The recent discovery of brine flows on the surface of Mars has intensified interest in the extremophilic character of extremely halophilic microorganisms in relation to astrobiology (Mancinelli et al. Reference Mancinelli, Fahlen, Landheim and Klovstad2004; DasSarma Reference DasSarma2006; Horneck et al. Reference Horneck, Klaus and Mancinelli2010; Leuko et al. Reference Leuko, Rettberg, Pontifex and Burns2014). Flowing brines were first noted as seasonal dark streaks or recurring slope lineae (RSL) on the walls of Garni crater observed from images captured by NASA's Mars Reconnaissance Orbiter (McEwen et al. Reference McEwen, Ojha, Dundas, Mattson, Byrne, Wray, Cull, Murchie, Thomas and Gulick2011). The occurrence of RSLs at subzero temperatures indicated that frozen brines melt and flow seasonally on the Martian surface (Ojha et al. Reference Ojha, Wilhelm, Murchie, McEwen and Wray2015). This prediction was also supported by spectroscopic evidence for hydrated sodium and magnesium chloride, chlorate and perchlorate salts at the Phoenix lander site (Kounaves et al. Reference Kounaves, Chaniotakis, Chevrier, Carrier, Folds, Hansen, McElhoney, O'Neil and Weber2014; Toner et al. Reference Toner, Catling and Light2014). Interest in Mars Special Regions, such as the Phoenix site, is heightened by the existence of usable metabolic energy and its potential for enabling habitability (Stoker et al. Reference Stoker, Zent, Catling, Douglas, Marshall, Archer, Clark, Kounaves, Lemmon and Quinn2010; Schuerger et al. Reference Schuerger, Ulrich, Berry and Nicholson2013; Rummel et al. Reference Rummel, Beaty, Jones, Bakermans, Barlow, Boston, Chevrier, Clark, de Vera and Gough2014).
On Earth, hypersaline brines are nearly ubiquitous and generally thalassic, with high biological productivity promoted by sunlight driving pigment production and a variety of primary metabolic processes. A diversity of halophilic microorganisms flourish, originating from all three branches of life: Archaea, Bacteria and Eukarya (DasSarma & DasSarma Reference DasSarma and DasSarma2012). Halophilic Archaea (Haloarchaea) are able to tolerate high salinity primarily through the salt-in strategy, producing negatively charged proteins that remain soluble and compete successfully with ions for hydration (Karan et al. Reference Karan, Capes and DasSarma2012; DasSarma & DasSarma Reference DasSarma and DasSarma2015, see also Goh et al. Reference Goh, Jeon, Barrow, Neilan and Burns2011). Bacteria usually survive by the salt-out strategy, synthesizing or taking-up zwitterionic compatible solutes, which helps maintain osmotic balance with external salts, while Eukarya generally produce neutral polyols, e.g. glycerol, for the same purpose (DasSarma & DasSarma Reference DasSarma and DasSarma2012). Combinations of these mechanisms operate in some species (DasSarma & DasSarma Reference DasSarma and DasSarma2015).
Among the Haloarchaea, Halobacterium sp. NRC-1 has been extensively studied for its extremophilic character (DasSarma et al. Reference DasSarma, Berquist, Coker, DasSarma and Müller2006; DasSarma & DasSarma Reference DasSarma and DasSarma2012). This species is capable of tolerating high concentrations of sodium and potassium chlorides and perchlorates (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007; DasSarma et al. unpublished). In addition to high salinity, this strain is slightly thermotolerant with optimum growth at 42°C and survival at 49–50°C (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007) and is highly resistant to ultraviolet (UV) and ionizing radiation (McCready et al. Reference McCready, Müller, Boubriak, Berquist, Ng and DasSarma2005; DeVeaux et al. Reference DeVeaux, Müller, Smith, Petrisko, Wells and DasSarma2007). Genetic, genomic and transcriptomic studies have established a wide range of survival mechanisms operating in Halobacterium, including presence of highly acidic proteins and direct photorepair, double-stranded gap repair and nucleotide excision repair systems (Crowley et al. Reference Crowley, Boubriak, Berquist, Clark, Richard, Sullivan, DasSarma and McCready2006; Boubriak et al. Reference Boubriak, Ng, DasSarma, DasSarma, Crowley and McCready2008; Karan et al. Reference Karan, DasSarma, Balcer-Kubiczek, Weng, Liao, Goodlett, Ng and DasSarma2014).
