ASTROBIOLOGY Volume 9, Number 1, 2009 © Mary Ann Liebert, Inc. DOI: 10.1089/ast.2007.0234 Special Paper Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria Sergiu Fendrihan,1 Attila Bérces,2 Helmut Lammer,3 Maurizio Musso,4 György Rontó,2 Tatjana K. Polacsek,1 Anita Holzinger,1 Christoph Kolb,3,5 and Helga Stan-Lotter1 Abstract The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibil- ity of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloar- chaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was ex- posed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated Ն 2 D37 (dose of 37 % survival) for Hcc. dombrowskii was 400 kJ/m . However, exposure of cells to UV flux while 2 in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m ); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of po- tential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation. Key Words: Halococcus dombrowskii—Simulated martian UV radiation—LIVE/DEAD staining— Halite fluid inclusions—UV transmittance and reflectance—Desiccation. Astrobiology 9, 104–112. Introduction 2001). These findings suggest the possibility of microbial long-term survival under conditions of desiccation, though EVERAL EXTREMELY HALOPHILIC ARCHAEA (haloarchaea) it is enigmatic as to which mechanisms might be used, since Shave been isolated from alpine rock salt deposits of Per- haloarchaea are not known to produce resting stages such mian and Triassic age, and some of them were described as as spores (Grant et al., 1998; McGenity et al., 2000). The ap- novel species (Denner et al., 1994; Stan-Lotter et al., 1999, parent longevity of the haloarchaeal isolates in dry salty en- 2002; Gruber et al., 2004; Fendrihan et al., 2006). In addition, vironments is of interest for the search for extraterrestrial life. amplification of 16S rRNA genes, via the polymerase chain Halite has been detected in meteorites, some of which stem reaction, indicated evidence for the presence of a large from Mars (Gooding, 1992; Zolensky et al., 1999; Treiman et haloarchaeal community in the salt deposits (Radax et al., al., 2000). Data from the martian rovers Spirit and Opportu- 1University of Salzburg, Division of Molecular Biology, Department of Microbiology, Salzburg, Austria. 2Research Group for Biophysics, Hungarian Academy of Sciences, Budapest, Hungary. 3Space Research Institute of the Austrian Academy of Sciences, Graz, Austria. 4University of Salzburg, Division of Materials Engineering and Physics, Department of Physics and Biophysics, Salzburg, Austria. 5On leave from the Space Research Institute of the Austrian Academy of Sciences, Graz, Austria. 104 SIMULATED MARTIAN UV RADIATION AND HALOARCHAEA 105 nity (Rieder et al., 2004; Squyres et al., 2006) suggest that some experiments with cells of Halococcus dombrowskii and two deposits on Mars formed from concentrated salt water. If noncoccoid haloarchaea (Halobacterium salinarum, Haloarcula Mars and Earth had a similar geological past (Nisbet and japonica). A comparison of the spectral intensity output of Sleep, 2001, Schidlowski, 2002), then there is the possibility the lamp between 200 and 400 nm with the theoretical flux that life occurred on Mars as well and that microbial life, or on Mars was published by Kolb et al. (2005). For the quanti- remnants of it, could still be present on Mars. tative determination of surviving haloarchaea, viable cells Since Mars lacks an ozone layer, strong UV radiation is were counted after staining with the so-called LIVE/DEAD inferred on the martian surface (Rontó et al., 2003; Patel et BacLight kit; the procedure has been described recently al., 2004). It is believed that, before the accumulation of oxy- (Leuko et al., 2004, Stan-Lotter et al., 2006). For some exper- gen and ozone, the early Earth environment experienced an iments, fluorescence microscopy was combined with growth influx of short-wavelength UV of high intensity (Bérces et al., studies and exposure in liquid culture and on semi-solid 2006; Westall et al., 2006). Early life on Earth, therefore, was medium; the effects of these experimental setups had not likely resistant to UV, and it follows that potential micro- been compared before. The results of the study described biota on Mars may have possessed, or may possess, resis- here indicate the potential for UV-resistant microscopic life tance to this type of radiation as well, which can produce