Introduction
If life ever appeared on Mars, it was during the pre-Noachian period when liquid water occurred on its surface, and then, when the climatic conditions degraded, the water-rich epoch was followed by a cold and semi-arid era and then by the transition into present-day arid and cold conditions (Fairén et al., Reference Fairén, Davila, Lim, Bramall, Bonaccorsi, Zavaleta, Uceda, Stoker, Wierzchos, Dohm, Amils, Andersen and McKay2010). The opportunistic colonization of habitable environments could have occurred at any time during the Mars' history if microbial life remained viable when transported from a niche that turned non-habitable, to a habitable one (Cockell et al., Reference Cockell, Balme, Bridges, Davilad and Schwenzer2012; Westall et al., Reference Westall, Loizeau, Foucher, Bost, Betrand, Vago and Kminek2013). However, if microbial life did refuge in sub-superficial environments when surface conditions turned too hostile, then it should have been periodically revived from the dormant state and have repaired the accumulated damage (Westall et al., Reference Westall, Loizeau, Foucher, Bost, Betrand, Vago and Kminek2013). Galactic cosmic rays continuously hit Mars that is unshielded by a thick atmosphere or a global magnetic field, thus severely limiting the long-term endurance of dried, frozen life forms (Kminek et al., Reference Kminek, Bada, Pogliano and Ward2003; Hassler et al., Reference Hassler, Zeitlin, Wimmer-Schweingruber, Ehresmann, Rafkin, Eigenbrode, Brinza, Weigle, Böttcher, Böhm, Burmeister, Guo, Köhler, Martin, Reitz, Cucinotta, Kim, Grinspoon, Bullock, Posner, Gómez-Elvira, Vasavada and Grotzinger2014). In the absence of an active repair mechanism, i.e. in the dormant state, even the radioresistant bacterium Deinococcus radiodurans and Bacillus subtilis spores would be killed in a few million years in the uppermost few meters, due to damage accumulation (Pavlov et al., Reference Pavlov, Blinov and Konstantinov2002; Kminek et al., Reference Kminek, Bada, Pogliano and Ward2003). However, in the post-Noachian era, clement climate episodes could have allowed the revival of dormant microbial forms so that they repaired the accumulated, radiation-induced damages and reset the ‘survival clock’ to zero for the next dormant phase, thus extending their resilience limit over geological timescales (Hassler et al., Reference Hassler, Zeitlin, Wimmer-Schweingruber, Ehresmann, Rafkin, Eigenbrode, Brinza, Weigle, Böttcher, Böhm, Burmeister, Guo, Köhler, Martin, Reitz, Cucinotta, Kim, Grinspoon, Bullock, Posner, Gómez-Elvira, Vasavada and Grotzinger2014).
The scenario of the opportunistic colonization of protected niches on Mars gained support from the exposure of dried cells of the desert cyanobacterium Chroococcidiopsis to Mars-like conditions by using the ESA facility EXPOSE-R2 installed outside the International Space Station (Rabbow et al., Reference Rabbow, Rettberg, Parpart, Panitz, Schulte, Molter, Jaramillo, Demets, Weiß and Willnecker2017). In fact, dried Chroococcidiopsis could survive a total UV-radiation dose corresponding to a few-hour exposure on the Martian surface when cells were UV-shielded by the mixing with minerals (Billi et al., Reference Billi, Verseux, Fagliarone, Napoli, Baqué and de Vera2019b) or when bottom-layer cells were protected by the top-layer cells of the biofilm structure (Billi et al., Reference Billi, Staibano, Verseux, Fagliarone, Mosca, Baqué, Rabbow and Rettberg2019a). However, during the EXPOSE-R2 space mission, samples received a total ionizing radiation dose of 500 mGy (Dachev et al., Reference Dachev, Bankov, Tomov, Matviichuk, Dimitrov, Häder and Horneck2017). Therefore, in view of the ionizing radiation dose of 76 mGy year−1 measured at Gale Crater's surface (Hassler et al., Reference Hassler, Zeitlin, Wimmer-Schweingruber, Ehresmann, Rafkin, Eigenbrode, Brinza, Weigle, Böttcher, Böhm, Burmeister, Guo, Köhler, Martin, Reitz, Cucinotta, Kim, Grinspoon, Bullock, Posner, Gómez-Elvira, Vasavada and Grotzinger2014), the simulation of radiation doses equivalent to millions of years on Mars is extremely complicated.