Another haloarchaeal strain relevant to astrobiology, Halorubrum lacusprofundi, was isolated from Deep Lake in the Vestfold Hills of Antarctica (Franzmann et al. Reference Franzmann, Stackebrandt, Sanderson, Volkman, Cameron, Stevenson, McMeekin and Burton1988). Deep Lake is perennially cold, with the temperature remaining subzero for more than 6 months of the year. However, Deep Lake does not freeze, even when temperatures drop to −18°C due to freezing-point depression from high (28%) salinity. H. lacusprofundi is capable of growth down to −2°C and is well-adapted to this environment (Reid et al. Reference Reid, Sparks, Lubow, McGrath, Livio, Valenti, Sowers, Shukla, MacAuley and Miller2006). H. lacusprofundi biofilms have been reported at the lowest growth temperatures and may serve to enhance survival. The H. lacusprofundi genome has been completely sequenced and its proteins exhibit reduced surface acidity and internal amino acid substitutions characteristic of enhanced internal flexibility (Anderson et al. Reference Anderson, DasSarma, Lucas, Copeland, Lapidus, Del Rio, Tice, Dalin, Bruce and Goodwin2016; DasSarma et al. Reference DasSarma, Capes, Karan and DasSarma2013; Karan et al. Reference Karan, Capes and DasSarma2013).
In order to evaluate the astrobiological potential of Haloarchaea, we sought to compare the ability of Halobacterium sp. NRC-1 and H. lacusprofundi to survive trips into the stratosphere. While Earth's stratosphere exhibits multiple extremes not dissimilar to those found on the surface of Mars (Smith et al. Reference Smith, Griffin, McPeters, Ward and Schuerger2011; Smith Reference Smith2013), including cold temperatures, high radiation fluxes and low pressures, our primary interest in this study was to assess the importance of tolerance to extremely cold temperatures in survival. Our results show that while both halophiles are able to survive trips into the stratosphere, H. lacusprofundi exhibits greater viability and a more robust transcriptomic response in the extremely cold temperatures experienced.
Materials and methods
Helium balloon launching and recovery
Natural rubber 1.6 kg balloons filled with 5.66 m3 of helium were used to provide the free lift required to pull a 2.2 kg capsule to the stratosphere at a rate of approximately 300 m min−1. Helium balloons were used for high-altitude launches from the Sierra Nevada Mountains of central California between February and July 2015. Items mounted on the outside of the payload included a cryogenic temperature probe, GPS ‘SPOT’ trackers and a GPS altimeter. Tubes containing Haloarchaea were affixed to the exterior of the payload in order to fully expose them to environmental conditions from ground level to altitudes of 33–36 km above sea level. When balloons reached peak altitude, they exploded and the payload plummeted towards Earth. A parachute slowed the descent, resulting in a soft landing. Each payload was recovered within about an hour of landing, by following the SPOT tracker GPS coordinates to the landing site.
Haloarchaeal culturing and laboratory analysis
Halobacterium sp. NRC-1 was cultured in CM+ medium at 42°C while H. lacusprofundi ATCC 49239 was cultured in ATCC1682 Deep Lake medium at 30°C, as previously described (Berquist et al. Reference Berquist, Müller, DasSarma, Oren and Rainey2006; Reid et al. Reference Reid, Sparks, Lubow, McGrath, Livio, Valenti, Sowers, Shukla, MacAuley and Miller2006; Anderson et al. Reference Anderson, DasSarma, Lucas, Copeland, Lapidus, Del Rio, Tice, Dalin, Bruce and Goodwin2016). Cultures were grown to late-log phase and aliquoted into cryovials for shipments by courier from Maryland to California and launching into the stratosphere (Fig. 1). To determine colony forming units, cultures were diluted and plated on agar plates and colonies counted after 10 days incubation at the optimal growth temperatures. In each case, experiments were done in triplicate and standard deviations calculated using Microsoft Excel. Cells were photographed in a Nikon Labophot phase contrast microscope using a Nikon D90 DSLR. Pigment analysis was conducted after pelleting cells by centrifugation (10 min, 10 000 × g ), hypotonic lysis in deionized water and spectroscopy using a Shimadzu UV-1601 spectrophotometer. For viability assays after freeze-thaw, cultures in cryotubes were placed in a −70°C freezer for 2 h, followed by thawing at room temperature and determining colony forming units, as described above.
