Isolation of UVC Tolerant Bacteria from the Hyperarid Atacama Desert, Chile
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(2) 326. contain chemically aggressive sulfates, chlorides, and perchlorates in concentrations well above the limits tolerated by mesophilic microorganisms [2]. Annual average sunlight in the Atacama core is 335 Wm−2 with a consistent daily maximum over 1,000 Wm−2 [16]. Maximum values for photosynthetic active radiation (PAR) recorded in 2006 summer at Yungay and Salar Grande were as high as 2.37 and 2.21 mmolm−2 s−1, respectively [6]. These harsh physical and chemical environmental constraints apparently caused those extensive regions of the Atacama Desert to be almost devoid even of microbial life, with abundances of one or two orders of magnitude below those found in any other arid region on Earth [19]. Several studies have been conducted on the Atacama Desert in order to obtain a better profiling of its extremophilic microbial communities [2, 17, 19, 24, 26]. Navarro-Gonzalez et al. [19] were among the first to report the unique properties of soils in the extreme arid core region of the Atacama Desert. Samples from this region exhibited only trace levels of organics and variable scores of culturable and nonculturable bacteria [10]. However, little has been published on indigenous UV radiation-tolerant microbial populations of the Atacama Desert. An impressive silica-encased filamentous cyanobacteria was recently found in El Tatio geothermal fields at the Andes Mountains in the Atacama. The silica matrices were suggested to provide cells with an efficient UV shield, without PAR attenuation [22]. In a different study conducted in the dry core (Yungay) of the Atacama Desert, nonnative microorganisms quickly died upon exposure to solar radiation at the soil surface [4, 9]. However, when protected by gypsum and mineral grain coverings, survival was extended up to 8 days, suggesting that UV-tolerant microorganisms may be able to survive below the soil surface of the Atacama. One well-known UV-tolerant microorganism which members have been found across the Atacama Desert is Chroococcidiopsis. These cyanobacterial species have been found in endolithic and hypolithic habitats [1, 26]. Besides this Cyanobacterium, there are no reports on the description and further enumeration of UV-tolerant microorganisms sampled from this Desert. Here, we report on novel bacterial isolates (mainly from the Bacillus genus) collected from different sites of the Atacama. The UV tolerance of one of these isolates is about tenfold that of a wild-type Escherichia coli strain.. I. G. Paulino-Lima et al.. Except for site 3 in which an epilithic biofilm was sampled, at the remaining sites, subsurface soils were collected by first removing and discarding the topmost 5 cm of the soil surface. These samples were then immediately shipped to the Molecular Radiobiology Lab at Carlos Chagas Filho Institute of Biophysics, Rio de Janeiro Federal University, Brazil. This sampling was performed in July of 2008, which is the wet season in this area of the Atacama Desert. The sample from site 1 was collected at the shores of the Salar de Llamara, a hypersaline environment in the Central Valley, northeast of the city of Iquique, which is known for its microbial mats formed by several species of bacteria algae and archaea found at the bottom of its hypersaline lagoons and beneath the surrounding salt crusts (Fig. 1a) [7]. This sample was composed of a mixture of clay and salt crusts. The sample from site 2 was collected in a quartz-covered field at the Coastal Range of the Atacama, south of the city of Antofagasta and close to the Pacific Ocean (Fig. 1b). This sample was mostly soil and small pieces of rocks. Sample 3 was collected from the region of La Portada, north of Antofagasta, from biofilm-covered rocks directly confronting the Pacific Ocean (Fig. 1c). This sample was composed of brittle chips of petrified sea shells covered by black biofilms. Sample 4 was collected from a field covered with dispersed gypsum patches and basaltic rocks, adjacent to site 2 (Fig. 1d). Sample from site 4 included both gypsum chips and the surrounding soils. Sample 5, a different quartz-covered field was collected close to site 2. This sample was mostly soil, similar to sample taken from site 2. Microbial Enumerations A one-gram sample from each site was placed separately in five conic flasks containing 10 ml saline solution (0.9 % NaCl) and glass beads. After shaking (100 rpm) for 3 h at room temperature (~22 °C), each mixture was submitted to serial dilution and plating on three different culture media: LB (1.0 % tryptone, 0.5 % NaCl, 0.5 % yeast extract, 1.5 % agar), TGY (0.5 % tryptone, 0.3 % yeast extract, 0.1 % glucose, 1.5 % agar), and Marine Agar (MA) 2216 (Difco) for enumeration of cultivable microorganisms. Microbial Screening of UV Tolerance. Material and Methods Sampling Sites Five different sites in the Atacama Desert (Chile) were sampled (Fig. 1) using sterile 50-ml Falcon tubes.. As the focus of this work was the isolation of microorganisms tolerant to UV radiation, the same soil suspensions used for microbial enumeration were exposed separately to UVC radiation using a germicidal lamp emitting a flux of 2.0 J m−2 s−1, as measured with a.