New insights into Mars-relevant ionizing-radiation doses were obtained with the STARLIFE project that exposed a selection of extremophiles to X-, gamma rays and heavy ions, representing major parts of the galactic cosmic radiation (Moeller et al., Reference Moeller, Raguse, Leuko, Berger, Hellweg, Fujimori, Okayasu and Horneck2017). Different types of ionizing radiation differ in their biological effectiveness depending on the linear energy transfer (LET) that represents the average energy loss of a particle per unit length of its track. Hence low-LET radiations, such as X- or gamma-rays, produce ionizations sparsely along their track, whereas high-LET radiation, such as heavy ions, induces more complex damage (Terato and Ide, Reference Terato and Ide2004; Horneck et al., Reference Horneck, Klaus and Mancinelli2010).
In the context of the STARLIFE project, it was shown that dried cells of the radioresistant cyanobacterium Chroococcidiopsis sp. CCMEE 029 survived up to 24 kGy of gamma radiation and 2 kGy of Fe-ion radiation, the highest high-LET dose tested (Verseux et al., Reference Verseux, Baqué, Cifariello, Fagliarone, Raguse, Moeller and Billi2017). Indeed, even though high atomic number and energy particles represent only the 1.5% of the galactic cosmic rays (Ferrari and Szuszkiewicz, Reference Ferrari and Szuszkiewicz2009), they pose the ultimate limit for life (Horneck et al., Reference Horneck, Klaus and Mancinelli2010). Therefore, unravelling how life, as we know it, can cope with high-LET doses is of paramount importance. The STARLIFE project allowed for the first time the exposure of dried Chroococcidiopsis cells to high-LET radiation. However, the lack of the genome sequence of the investigated Chroococcidiopsis strain did not allow a deeper investigation of the accumulated DNA damage under high-LET radiation as well as of the repair mechanisms taking place upon rehydration. Recently, the sequencing of Chroococcidiopsis sp. CCMEE 029's genome allowed to unravel the role of the photoreactivation, nucleotide excision repair (NER) and UV damage endonuclease (UvsE)-dependent excision repair (UVER) in the recovery of dried biofilms exposed to a Mars-like UV flux (Mosca et al., Reference Mosca, Rothschild, Napoli, Ferré, Pietrosanto, Fagliarone, Baqué, Rabbow, Rettberg and Billi2019). Similarly, the involvement of the homologous recombination RecF pathway and base excision repair (BER) was highlighted during the revival of dried cells exposed to space vacuum and cosmic ionizing radiation (Mosca et al., Reference Mosca, Fagliarone, Napoli, Rabbow, Rettberg and Billi2021).
In the present work, we have taken advantage of the previously identified DNA repair pathways of Chroococcidiopsis sp. CCMEE 029 to investigate during the rehydration of dried cells exposed to high-LET radiation, the role of DNA repair mechanisms in the restoration of the induced complex DNA damage. After the exposure to 2 kGy of Fe-ion radiation, cell viability was verified using a quantitative polymerase chain reaction (qPCR) coupled with propidium monoazide (PMA) treatment, while DNA damage was determined using a PCR-stop assay. Then, an in-silico search for the uvrD gene, previously not identified, was performed. Finally, the expression of the genes involved in the RecF, BER, NER and UVER pathways was monitored by real-time quantitative polymerase chain reaction (RT-qPCR) during 1, 6 and 12 h rehydration.