Genome-wide transcriptomic analyses
Custom whole genome DNA oligonucleotide microarrays for Halobacterium sp. NRC-1 and H. lacusprofundi were manufactured by Agilent Corporation (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007). To compare transcriptional profiles, triplicate cultures of Halobacterium sp. NRC-1 grown at 42 or 15°C and H. lacusprofundi grown at 30 or 4°C, both to an OD600 of 0.3. The temperatures reflected either optimal or minimal growth temperatures for transcriptomic analysis. After collecting cells by centrifugation, RNA was isolated and pooled, Cy3 and Cy5-labelled cDNA was synthesized and labelled cDNA was hybridized to duplicate microarrays, washed and scanned on an Agilent DNA microarray scanner (model no. G2565BA), as previously described (Müller & DasSarma Reference Müller and DasSarma2005; Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007). Agilent Feature Extraction software was used for image analysis and processing of the microarray image file. Statistical analyses were performed as previously described using a 1.5-fold change threshold cutoff. Expression data were aligned with protein families (conserved Haloarchaeal orthologous genes (cHOGs)) using MS Excel (DasSarma et al. Reference DasSarma, Capes, DasSarma and DasSarma2010; Capes et al. Reference Capes, DasSarma and DasSarma2012).
Flights into the stratosphere
Halobacterium sp. NRC-1 and H. lacusprofundi grown in liquid cultures in the laboratory in Maryland were aliquoted and shipped to California by overnight courier (Fig. 1). Experimental samples were launched to the stratosphere affixed to the payloads of Earth to Sky Calculus high-altitude helium balloons while control aliquots were either stored on the ground in Maryland or shipped to and returned from California without balloon flights. Experimental vials were launched to heights of 36 km, with flight times of 2.5 h from launch to landing, including 1 h in the stratosphere. The minimum temperature recorded was −65°C, with UV-C doses of 18 J m−2 and air pressure as low as 230 Pa (Jacob Reference Jacob1999). Samples were recovered typically within an hour and shipped by overnight courier to Maryland for analysis.
Experimental and control samples of Halobacterium sp. NRC-1 and H. lacusprofundi were analysed visually, spectrophotometrically and microscopically. Balloon-flown samples were found to be partially cleared and bleached upon visual inspection compared with the courier flown and ground-bound control samples. Examination of cells by phase contrast microscopy indicated that Halobacterium sp. NRC-1 cells had lost their refractive gas vesicles (data not shown), which normally provide buoyancy and promote cell flotation for phototrophic growth in their hypersaline environment (DasSarma et al. Reference DasSarma, Zamora, Müller and DasSarma2012). Morphological differences were less pronounced for H. lacusprofundi cells, which naturally lack gas vesicles and exhibit a higher level of pigmentation. The partial loss of pigmentation for both organisms after flights to the stratosphere was confirmed by UV-VIS spectroscopy, with reduced absorption detected at wavelengths characteristic of bacterioruberins (Fig. 2).
Survival difference after stratospheric flights corresponds to freeze-thaw tolerance
In order to determine the survival of Halobacterium sp. NRC-1 and H. lacusprofundi, dilutions of cultures remaining in Maryland and cultures that were shipped to and from California with or without experiencing the stratosphere from launches on balloons, were plated in triplicate. Cultures with or without shipping showed no statistical difference in survival, indicating that there was no significant effect on viability of transit by courier (Fig. 3). Interestingly, cultures of both Halobacterium sp. NRC-1 and H. lacusprofundi flown to the stratosphere appeared to show a significant degree of survival, although the Antarctic species showed relatively better survival than the mesophile (Fig. 3). Quantitative analysis indicated 50% survival for H. lacusprofundi and 5% for Halobacterium sp. NRC-1.
In order to determine whether freeze-thaw tolerance was responsible for the observed difference in survival, we froze both Halobacterium sp. NRC-1 and H. lacusprofundi cultures in the laboratory. After thawing, survival of each Haloarchaeaon was quantified by serial dilution and plating. H. lacusprofundi was found to be considerably more tolerant to freeze-thaw than Halobacterium sp. NRC-1 (88 versus 16% survival, respectively) (Fig. 4). These findings showed that the better freeze-thaw survival of the cold-adapted Antarctic isolate, H. lacusprofundi, compared with the mesophilic Halobacterium sp. NRC-1, parallels the observed higher survival in the stratosphere (Fig. 3). Interestingly, freeze-thaw also led to partial loss of pigmentation in both Haloarchaea and loss of intracellular gas vesicles in Halobacterium sp. NRC-1 (data not shown).
Genome-wide transcriptomic analysis
In order to address the difference in cold-survival of Halobacterium sp. NRC-1 and H. lacusprofundi at the transcriptomic level, we compared genome-wide transcriptomes of the two microorganisms at low, versus optimum growth temperatures using DNA microarrays. For Halobacterium sp. NRC-1, DNA microarrays comparing the optimal temperature of 42°C to the low temperature of 15°C had been conducted earlier (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007). We compared these data to transcriptomic data for H. lacusprofundi near its optimal growth temperature of 30°C and low growth temperature of 4°C. The numbers of genes with significantly changed transcription levels are shown in Table 1.