(3) UVC-Tolerant Bacteria from the Hyperarid Atacama Desert. 327. Fig. 1 a Geographical location of the sampled sites (modified from a NASA MODIS image). Site labeling: b Salar de Llamara (21°16′ 07.54″S, 69°37′03.90″W. Altitude: 751 m); c quartz field 1 (23°48′ 59.15″S, 70°29′25.59″W. Altitude: 538 m); d cliff biofilms, Capa. Negra La Portada (23°29′58.61″S, 70°25′42.10″W. Altitude: 27 m); e soil gypsum field (23°49′01.14″S, 70°29′22.98″W. Altitude: 531 m); f quartz field 2 (23°49′10.76″S, 70°28′36.77″W. Altitude: 736 m). Vilber Lourmat radiometer using a photocell CX-254. A volume of 5 ml from each soil suspension was placed in 9 mm diameter glass Petri dishes and exposed to a total fluency of 300 J m−2 UVC (λ 0254 nm). This treatment was performed under continuous shaking to homogenize the cell population being irradiated and to minimize the protection effects caused by the particulate material of the soil suspension. After the UVC. irradiation, samples were subjected to serial dilution and plating on culture media (LB, TGY, and MA) for microbial enumeration of survivors (A in Fig. 2). After up to 7 days of incubation at 28 °C, the number of colony-forming units (CFU) corresponding to the irradiated aliquots (N) was divided by the number of CFU corresponding to the nonirradiated control (N0), resulting in survival fractions (N/N0) for each sample..
(4) 328. I. G. Paulino-Lima et al.. Fig. 2 Scheme of the isolation and irradiation procedures used in this study. A Irradiation of the liquid phase of each sample with 300 Jm−2 of UVC radiation. B Irradiation of sample from site 4 with several doses of UVC radiation. C Inoculation of soil in natura on agar plates. D Visual inspection and selection of pigmented morphotypes. E Irradiation of isolate S3.300-2 with several doses of UVC radiation. In order to check the efficiency of the irradiation treatment, the sample from site 4 was subjected to an additional irradiation experiment with fractionated fluencies (Joule per square meters) of 0, 25, 50, 100, 200, 300, and 400 (B in Fig. 2). This sample was chosen for this procedure because it showed a higher number of pigmented morphotypes after the single exposure irradiation experiment. Survival fractions (N/N0) were determined as described above. A third approach for screening UV-resistant microorganisms was applied by sprinkling about 100 mg samples from each site onto three agar plates (LB, TGY, and MA). After up to 7 days of incubation at 28 °C, pigmented morphotypes were isolated for subsequent analysis (C in Fig. 2).. 28 °C. The isolates able to grow under these conditions were subjected to a new step of UVC irradiation, this time in saline solution without the interference of the particulate material from the soil samples. For this, cultures of each isolate were grown under shaking at 150 rpm, 28 °C for up to 48 h, until reaching OD600 ~0.8. A volume of 5 ml from each culture was washed twice in saline solution through centrifugation at 8,000× g for 5 min at 4 °C, followed by replacement of supernatant by a new saline solution and vortexing for 10 s. The resulting cell suspension was placed in 9 mm diameter glass Petri dishes, exposed to UVC irradiation (300 Jm−2) and recovered for survival fraction analysis, as described previously. Results were plotted on a graph showing different survival fractions.. Determination of UVC Tolerance Survival Curve Since pigmentation is a known cellular protection mechanism against UV radiation [14], different colored morphotypes were chosen among the survivors for subsequent analysis (D in Fig. 2). These isolates were grown in liquid culture medium (LB, TGY, and Marine Broth (MB) 2216, Difco) under shaking at 150 rpm,. The most UV-tolerant isolate (S3.300-2) was grown in MB under shaking at 150 rpm, 28 °C for up to 48 h, until reaching OD600 ~0.8. A volume of 5 ml from each culture was washed twice in saline solution as described above. The resulting cell suspension was placed in 9 mm diameter glass.