Material and methods
Organism and culture conditions
Chroococcidiopsis sp. CCMEE 029 was isolated by Roseli Ocampo-Friedmann from cryptoendolithic growth in sandstone in the Negev Desert (Israel) and it is now kept at the University of Rome Tor Vergata, as part of the Culture Collection of Microorganisms from Extreme Environments (CCMEE) established by E. Imre Friedmann (Friedmann and Ocampo-Friedmann, Reference Friedmann and Ocampo-Friedmann1995). Cultures were grown under routine conditions at 25 °C in BG-11 liquid medium (Rippka et al., Reference Rippka, Deruelles, Waterbury, Herdman and Stanier1979), under a photon flux density of 40 μmol m−2 s−1 provided by fluorescent cool-white bulbs.
Cell drying, irradiation and recovery
Triplicates of dried samples were prepared by filtering on Millipore filters about 5 × 108 cells obtained from cultures in early stationary phase. Filters were air-dried overnight under a sterile hood and shipped to the Heavy Ion Medical Accelerator in Chiba (HIMAC, Gunma University Heavy Ion Medical Center, Japan) for the irradiation campaign performed in June 2018. Samples were irradiated with 198 keV μm−1 Fe ions with a dose rate of 12.1 Gy min−1 at 23 °C ± 1 °C. Dried-irradiated samples were shipped back with non-irradiated dried cells that were used as control. After the irradiation, samples were stored for one year in the dried state until the analyses were performed. For the recovery, each filter was cut in four pieces, and each piece transferred in a polystyrene falcon with 1 ml of BG-11 medium and incubated under routine conditions. Afterwards 1, 6 and 12 h recovery cells were centrifuged and pellets harvested in ice for RNA extraction (see below).
Cell viability
The viability of dried-irradiated cells was evaluated according to the PMA-qPCR assay (Nocker et al., Reference Nocker, Cheung and Camper2006). About 2.5 × 108 cells from each sample were harvested with 500 μl of PBS 1× containing 50 μM PMA (Biotium Inc., Hayward, WI, USA). After incubation for 10 min in the dark, at room temperature, samples were placed on ice and exposed to a 650 W halogen lamp for 15 min, to allow the PMA photoactivation and cross-link to DNA. Genomic DNA was extracted and amplified by qPCR as described (Baqué et al., Reference Baqué, Scalzi, Rabbow, Rettberg and Billi2013). Liquid cultures and dried cells were used as control.
DNA damage evaluation
DNA was extracted from about 2.5 × 108 cells harvested from dried samples with 1 ml of PBS 1×, as described (Baqué et al., Reference Baqué, Scalzi, Rabbow, Rettberg and Billi2013), and quantified with a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). PCR-stop assay with a 4 kbp target was performed as previously reported (Mosca et al., Reference Mosca, Rothschild, Napoli, Ferré, Pietrosanto, Fagliarone, Baqué, Rabbow, Rettberg and Billi2019). Liquid cultures and dried cells were used as control.
In-silico search for DNA repair genes
The sequence of Chroococcidiopsis sp. CCMEE 029's genome was already available and the previously unidentified uvrD gene (Mosca et al., Reference Mosca, Rothschild, Napoli, Ferré, Pietrosanto, Fagliarone, Baqué, Rabbow, Rettberg and Billi2019, Reference Mosca, Fagliarone, Napoli, Rabbow, Rettberg and Billi2021) was annotated by using BlastKOALA (Kanehisa et al., Reference Kanehisa, Sato and Morishima2016). The sequence of the uvrD gene was deposited in GenBank under the accession number MT813437.