The findings showed that gene expression in H. lacusprofundi is much more responsive to temperature differences than in Halobacterium sp. NRC-1 (Table 1). Nearly 60% of H. lacusprofundi genes were up- or down-regulated by ≥1.5-fold in this cold-adapted haloarchaeon with nearly equal numbers of genes changing in each direction, 31% up-regulated and 29% down-regulated. By contrast, <20% of genes in the mesophile Halobacterium sp. NRC-1 were regulated to the same extent, with twice as many down-regulated as up-regulated genes. Down-regulated genes are likely primarily growth rate-dependent since colder temperatures slow growth (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007).
Regulated genes in the two halophiles were determined for conserved genes using the cHOGs database (DasSarma et al. Reference DasSarma, Capes, DasSarma and DasSarma2010; Capes et al. Reference Capes, Coker, Gessler, Grinblat-Huse, DasSarma, Jacob, Kim, DasSarma and DasSarma2011, Reference Capes, DasSarma and DasSarma2012). The results for the subset of cHOGs were very similar to the genome as a whole, with 55% of H. lacusprofundi compared with 20% of Halobacterium sp. NRC-1 orthologs significantly changed (Table 1). Most of the up-regulated cHOGs in Halobacterium sp. NRC-1 were also similarly perturbed in H. lacusprofundi (29 out of 43). Interestingly, some of the most highly up-regulated genes were conserved transcriptional regulators, general transcription factors and chaperones (Table 2). For transcription factors and regulators, there were a larger number of genes up-regulated in H. lacusprofundi compared with Halobacterium sp. NRC-1, while the number was nearly the same for chaperones. Orthologs of nearly all of the perturbed Halobacterium sp. NRC-1 genes were also found to be changed in H. lacusprofundi. These findings are consistent with a common core response to cold-temperature, as reflected by changes observed in both Haloarchaea, with an additional strong and diverse response to cold and freezing conditions specifically in the Antarctic H. lacusprofundi.
Halophilic Archaea have been proposed to be excellent candidates for astrobiology (DasSarma Reference DasSarma2006). We tested effects of stratospheric conditions on survival of live, metabolically active Haloarchaea by launching two species on helium balloons. Our results showed that both species, Halobacterium sp. NRC-1 and H. lacusprofundi, can survive stratospheric flights, with the latter cold-adapted Antarctic species displaying superior survival ability compared with the former mesophilic species. H. lacusprofundi therefore appears better suited for stratospheric conditions.
Enhanced survival of H. lacusprofundi over Halobacterium sp. NRC-1 in the stratosphere is primarily due to its better adaptation to cold temperatures (Fig. 4). The stratosphere is very cold, between −10 and −65°C (Jacob Reference Jacob1999), with the lower temperature resulting in freezing of the hypersaline culture media. Upon return to the troposphere or after landing, Haloarchaeal cultures thawed. Since H. lacusprofundi experiences frigid subzero temperatures for 8 months of the year in its natural environment of Deep Lake in the Vestfold Hills of Antarctica, it is better capable of tolerating the cold freeze occurring in the stratosphere (Franzmann et al. Reference Franzmann, Stackebrandt, Sanderson, Volkman, Cameron, Stevenson, McMeekin and Burton1988; Reid et al. Reference Reid, Sparks, Lubow, McGrath, Livio, Valenti, Sowers, Shukla, MacAuley and Miller2006). In contrast, the mesophilic species isolated from a temperate climate does not appear to be able to withstand cold temperatures well and loses considerably more viability from the journey into the stratosphere (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007).
The cold-adaptive mechanisms operating in H. lacusprofundi and Halobacterium sp. NRC-1 were found to have similarities and differences using genome-wide transcriptomic analysis (Tables 1 and 2) (Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007). H. lacusprofundi exhibited significantly higher percentage and numbers of cold-induced genes, including general transcription factors and transcriptional regulators. In Halobacterium sp. NRC-1, our prior work had shown that three transcription factors (TbpD, TfbA and TfbG) were involved in, or were responsive to cold temperatures, with only a single one, TfbG, displaying expression level perturbation by cold temperature (Coker & DasSarma Reference Coker and DasSarma2007; Coker et al. Reference Coker, DasSarma, Kumar, Müller and DasSarma2007). H. lacusprofundi showed five transcription factors with gene expression responsive to cold temperatures, two Tbps and three Tfbs. Similarly, H. lacusprofundi also exhibited a larger number of transcriptional regulators responding to cold temperatures, twelve versus only three in Halobacterium sp. NRC-1. For stress protein and chaperone genes, five orthologous genes were up-regulated in both, with only three non-orthologs varying between the two (two in H. lacusprofundi and one in Halobacterium sp. NRC-1). Together, these findings showed that while the core cold-stress response is quite similar in both Haloarchaea, the Antarctic halophile has a much larger and diverse set of cold-responsive genes compared with the mesophile. These findings likely account for H. lacusprofundi’s better survival under cold and freezing temperatures encountered in the stratosphere.