(5) UVC-Tolerant Bacteria from the Hyperarid Atacama Desert. Petri dishes, exposed to UVC radiation using fractionated fluences (Joule per square·meters) of 0, 50, 100, 200, 300, 400, 500, and 600 (E in Fig. 2). Aliquots of each treatment were recovered for survival analysis, as described previously. In order to compare its survival profile with other biological models, aliquots of UVCtolerant Deinococcus radiodurans and UV-sensitive E. coli K12 wild-type strain (StrR) [8] were also submitted to the same protocol. Results were plotted on a graph showing survival curves. Molecular Identification of UV-Tolerant Isolates Total genomic DNA was extracted from colonies using the Microbial DNA Isolation Kit (Mo Bio Laboratories, Solano Beach, CA, USA) according to the manufacturer's instructions. 16S rDNA sequences present in the total extracted DNA were amplified using universal oligonucleotide primers 515Fw (5′-GTG CCA GCA GCC GCG GTA A-3′) and 1492Rv (5′-GGT TAC CTT GTT ACG ACT T-3′). For the amplification of template DNA, the GoTaq colorless Master Mix (Promega Corporation, Madison, WI, USA) was used according to the manufacturer's instructions. PCR conditions were as follows: an initial denaturing step at 94 °C for 5 min followed by four consecutive cycles of 94 °C for 1 min, n degrees Celsius for 1 min, and 72 °C for 1 min, in which n048–49–50–51 °C. This was then followed by 31 cycles of 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min, and a final extension step at 72 °C for 15 min, thus totaling 35 cycles of amplification. PCR products were then separately digested with restriction enzymes AluI and MspI. PCR products with unique restriction patterns were stored and ligated to the pGEM-T Easy Vector System (Promega Corporation, Madison, WI, USA), and cloned in E. coli XL1-Blue cells. The resulting plasmid vectors were isolated and purified using the Invisorb Spin Plasmid Mini Two (Invitek GmbH, Berlin, Germany) according to the manufacturer's instructions. The automated sequencing of the clones was conducted by Macrogen DNA Sequencing Inc. (Seoul, South Korea) using the M13 forward primer site of the pGEM-T Easy Vector. Strains with unique 16S rDNA restriction patterns were identified and maintained on LB or Marine Broth agar plates. To identify the closest relatives of the queried 16S rRNA gene sequences, the nucleotide sequence of the isolated 16S rRNA genes were analyzed using the Mega BLAST option for highly similar sequences of the BLASTn algorithm against the National Centre for Biotechnology Information nonredundant database (www.ncbi.nlm.nih.gov). Phylogeny was assigned by comparing the maximum identity values of known species in the list of results with the corresponding new clone.. 329. DGGE Analysis of Soil Communities Total genomic DNA was extracted from 0.5 g of soil/ biofilm from each site using the FastDNA Spin Kit for soil (Qbiogene, Carlsbad, CA) following the manufacturer's instructions. A 16S rRNA gene fragment corresponding to nucleotide positions 968–1401 (E. coli numbering) was amplified using the universal bacterial primers: 968f (5′-AAC GCG AAG AAC CTT AC-3′), which contains a 40-bp GC clamp (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG G-3′) attached to its 5′ end, making it suitable for denaturing gradient gel electrophoresis (DGGE) and 1401r (5′-CGG TGT GTA CAA GAC CC-3′). The PCR mixture consisted of 1 μl DNA (10–30 ng), 25 pmol of each primer, 5 μl 10× PCR buffer (Fermentas), 2 .5 m M Mg C l 2 , 2 . 5 U Ta q D N A p o l y m e r a s e (Fermentas), 0.2 mM of each deoxynucleoside triphosphate (Promega), 1 % formamide, 5 μg bovine serum albumin (BSA) and sterile, filtered MilliQ water to a final volume of 50 μl. Negative controls consisted of sterile MilliQ water instead of sample. PCR amplification was performed in a DNA thermocycler (Mastercycler Personal, Eppendorf, Hamburg, Germany). The temperature profile included an initial denaturation step at 94 °C for 2 min, 35 cycles of a denaturation step at 94 °C for 1 min, a primer annealing step at 55 °C for 1 min, and an extension step at 72 °C for 2 min, followed by a final step of 72 °C for 10 min. Before DGGE analysis, the presence of PCR products was confirmed by electrophoresis in a 1.2 % agarose gel run at 80 V in Tris–borate–EDTA buffer. The gel was stained for 15 min with 0.5 μg.ml−1 ethidium bromide and viewed under short-wavelength UV. A 100-bp DNA ladder (Fermentas) served as the molecular size standard. DGGE of the PCR products generated with the 968fGC/1401r primer set was performed using the DCode universal mutation detection system (Bio-Rad DCode, Richmond, VA, USA) at 75 V and 60 °C for 16 h in 0.5× TAE buffer. PCR products (30 μl) were loaded on 6 % (w/v) polyacrylamide gels containing a linear gradient ranging from 45 to 65 % denaturant (100 % denaturant corresponded to 7 M urea and 40 % (v/v) formamide), increasing in the direction of electrophoresis. A 10-ml stacking gel without denaturant was added on top. After electrophoresis, the gels were stained with SYBR green I nucleic acid gel stain (Molecular Probes, Leiden, The Netherlands) for 40 min and were then scanned using a Storm PhosphorImager (Amersham Biosciences, Uppsala, Sweden). Analysis of the DGGE profiles was performed using the BioNumerics version 5.10 software package (Applied Maths). A.