Expression of DNA repair genes
Total RNA was extracted from about 5 × 108 cells from dried, irradiated samples after 1, 6 and 12 h of rehydration by using the TRI Reagent (Sigma Aldrich, Saint Louis, MO, USA). The reverse transcription of the RNA into cDNA was performed by using the SensiFAST™ cDNA Synthesis Kit (Bioline, Meridian Life Science, Memphis, TN, USA) as previously described (Mosca et al., Reference Mosca, Rothschild, Napoli, Ferré, Pietrosanto, Fagliarone, Baqué, Rabbow, Rettberg and Billi2019). Each real-time PCR reaction was performed on StepOnePlus Real Time PCR System (Thermo Fisher Scientific) with equal volumes of cDNA synthesis mixture in 12 μl reaction volume, including 6 μl of iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and 0.4 μM of forward and reverse primers (Table 1). PCR conditions were as described (Mosca et al., Reference Mosca, Rothschild, Napoli, Ferré, Pietrosanto, Fagliarone, Baqué, Rabbow, Rettberg and Billi2019) and each run was completed with a melting curve analysis to validate the specificity of amplification. Each experiment contained a no-template control and the 16S rRNA was used as housekeeping gene. Relative mRNA levels were calculated by the comparative cycle threshold (Ct) method. Values obtained for dried-irradiated cells (0-recovery) were set as 1 and values of re-hydrated, irradiated, dried cells were considered up-regulated (>1) or down-regulated (<1).
Table 1. List of gene and primer sequences used in the RT-qPCR analysis
Results
Viability and evaluation of DNA damage in dried-irradiated cells
The viability of dried cells of Chroococcidiopsis sp. CCMEE 029 irradiated with 2 kGy of Fe-ion radiation was evaluated by using the PMA-qPCR assay (Fig. 1), that is based on fact that the cell membrane-impermeable PMA dye stains only dead cells and by binding the genomic DNA, it prevents the PCR target amplification (Nocker et al., Reference Nocker, Cheung and Camper2006). Results showed a non-significant reduction of the amplified copy numbers in dried-irradiated cells compared to dried cells. While dried cells did not show any significant difference in the PCR target amplification compared to liquid cultures (Fig. 1(a)).
Fig. 1. Cell viability of Chroococcidiopsis sp. CCMEE 029 according to PMA-qPCR assay (a). DNA damage evaluated with the PCR-stop assay (b). Liq, liquid culture; D, dried cells; DIR, dried cells irradiated with 2 kGy of Fe-ion radiation; M, hyperladder 1 kbp.
The PCR-stop assay with a 4 kbp target revealed an increased amount of DNA lesions in the target sequence that resulted in a progressive reduction of the amplicon intensity in dried cells and in dried-irradiated cells compared to liquid cultures (Fig. 1(b)).
Expression of RecF pathway genes during the rehydration of dried-irradiated cells
Upon rehydration, dried-irradiated cells of Chroococcidiopsis sp. CCMEE 029 showed a different over-expression of genes involved in the RecF DNA repair pathway (Fig. 2). The recF gene was up-regulated by 2.40-, 2.89- and 3.01-fold during 1, 6 and 12 h rehydration, respectively. Among the two recJ genes, only the reJ1 gene was over-expressed with a 2.67-, 2.72- and 2.94-fold increase after 1, 6 and 12 h, respectively. The recN gene was over-expressed by 2.43-, 2.83- and 3.02-fold, at 1, 6 and 12 h, respectively. The recQ1 and recQ2 genes were up-regulated by about twofold after 1 h, and by 2.98- and 3.64-fold, respectively, after 6 h and by 2.81- and 3.71-fold, respectively, after 12 h. The recO and recR genes were weakly over-expressed during 1 and 6 h rehydration, while after 12 h they were up-regulated by about twofold. While the expression of the ssb gene was fourfold increased after 12 h rehydration (Fig. 2).
Fig. 2. Expression of genes of the RecF repair pathway during the rehydration of dried cells of Chroococcidiopsis sp. CCMEE 029 exposed to 2 kGy of Fe-ion radiation. Values from non-rehydrated, irradiated cells were considered as control values and set to 1.
