The assessment of the ability of terrestrial microorganisms to survive and thrive in conditions mimicking the space environment is a relevant topic for Astrobiology. As recently underlined by the “AstRoMap European Astrobiology Roadmap” (Horneck et al.2016) the identification of microorganisms that could adapt to live in space on other planets is one of the main search topics. Therefore, among the main tasks to be achieved, the AstRoMap considers the implementation of microbiological experiments, to be carried out either in space or on ground by simulating the harsh extraterrestrial conditions. In this context significant attention has been paid to the extremophiles i.e. microorganisms that are capable to live in conditions incompatible with life from an anthropocentric point of view. Indeed, their ability to resist in extreme environmental conditions make them a good biological model for astrobiology and for the study of the origin of life on Earth (Moissl-Eichinger et al.2016). According to thepanspermia hypothesis (Zagorski2007), life could have originated after transfer on Earth of extraterrestrial life forms (most probably microorganisms) that could have been transported by radiation pressure or by meteorites on the Earth’s surface. The transfer from one planet to another requires the ability to survive to several extreme environmental parameters (absence of oxygen, of water and of gravity; exposition to ionizing radiations; extreme temperature’s variations et cetera). Extremophiles can survive to a variety of harsh physical and chemical conditions including high pressure, dehydration, desiccation, UV and gamma radiations, extreme temperatures and pH values, high salt concentrations. Their ability to live in extreme conditions suggests that most probably extremophiles have been among the first living organisms that colonised the Earth. For these reasons they are the object of attention of astrobiology researches, indeed several extremophilic bacteria have been shown to be able to survive to the exposition to space conditions (Moissl-Eichinger et al.2016). The investigation of extremophiles’ behaviour in space or by on-ground facilities mimicking the space parameters, is crucial for the identification of the terrestrial life forms that could be able to resist the transport across the space as dormant or viable cells in meteorites, and then could be able to start growing and proliferate on Earth. In this line, in a previous work we investigated the ability of extremophiles to resist in simulated extraterrestrial conditions (Mastascusa et al.2014) and to restart cellular growth after the stress exposition. The cells of four extremophilic species were subjected to Mars simulated pressure and humidity, and to temperatures’ variation and UV irradiation mimicking the harsh space environments during the interplanetary transport. Among the investigated bacteria we have selected the thermophilic microorganismGeobacillus thermantarcticus, isolated from Mt. Melbourne, an active volcano in Antarctica. The cells ofG. thermantarcticus after the exposition to space simulating conditions, still kept their proliferative activity since they were able to grow as much as the cells stored and grown in their optimal conditions (Mastascusa et al.2014). Indeed, the cells ofG. thermantarcticus exhibited a significant resistance to the exposition to some environmental parameters that could be experienced during travelling across the space such as extreme temperature’s variation, desiccation, exposition to UVC rays irradiation. (Mastascusa et al.2014) In order to get more insights into the ability of this species to resist to space environment, in this work we investigated the survival rate after exposition to other parameters that likely could be experienced during the interstellar transport i.e. X-rays irradiation and absence of water. The cellular mechanisms underlying the cellular resistance were studied by assessing the biological effects after exposition to all the selected parameters i.e. extreme temperature, UV radiation, X-rays radiation and desiccation. Then the morphology, the membrane composition, the protein pattern and the DNA ofG. thermantarcticus cells grown after the stresses were investigated in order to assess the how this species adapted to the space simulated conditions.G. thermantarcticus is a sporulating species and since sporulation is triggered by stressing environmental conditions like UV space conditions, also spores were investigated for their resistance.

Culture Conditions

Geobacillus thermantarcticus (strain M1) (Lama et al.2012) was grown in static conditions at 60 °C (Heareus Instruments incubator) for 18 h in the following medium (g L⁻¹): 6.0 yeast extract, 3.0 NaCl, in tap water. The pH was adjusted to 5.6–5.8 by adding HCl 6 M. Cell growth was monitored by measuring optical density (O.D.) at λ = 540 nm. At the end of logarithmic phase (i.e. at O.D.⁵⁴⁰ = 0.700) an aliquot of the cell culture was taken to be subjected to the stressing conditions described in the following sections. After each stressing experiment the cells were recovered by centrifugation and used asinoculum in the standard conditions above described. For each experiment the cell growth was prolonged up to 18 h of incubation, then the final O.D. value was registered to determine the ability to restart growth after space’s conditions simulation. The cells so obtained (named “second generation cells”) were recovered by centrifugation for the analysis of morphology, proteins, lipids and DNA. All experiments were carried out in triplicate.

Temperature Stress

1.5 mL of cell culture (O.D.⁵⁴⁰ = 0.700) were centrifuged at 10,000 rpm for 15 min and the recovered cell pellet was stored without washing at temperature values of −196 °C, −80 °C, −20 °C, 0 °C, 4 °C, 10 °C, 25 °C, 50 °C, 70 °C, 85 °C for 3 months as previously described (Mastascusa et al.2014). After each experiment the cell pellets were re-suspended in the standard growth medium and used asinoculum in the standard conditions as described above to produce the second generation cells. The latter were used to assess the growth ability and for the analysis of proteins, lipids and DNA.