(6) 330. dendrogram was constructed using Pearson correlation coefficients (r) and cluster analysis performed by the unweighted pair group method with average linkages (UPGMA).. I. G. Paulino-Lima et al.. incubation at 28 °C. The color of some of those colonies varied depending on the culture medium used. The highest number of pigmented colonies was observed for site 4. Screening of Microbial UV Tolerance. Results Microbial Enumerations CFU of all sampled sites were surprisingly high and changed depending on the culture medium used (Fig. 3). For example, no growth was observed in LB plates from site 1, neither in TGY plates from site 3, after up to 7 days of incubation at 28 °C. Marine Agar was the best culture medium for microbial enumeration of samples from sites 1, 3, and 5, whereas LB medium was the best for sites 2 and 4. Samples from sites 2 and 4 showed the highest colony-forming units per gram of soil as evidenced in all types of culture media. The sample from site 3 showed the lowest colonyforming units per gram of soil on average, although the lowest score was observed for site 1 plated on TGY. For all samples, the highest number of pigmented colonies appeared on MA plates after up to 7 days of. Fig. 3 Total number of colonyforming units (CFU) of nonirradiated sites samples grown on Marine Agar (MA), Luria Broth (LB), or TGY plating media. As shown in Fig. 4, the highest survival fraction was observed for the sample taken from site 2. However, sample from site 4 exhibited the highest number of different pigmented morphotypes and the largest abundance of cultivable microorganisms in all tried media. For all samples, the survival fraction varied depending on the culture medium used. A total of 39 different pigmented morphotypes were obtained after visual inspection of all irradiated plates. From these, 33 morphotypes were able to grow in at least one of the tried culture media (LB, MA, or TGY) under shaking at 150 rpm, 28 °C. In addition, another 29 different pigmented morphotypes were obtained through the sprinkling method, from which only seven were able to grow under shaking at 150 rpm, 28 °C, for subsequent irradiation experiments. The selected UVC-tolerant isolate exhibited a wide range of colors, from pale orange to red (Fig. 5)..
(7) UVC-Tolerant Bacteria from the Hyperarid Atacama Desert. 331. Fig. 4 Survival scores (CFU/g soil) of microbial cells from each site diluted in saline solution (0.1 gml−1) after 300 J m2 UVC (λ0254 nm) irradiation and incubation in three types of culture media. On top of each bar, the percent survival relative to the initial unirradiated scores are shown. Since irradiation of soils was performed in aqueous solution, it could be argued that the particulate material of the soil could protect the cells. However, as shown. Fig. 5 Isolation and growth of several pigmented morphotypes from Atacama Desert in MA (top row), LB (middle row), and TGY (bottom row) media. in Fig. 6, the survival fraction decreases as the doses increase, suggesting that such particulate material has minimal effect on cell survival against the irradiation.