Expression of BER genes during the rehydration of dried-irradiated cells
During the rehydration of dried-irradiated cells of Chroococcidiopsis sp. CCMEE 029, the genes involved in the BER DNA repair pathway were weakly over-expressed, exception made for the mutY gene that was up-regulated by 2.92-fold after 1 h, and by 6.38- and 5.91-fold after 6 and 12 h, respectively (Fig. 3). The expression of the alkA gene showed a 2.14-fold increase after 6 h rehydration. The fpg was over-expressed by 1.81-, 2.26- and 2.11-fold after 1, 6 and 12 h, respectively. The mpg was up-regulated by 2.53- and 2.38-fold after 6 and 12 h, respectively. While the mug gene was up-regulated by 2.04-, 3.15- and 3.01-fold after 1, 6 and 12 h of rehydration, respectively. There was no over-expression of the nth gene, while the udg gene was up-regulated by 2.35- and 2.03-fold after 6 and 12 h, respectively. Finally, the xth gene was over-expressed by 2.2-, 3.11- and 2.59-fold after 1, 6 and 12 h rehydration, respectively (Fig. 3).
Fig. 3. Expression of genes of the BER repair pathway during the rehydration of dried cells of Chroococcidiopsis sp. CCMEE 029 exposed to 2 kGy of Fe-ion radiation. Values from non-rehydrated, irradiated cells were considered as control values and set to 1.
Expression of NER and UVER genes during the rehydration of dried-irradiated cells
Upon rehydration, dried-irradiated cells of Chroococcidiopsis sp. CCMEE 029 did not show any over-expression of the uvrA and uvrB genes involved in the NER repair pathway (Fig. 4). The uvrC gene was up-regulated by 2.34-fold only after 12 h rehydration, while the uvrD gene was up-regulated by 1.86-, 3.00- and 3.10-fold after 1, 6 and 12 h, respectively. Finally, the uvsE showed a weak over-expression only at 1 h rehydration (Fig. 4).
Fig. 4. Expression of genes of the NER and UVER repair pathways during the rehydration of dried cells of Chroococcidiopsis sp. CCMEE 029 exposed to 2 kGy of Fe-ion radiation. Values from non-rehydrated, irradiated cells were considered as control values and set to 1.
Table 2 shows an overview of the role of DNA repair pathways in the repair of damages induced by 2 kGy of Fe-radiation during the rehydration of Chroococcidiopsis sp. CCMEE 029.
Table 2. Involvement of DNA repair pathways in the repair of damages induced by 2 kGy of Fe-radiation during the rehydration of Chroococcidiopsis sp. CCMEE 029
RecF, RecF homologous recombination; BER, base excision repair; NER, nucleotide excision repair; UVER, UV damage endonuclease (UvsE)-dependent excision repair.
a Over-expressed genes are highlighted in dark grey, not over-expressed genes are highlighted in grey.
Discussion
The measurement of Mars' surface radiation environment has allowed the modelling of subsurface radiation environment with paramount implications for microbial survival times (Hassler et al., Reference Hassler, Zeitlin, Wimmer-Schweingruber, Ehresmann, Rafkin, Eigenbrode, Brinza, Weigle, Böttcher, Böhm, Burmeister, Guo, Köhler, Martin, Reitz, Cucinotta, Kim, Grinspoon, Bullock, Posner, Gómez-Elvira, Vasavada and Grotzinger2014). Any putative dormant life occurring in near-surface frozen habitats would accumulate high doses of cosmic rays over geological timescales and finally eradicated (Dartnell et al., Reference Dartnell, Desorgher, Ward and Coates2007). Nevertheless, during the post-Noachian, brief periods of clement conditions might have occurred on timescales of several hundred thousand to a few million years era that allowed the metabolic activity recovery and repair of the radiation-induced damage (Hassler et al., Reference Hassler, Zeitlin, Wimmer-Schweingruber, Ehresmann, Rafkin, Eigenbrode, Brinza, Weigle, Böttcher, Böhm, Burmeister, Guo, Köhler, Martin, Reitz, Cucinotta, Kim, Grinspoon, Bullock, Posner, Gómez-Elvira, Vasavada and Grotzinger2014). In this scenario, the exposure of dried, radioresistant microorganisms to ionizing radiation doses mimicking long-term irradiation is fundamental to get insights into the resilience limit of putative dormant microbial life in the Martian subsurface over geological timescales.