This is the first study of survival of live Haloarchaeal cultures under stratospheric conditions. Prior studies have examined the survival ability of a related species, Halorubrum chaoviatoris, from a Mexican intertidal marine area during a flight aboard ESA's Biopan 1 facility (Mancinelli et al. Reference Mancinelli, White and Rothschild1998). This experiment exposed desiccated cells to deep space conditions with considerably higher dose of radiation, leading to lowering of viability by 106-fold. Clearly, exposure of desiccated cells to space conditions is significantly more challenging for the microbes. The current study is more likely to reflect potential survival of Haloarchaea under low temperatures, in surface or subsurface brines, than unprotected exposure to the surface of Mars or deep space. Additional studies are needed to determine the combinatorial effects of several stressors, including cold temperature, UV-C and cosmic radiation and low pressures.
A variety of other microorganisms have been tested for survival under space and simulated Mars conditions and in the stratosphere. In one study of nearly two-dozen bacterial species exposed to cold, low pressure and anoxic conditions in the laboratory, only a single microbe, Serratia liquefaciens was found to survive all of the extremes (Schuerger et al. Reference Schuerger, Ulrich, Berry and Nicholson2013). In this study, two extremophiles, Deinococcus radiodurans and Psychrobacter cryohalolentis, were not capable of survival under these conditions. Other studies have focused on spore forming bacteria, such as Bacillus subtilis (Nicholson et al. Reference Nicholson, Moeller and Horneck2012; Wassmann et al. Reference Wassmann, Moeller, Rabbow, Panitz, Horneck, Reitz, Douki, Cadet, Stan-Lotter and Cockell2012) and B. pumilus SAFR-032. The quiescent spore forms of B. pumilus are highly resistant to UV radiation and cold temperatures (Calcott & MacLeod Reference Calcott and MacLeod1975; Vaishampayan et al. Reference Vaishampayan, Rabbow, Horneck and Venkateswaran2012).
Recently, several commercial bacterial cultures (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli and Salmonella typhimurium) and human-associated bacteria (Proteus mirabilis and Pseudomonas aeruginosa) were launched into the stratosphere on helium balloons (Chudobova et al. Reference Chudobova, Cihalova, Jelinkova, Zitka, Nejdl, Guran, Klimanek, Adam and Kizek2015). The survival of these bacteria was found to be significantly poorer than for the Haloarchaea in our study, especially H. lacusprofundi. Moreover, this study reported survival to be lower in the stratosphere than for a similar dose exposure to UV-C in the laboratory. However, the effects of temperature were not investigated in this study.
While the focus in our study has been survival in cold temperatures, we have also examined the effect of UV-C on the two Haloarchaea in the past (McCready et al. Reference McCready, Müller, Boubriak, Berquist, Ng and DasSarma2005; Crowley et al. Reference Crowley, Boubriak, Berquist, Clark, Richard, Sullivan, DasSarma and McCready2006; Boubriak et al. Reference Boubriak, Ng, DasSarma, DasSarma, Crowley and McCready2008; our unpublished data). Our findings have shown that Halobacterium sp. NRC-1 and H. lacusprofundi are both quite tolerant to UV-C, with an LD50 of ~50 J m−2. Additional experiments are needed to determine the relative importance of UV-C, low pressure and other stressors in the partial loss of viability of Haloarchaea in the stratosphere. However, such studies would require longer flights.
Halobacterium sp. NRC-1 and H. lacusprofundi are able to withstand exposure to stratospheric conditions. H. lacusprofundi, an isolate from a perennially cold, hypersaline lake in Antarctica retains greater viability, at least for the time periods experienced using helium balloons, a property likely resulting from better cold and freeze adaptation. Most of the genes in the genome of the Antarctic species are significantly regulated at cold temperatures, which likely results in its better adaptation to the cold conditions in its natural environment and in the stratosphere.
Our work was supported by National Aeronautics and Space Administration grant NNX15AM07 G to S.D. We thank Sam Johnson, Justin Gilpin, Michael White, Bronwyn Stephenson and Jordan Herbst for assistance with balloon flights.
Author Disclosure Statement
No competing financial interests exist.