(8) 332. I. G. Paulino-Lima et al.. UVC Survival Forty isolates were able to grow in liquid medium under shaking at 150 rpm, 28 °C. They were subjected to an acute irradiation of 300 Jm−2. In order to better track their origin, isolates were numbered according to their site of origin (1 to 5) as S1 to S5, followed by the highest dose (Joule per square·meters) that they survived (100 to 400) and the actual number of the isolate (1, 2, 3, etc.) (Fig. 7). Some isolates were obtained through the sprinkling method (with no previous irradiation treatment) and did not follow this nomenclature. These were isolates AT02-08 from site 2 (quartz field); LPMT11, LPMT13, LPMT14, and LPMT17 from site 3 (La Portada); and S4.2 and S4.4 from site 4 (gypsum field). The most tolerant isolate to UVC was the yellow/ orange colored strain S3.300-2, with a survival fraction of 4 % after acute exposure of 300 Jm−2. A LD10 of 318 Jm−2 was obtained through the survival curve experiment (Fig. 8). Fig. 6 Decrease of microbial total scores following irradiation with different UVC doses of soil sample from site 4. procedure. It is noteworthy that all irradiation treatments were performed under continuous shaking to enhance the exposure of the cell population to the radiation source.. Identification of UV-Tolerant Isolates The 14 most tolerant isolates were identified by partial amplification of their 16S rRNA genes followed by sequencing and comparison with the NCBI database (Table 1). Most isolates showed high similarity to Bacillus cereus and Bacillus thuringiensis. One isolate was identified as closely related to Pseudomonas stutzeri and. Fig. 7 UVC tolerance (300 Jm−2) profiles of isolates obtained from five sites of the Atacama Desert. Columns were colored according to the pigmentation of each microbial isolate.
(9) UVC-Tolerant Bacteria from the Hyperarid Atacama Desert. 333. Discussion. Fig. 8 Survival curve of the top UVC-tolerant isolate S3.300-2 (closed triangles) in comparison to survival curves of E. coli (open circles) wild-type strain K12, and D. radiodurans wild-type strain R1 (closed circles). the most tolerant isolate (S3.300-2) was identified as a Bacillus sp. DGGE Analysis of the Soil Communities DGGE analysis revealed the presence of complex microbial communities in all sites, with a number of bands ranging from 16 (site 5) to 32 (site 1) (Fig. 9). Samples from sites 2, 3, and 4 were represented by 23, 25, and 20 bands, respectively. Calculation of differences in the bacterial community composition based on these band patterns using the Pearson's correlation index showed that the five analyzed soils are distinct. According to this profiling, site 1 and site 3 are closely related, and site 2 is closely related to site 5. The band profile shown by site 4 clustered separate from all others. Sites 3, 2, and 4 (25, 23, and 20 DGGE bands, respectively) appear to be grouped in terms of environmental origin and community structure, displaying more diverse 16S sequences. These associations make sense, as sites 2 and 5 are represented by soil samples taken at closeby quartz fields. Interestingly, sites 1 and 3, represented by a soil sample taken at the shore of a hyperarid lagoon in the first case and epilithic biofilms in the second, appear to be related according to the Pearson's index. This similarity may be explained as in both cases, salty (NaCl) water is the main source of this vital element, and the similar source of ionic–osmotic stress could explain comparable community structures. Site 4, being represented by a sample taken at gypsumrich soil not surprisingly clusters separate as the chemical composition of this type of soils is quite different from the rest of the sampled sites.. The search for life on Mars will be greatly aided by the understanding of the microbial life present in harsh terrestrial analog environments [3, 15, 23] such as the Atacama Desert in northern Chile [5, 6, 11, 19]. Desertification in this region is mainly driven by anticyclonic subtropical atmospheric phenomena. This is reinforced by the presence of the cold Humboldt Current along the west coast of South America, which prevents precipitation in coastal regions. An additional restrictive factor is the presence of the Andes Mountains, which causes precipitation to fall on the east side and keeps humidity near zero on the west side [25]. These factors help to explain the absence of clouds, thus enhancing solar radiation over most of the Atacama (Fig. 1). In this study, subsurface soil samples (sites 1, 2, 4, and 5) and an epilithic biofilm (site 3) were collected from different regions of the Atacama Desert in order to test the effects of UV on their microbial inhabitants (Fig. 1). Despite the inhospitable conditions prevailing in these locations, our data revealed a surprising microbial abundance in all samples, since total CFU showed a relatively large number of cultivable microorganisms after up to 1 week of incubation at 28 °C (Fig. 3). Since cultivation techniques cannot assess total microbial diversity, molecular techniques were also used in order to get a better profile of the analyzed samples. Thus, DGGE profiles greatly helped in the initial screening of the studied microbial communities. The profiles obtained followed the expected abundance Table 1 Molecular assignments of the UVC most tolerant isolates. All E values scored 0.0 for the shown 16S DNA sequences Code. Species. Origin. S3-300-2. Bacillus Sp.. Capa Negra La Portada. S4.300-3 S4.300-8 LP-MT13 S4.400-2 S4.200-5 S4.200-3 S4.200-9 S4.200-11 S4.100-3 S4.300-2 S4.2 S4.400-1 AT02-08. Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Bacillus cereus Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Pseudomonas stutzeri Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis Bacillus cereus/thuringiensis. Sitio 2 Gypsum Sitio 2 Gypsum La Portada cave Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio 2 Gypsum Sitio cuarzos.