Here the capability of repairing the DNA damage accumulated under high-LET Fe-ion radiation was investigated by irradiating dried cells of the radioresistant cyanobacterium Chroococcidiopsis sp. CCMEE 029 to accelerate Fe ions up to the final dose of 2 kGy. As revealed by the PMA-qPCR assay, this exposure did not significantly affect its survivability. This result is relevant if compared to the previously reported reduced survival of dormant microorganisms exposed to Fe irradiation. For example, 500 mGy of Fe-ion radiation caused a twofold reduction of B. subtilis spore survival (Moeller et al., Reference Moeller, Setlow, Horneck, Berger, Reitz, Rettberg, Doherty, Okayasu and Nicholson2008) and a fivefold reduction in four Bacillus spp. from hot and cold environments (Zammuto et al., Reference Zammuto, Rizzo, De Plano, Franco, Guglielmino, Caccamo, Magazù, Fujimori, Giudice, Guglielmin, McAlpin, Moeller and Gugliandolo2020). While 1 kGy of Fe-ion radiation reduced the viability of dried colonies of the Antarctic fungus Cryomyces antarcticus to 13% (Aureli et al., Reference Aureli, Pacelli, Cassaro, Fujimori, Moeller and Onofri2020).
There might be a number of possible explanations for the enhanced radiation-tolerance of dried Chroococcidiopsis sp. CCMEE 029. First, an efficient stabilization of dried cellular components achieved via the accumulation of sucrose and trehalose, the latter acting also as free-radical scavenger (Fagliarone et al., Reference Fagliarone, Napoli, Chiavarini, Baqué, de Vera and Billi2020). Second, an efficient DNA repair mechanism of the double strand break (DSB) taking advantage of the presence of multiple genome copies and absence of the low-fidelity non-homologous end joining system (Mosca et al., Reference Mosca, Fagliarone, Napoli, Rabbow, Rettberg and Billi2021).
It was estimated that a timescale longer than 10 million years would be required to accumulate a dose of 1 kGy of Fe-ion radiation in the Martian surface (Aureli et al., Reference Aureli, Pacelli, Cassaro, Fujimori, Moeller and Onofri2020). Therefore, the non-significant reduction in the survival of dried Chroococcidiopsis irradiated with 2 kGy of Fe-ion radiation further expanded our appreciation of the long-term resilience of a putative dormant microbial life in the Martian subsurface. Moreover, putative Martian microbial life forms could have evolved even a greater radiotolerance than Earth radioresistant microorganisms (Dartnell et al., Reference Dartnell, Desorgher, Ward and Coates2007).
In the present work, the absence of a significant reduction in the survival of dried, irradiated Chroococcidiopsis was accompanied by DNA damage accumulation. An increased amount of DNA lesions was highlighted by PCR-stop assay with a 4 kbp target in dried, irradiated cells compared to dried cells. This is in agreement with the previously reported lack of DNA damage tested with shorter-PCR targets (Verseux et al., Reference Verseux, Baqué, Cifariello, Fagliarone, Raguse, Moeller and Billi2017), because there is a higher likelihood of encountering lesions that impair DNA polymerase progression in longer DNA sequences than in the shorter ones (Rudi et al., Reference Rudi, Hagen, Johnsrud, Skjefstad and Tryland2010).
