction Robb 2004; Kelman and White 2005). Homologs of the conserved mismatch proteins MutS and MutL have only Cultures were centrifuged at 4,000g for 4 min at 4°C and been found in the genomes of a few archaea (Grogan RNA was extracted from the pellet using the TRI re- 2004), and while most archaeal genomes contain ho- agent (SIGMA T9424, St Louis, MO) as previously mologs of eukaryal nucleotide excision repair proteins, described (Chomczynski and Sacchi 1987). RNA sam- several archaea also have bacterial UvrABC homologs ples were analyzed with a Beckman DU640 spectro- (Kelman and White 2005). Archaeal proteins involved in photometer (Beckman Coulter, Fullerton, CA) and gel homologous recombination, one of the major pathways electrophoresis for quality and quantification. for the repair of double-strand breaks, are structurally and functionally similar to eukaryotic proteins (Ishino et al. 2006) but several proteins involved in key steps of Array design and printing this pathway remain to be identified in both the Archaea and Eukarya (DiRuggiero and Robb 2004; Ishino et al. The P. furiosus full-genome microarray represented 2006). 2,065 protein encoding genes. DNA fragments for In this work we investigated the genes and regulatory spotting were produced by PCR. The array was con- pathways in P. furiosus involved in the repair of DNA structed by designing oligonucleotide primers to span strand breaks and oxidative damage produced by ion- the most unique portions of each gene, averaging 350 izing irradiation, using a full-genome microarray. We nucleotides in length. A custom Perl program was found that the physiological response of P. furiosus written to select primers with the most desirable ther- during recovery from gamma irradiation involved sev- modynamic conditions possible for each gene using the eral DNA repair processes, a down-regulation of meta- Whitehead Institute’s Primer3 program (version 3.09) as bolic functions and a concerted attempt to reverse and its core analysis engine. The polymerase chain reaction minimize oxidative damage. Surprisingly, many of the was performed in 100 ll reactions with 1 lM of each oxygen detoxification systems appeared to be constitu- primer, 2 mM MgCl2, 1.0 mM dNTPs, 1.75 U Amp- tively expressed. liTaq DNA polymerase (Applied Biosystems, Foster City, CA) 1x AmpliTaq buffer, and 20 ng genomic DNA for 30 cycles (94°C30s,50°C 1 min, 72°C 2 min). All Materials and methods PCR products were checked by agarose gel electropho- resis and failures were repeated or new primers designed. Cell growth and treatments Each PCR amplicon was spotted on poly-L-lysine coated glass slides twice, each time by a different print P. furiosus strain (DSMZ 3638) was grown in 100 ml pin (print plates were rotated 180° for second spotting) serum bottles at 90°C under anaerobic conditions in the using a 16-pins ArrayMaker Version 1.0 (http:// absence of sulfur and with 100 lMNa2WO4 and 0.5% www.cmgm.stanford.edu/pbrown/mguide/index.html). (wt/vol) maltose (Robb et al. 1992). Cultures grown to approximately 5 · 106 cells/ml were chilled to 4°C and exposed to 2,500 Gy of gamma radiation using a 26,000- Labeling and hybridization curie (9.6 · 1014 Bq) 60Co gamma source at the Uni- versity of Maryland College Park Gamma Test Facility, First strand cDNA synthesis and labeling was carried at a dose rate of 73 Gy/min. Cultures were further out with total RNA (3 lg), 200 U Superscript II reverse incubated at 90°C and samples for RNA extraction were transcriptase (Invitrogen, Carlsbad, CA), random taken at 0, 20, 40, 60, 120 and 240 min after the tem- hexamers, 0.5 mM dATP, dGTP, dCTP, 0.2 mM dTTP perature of the cultures reached 90°C (DiRuggiero et al. and 0.3 mM Cy3-dUTP or Cy5-dUTP (Amersham 1997). Cells were treated with H2O2 and ethyl me- Biosciences, Piscataway, NJ) for 110 min at 42°C. Labeled cDNAs were cleaned up with alkaline treatment product was spotted at two spatially different locations and Cyscribe GFX columns (Amersham Biosciences) onto the slides, (2) biological replicates: each compari- according to the manufacturer’s instructions. Hybrid- son was performed from four independently processed ization was carried out in a sealed Corning hybridization cultures and hybridized to four different slides to ac- chamber (Acton, MA) at 60°C for 18 h. For each count for inherent biological variation occurring inde- experimental time point four slides, with at least two pendently of the experimental perturbations, giving a features per open reading frame (ORF), were hybridized total of eight data points per condition per gene. All producing eight replicates or more per ORF. RNA samples were compared with a reference RNA extracted from an aliquot of the same culture prior to treatment.