ex- in small crystals, which could be present in martian dust. tensive DNA damage (Cockell, 1998; Cadet et al., 2005). Previous experiments of microbial exposure to the space en- Materials and Methods vironment and its radiation were carried out mainly with Bacillus spores (Horneck, 1993; Horneck et al., 2001). More Growth of halophilic microorganisms recently, lichens and cyanobacteria were exposed to space Strains Halococcus dombrowskii H4 DSM 14522T and Haloar- conditions by Sancho et al. (2007) and Cockell et al. (2007). cula japonica DSM 6131T were grown in M2 culture medium To date, one exposure of an extremely halophilic archaeon, (Tomlinson and Hochstein, 1976) that contained (g ϫ LϪ1): Haloarcula sp., to space conditions was performed in the casamino acids (Hycase, Sigma), 5.0; yeast extract (Difco), Biopan experiments by Mancinelli et al. (1998), who demon- 5.0; Tris (ICN Biochemicals), 12.1; NaCl, 200.0; MgCl ϫ strated considerable survival of cells after 14 days in space. 2 6H O, 20.0; KCl, 2.0; CaCl ϫ 2H O, 0.2; in side arm flasks Haloarchaea, including Halobacterium salinarum strain 2 2 2 at 37°C with shaking at 150 rpm in an incubator (Innova NRC-1, whose total genome sequence is known (Ng et al., 4230). The optical density (OD) of cultures was measured at 2000), have been investigated for the activity of DNA repair 600 nm with a Novaspec II photometer (Pharmacia). When systems after exposure to UV radiation (McCready, 1996; the cultures reached an OD of 0.8 to 1.0, which corresponded Baliga et al., 2004; Zhou et al., 2007); therefore, some data on to cell densities of 2.5–4 ϫ 108 per ml, cells were harvested their resistance to UV are available. However, these studies by centrifugation at 5000 ϫ g for 5 minutes, and the pellets are generally performed with cells in a liquid salt-contain- were resuspended in Tris-NaCl buffer, which consisted of 4 ing medium during exposure to UV radiation. The presence M NaCl (Sigma) and 100 mM Tris. The pH was adjusted to of liquid saline pools on Mars is not likely, but solid halite 7.4 with dilute HCl. Halobacterium salinarum NRC-1 ATCC- has been discovered in martian meteorites (Gooding, 1992; 700922 was purchased from LGC Teddington, UK, grown as Treiman et al., 2000). Therefore, the haloarchaeal survival ca- described previously (Gruber et al., 2004) and prepared as pacity, while enclosed in salt crystals, is of interest for as- described for the haloarchaea mentioned before. trobiological studies. The experiments performed here with Halococcus dombrowskii are part of the ground investigations Exposure of cells to UV irradiation of the ADAPT project (Principal Investigator P. Rettberg; for more information see http://www.nasa.gov/mission_pages/ Forty microliters and 28 microliters, respectively, of the station/science/experiments/Expose.html#overview), cell suspensions were placed on quartz discs of 11 or 7 mm which was selected for exposure on the International Space diameter (Aachener Quarzglas Technologie, Aachen, Ger- Station (ISS). The exposure facility was developed by ESA many). Discs either contained approximately 7 ϫ 105 or 4.8 ϫ and is described elsewhere, along with details of trays and 105 haloarchaeal cells. The discs were inserted in the wells the localization on the ISS (Baglioni et al., 2007). of 24-well plates (Becton Dickinson, USA) and dried While haloarchaeal strains obtained from rock salt that is overnight in a sterile working bench (Holten LaminAir 2010, believed to be millions of years of age should be eminently Denmark) under laminar flow at ambient temperature. This suitable model organisms for exposure studies, there are also led to the formation of flat halite crystals with embedded some drawbacks. In particular, the isolates grow rather haloarchaeal cells. For each exposure time, three quartz discs slowly, and some of them possess generation times on the of each sample were used (placed in plastic petri dishes of order of weeks or even months (Gruber et al., 2004; Fendri- 55 mm diameter) and exposed to the Mars-UV simulator han, unpublished results), which complicates the determi- lamp at the Austrian Academy of Sciences (Kolb et al., 2005) nation of various growth parameters following potentially for times ranging from 1 to 3600 s. The spot of the lamp had lethal treatment of cells, especially when only a few samples a 50 mm diameter at a distance of 300 mm.