The monitoring of the expression of genes involved in DNA repair pathways revealed the capability of dried, irradiated Chroococcidiopsis to revive upon rehydration and to repair complex, accumulated DNA damage. The fact that DNA repair genes were still expressed after 12 h rehydration highlighted the time needed to repair complex DNA damage as previously reported for the repair of the severe DNA damage induced by 5 kGy of X-rays and restored after 24 h of recovery (Billi et al., Reference Billi, Friedmann, Hofer, Grilli Caiola and Ocampo-Friedmann2000). In fact, when a high-LET radiation passes through DNA, it induces clustered damage, defined as two or more closely associated lesions consisting of DSBs along with oxidized bases and apurinic-apyrimidinic sites (Terato and Ide, Reference Terato and Ide2004; Okayasu, Reference Okayasu2012). Among clustered DNA damage, DSBs are the most severe ones, thus limiting the chance of survival (Spies and Kowalczykowski, Reference Spies, Kowalczykowski and Higgins2005).
During the rehydration of dried-irradiated Chroococcidiopsis, most genes associated with the RecF pathway were over-expressed, suggesting the role of this homologous recombination pathway in the DSB repair. In particular, the over-expression of the recN gene was interesting since in D. radiodurans the RecN protein showed a cohesin-like activity and stimulated RecA-mediated recombinational repair of DNA damage (Uranga et al., Reference Uranga, Reyes, Patidar, Redman and Lusetti2017).
The bioinformatics analysis revealed the presence in Chroococcidiopsis sp. CCMEE 029's genome of eight genes of the BER system encoding seven nucleotide glycosylases and one endonuclease (Mosca et al., Reference Mosca, Fagliarone, Napoli, Rabbow, Rettberg and Billi2021). Such a high number of DNA glycosylases was reported for the genome of D. radiodurans and ruled out as important for the DNA repair efficiency (Makarova et al., Reference Makarova, Aravind, Wolf, Tatusov, Minton, Koonin and Daly2001). However, upon rehydration dried-irradiated Chroococcidiopsis showed a weak over-expression of the BER genes, exception made for the mutY gene, thus suggesting a lack of DNA oxidative damage. In addition, the absence of an up-regulation of the uvrA and uvrB genes of the NER repair system suggested the lack of DNA lesions, like cyclobutane-pyrimidine dimers and 6–4 photoproducts that are generally induced by UV radiation (Rastogi et al., Reference Rastogi, Kumar, Tyagi and Sinha2010). Also the uvsE gene was not over-expressed, thus confirming its role in repairing DNA damage due to desiccation rather than to UV radiation (Mosca et al., Reference Mosca, Rothschild, Napoli, Ferré, Pietrosanto, Fagliarone, Baqué, Rabbow, Rettberg and Billi2019) and high-LET radiation. On the other hand, the over-expression of the uvrD gene suggested the involvement of the encoded DNA helicase in the DSB repair as previously reported for D. radiodurans (Bentchikou et al., Reference Bentchikou, Servant, Coste and Sommer2010).
In conclusion, our findings provided new insights into the resilience of radiotolerant, dried microorganisms exposed to a high-LET dose mimicking long-term exposure in the Martian subsurface. In addition, the tolerance of dried Chroococcidiopsis towards high-LET radiation is relevant when searching life in other astrobiology targets rather than Mars, like Europa and Enceladus, icy moon of Jupiter and Saturn, respectively, where the high radiation environments (Rettberg et al., Reference Rettberg, Antunes, Brucato, Cabezas, Collins, Haddaji, Kminek, Leuko, McKenna-Lawlor, Moissl-Eichinger, Fellous, Olsson-Francis, Pearce, Rabbow, Royle, Saunders, Sephton, Spry, Walter, Wimmer Schweingruber and Treuet2019) might critically affect the long-term survival of dormant, frozen life forms.
Financial support
This research was supported by the Italian Space Agency (Bio-Signatures and habitable niches_Cyanobacteria - BIOSIGN_Cyano; grant 2018-15-U.0 to DB). RM was supported by the DLR grant FuE-Projekt ‘ISS LIFE’ (Programm RF-FuW, Teilprogramm 475). AF and RM – MEXT Grant-in-Aid for Scientific Research on Innovative Areas ‘Living in Space’ (Grant Numbers: 15H05935, 15K21745).