(10) 334. I. G. Paulino-Lima et al.. Fig. 9 Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA sequences obtained from the studied microbial communities. patterns according to water availability for microbial colonization [16]. For example, site 1 (Salar de Llamara), besides holding the greatest 16S abundance, displays twice as high the number of banding profiled for site 5 (Fig. 9). Previous studies profiled several areas of the Atacama Desert, including the driest ones [10, 19]. Thus, considering that these different sites were able to harbor different microbial communities, the Atacama Desert represents an interesting environment in the search for new microbial species adapted to extreme environmental conditions. The irradiation procedure allowed the recovery of many pigmented colonies (Fig. 5). It is known that the presence of pigments in bacteria (such as carotenoids) provides tolerance to radiation by passively absorbing most of the stressing UV [14]. Noteworthy, only pigmented microorganisms were isolated and subsequently cultured from all sites as illustrated in Fig. 5. As a large number of pigmented microorganisms were recovered from site 4 after exposure to UVC, samples coming from this location underwent a secondcycle of fractionated irradiation, this time with doses up to 400 Jm−2 (Fig. 4). It is interesting to highlight that, according to the DGGE profiles and cluster analysis, site 4 was distinct from the other ones, including the fact that most survivors to the higher UV doses were better grown in MA, which has a large variety of salts and trace elements that may be required by UV-resistant microorganisms. This procedure resulted in increased total number of pigmented microorganisms, which were grown in different culture media. Although 40 pigmented isolates were fully cultivated and grown in liquid culture medium, a larger number of pigmented microorganisms could be obtained if additional media were used, as well as cultivation strategies, pH, temperature, oxygenation, moisture, etc. As shown in Table 1, most of the identified UV-tolerant isolates are closely related to B. cereus and B. thuringiensis, with the most UV-tolerant isolate (S3.300-2) ascribed to a Bacillus species. One of the isolates was identified as P. stutzeri. Interestingly, Pseudomonas species have already been found associated to rock. varnish in the hyperarid areas of the Atacama Desert [19]. The predominance of sporulating and pigmented microorganisms is not surprising given that all environments from where samples were collected for the study would favor the occurrence of spore formation as a protective mechanism to cope with such hyperarid conditions [18]. An extensive revision of UV resistance mechanisms in Bacillus can be found in Nicholson et al. (2005) [21]. The UV dose shown to leave 10 % survivors (LD10) in the vegetative stage cell population of isolate S3.3002 was 318 Jm−2, which is nearly tenfold higher compared to that of E. coli (30 Jm−2) and somewhat lower in the 0–300 Jm−2 range compared to that of D. radiodurans (Fig. 8). As this test was done in vegetative cells, the tolerance of the potential spores of this particular isolate to UV light is expected to be much higher than that reported by its vegetative form [20]. Altogether, our results revealed that the Atacama Desert harbor highly UV-tolerant species, confirming their pertinence as a Mars analog in this sense. We emphasize the importance of diverse cultivation strategies in order to expand our knowledge on the UV tolerance of microorganisms with diverse metabolisms, especially when extreme environments are studied. Future work will be focused only in the hyperarid sites of the Central Valley of this desert, where microorganisms even more tolerant to UV radiation are expected to be found.. References 1. Azúa-Bustos A, González-Silva C, Mancilla RA, Salas L, GómezSilva B, McKay CP, Vicuña R (2011) Hypolithic cyanobacteria supported mainly by fog in the coastal range of the Atacama Desert. Microb Ecol 61:568–581 2. Catling DC, Claire MW, Zahnle KJ, Quinn RC, Clark BC, Hecht MH, Kounaves S (2010) Atmospheric origins of perchlorate on Mars and in the Atacama. J Geophys Res 115:E00E11 3. Cockell CS (2001) The Martian and extraterrestrial UV radiation environment. Part II: further considerations on materials and design criteria for artificial ecosystems. Acta Astronaut 49:631–640 4. Cockell CS, McKay CP, Warren-Rhodes K, Horneck G (2008) Ultraviolet radiation-induced limitation to epilithic microbial.
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