UVC Irradiation in Isotonic Solution

5.0 mL of cell culture (O.D.⁵⁴⁰ = 0.700) were centrifuged at 10.000 rpm for 15 min; the cell pellet was washed with a sterile isotonic solution (IS) (3.0 g/L NaCl in tap water, pH 6.2) and then after centrifugation (10,000 rpm for 15 min) it was resuspended in 5 mL of IS and seeded on a Petri dish at room pressure and temperature. The sample was gently stirred (100 rotations per minute) during irradiation that was performed by a UV-C lamp (Spectroline model EF-280C/FE 230 V 50 Hz 0.34 AMPS) set at λ = 254 nm with a power of about 6.00 W/m². The experiment was carried out in sterile conditions for 7.5, 15, 30 min and 1 h thus the irradiation doses absorbed were respectively 125, 250, 500 and 1000 J/m². The UV flux at the surface of cells was measured with a UV digital radiometer (HD 2102.2 Delta Ohm). After each experiment the cell pellets were re-suspended in the standard growth medium and used asinoculum in the standard conditions as described above to produce the second generation cells. The latter were used to assess the growth ability and for the analysis of proteins, lipids and DNA.

Desiccation Resistance

For desiccation experiments, 1 mL of fresh culture (O.D.⁵⁴⁰ = 0.700) were seeded on a Petri dish and dried under air laminar flux for 10 h. The samples were stored for different time intervals (3 days, 7 days, 10 days, 15 days, 20 days, 30 days, 40 days, and 1 year) at room temperature and at −20 °C. After each experiment the cell pellets were re-suspended in the standard growth medium and used asinoculum in the standard conditions as described above to produce the second generation cells. The latter were used to assess the growth ability and for the analysis of proteins, lipids and DNA.

X-Rays Irradiation

1 mL of cell culture (O.D.⁵⁴⁰ = 0.700) were uniformly seeded on a sterile filter (Isopore ™ Membrane Filters 0.2 μm GTTP) by using a vacuum pump. The filter with the cells was kept in a sterile plastic Petri dish and exposed to X-rays using a tube with a maximum voltage of 300 kV/p (Siemens, Germany) in the following conditions: voltage 250 kV, filtering by 1-mm-thick copper foil, dose rate of 1 Gy/min. X-rays doses values ranged from 50 to 1000 Gy and were measured at the sample position by ionization chamber (Victoreen) for each exposition value. After each experiment the cell pellets were re-suspended in the standard growth medium and used asinoculum in the standard conditions as described above to produce the second generation cells. The latter were used to assess the growth ability and for the analysis of proteins, lipids and DNA.

Spores Isolation and Resistance to UVC Irradiation

Spores’ formation was induced by using the sporulating medium YNM (0.6% yeast extract, 0.3% NaCl and 0.001% MnSO₄, pH 5.6–6.8 in tap water). The strain was inoculated in 100 mL YNM (1:100 v/v) and incubated at 60 °C in static conditions for 72 h. The sporulation process was verified by means of microscope analysis. Vegetative cells and spores were harvested by centrifugation at 10,000 rpm for 15 min. The pellet was washed with sterile distilled water and spores were recovered by centrifugation at 4000 rpm for 15 min; the treatment was repeated until the spore suspension, as confirmed by phase-contrast microscopy, contained 99% phase-bright spores. 10 ml of spores’ suspensions, prepared in distilled water to a final concentration of 10⁸ CFU/mL, were placed in a sterile Petri dish (diameter 5 cm) at room temperature and gently stirred. Irradiation by UVC lamp was conducted as above described. Sample volumes of 1 mL were removed at specific time points, serially diluted, and spread on YN agar plates. Survivors after irradiation were determined by colony-forming ability.

Electron Microscopy

The cells generated after 18 h of incubation following the stressing experiments as described above, were recovered by centrifugation and then fixed overnight in glutaraldehyde (2.5%) in phosphate buffer (pH 7), washed in phosphate buffer, centrifuged at 6000 rpm for 10 min (3–4 times) and then dehydrated in ethyl alcohol series. The samples were dried at room temperature on aluminium stubs and coated with a homogeneous layer (18 ± 0.2 nm) of Au–Pd alloy using a coating device (MED 020, Ba Tec AG, Tucson, AZ, USA) before SEM (Quanta 200 Feg) observations.

Total Lipids Analysis

0.3 g of freeze-dried cells were extracted with CHCl₃/MeOH/H₂O (65:25:4 v/v/v) (Di Cristo et al.2010) to recover the total lipid fraction that was analysed by means of¹H–NMR with a Bruker DPX-400 instrument operating at 400 MHz. Spectra were recorded in CDCl₃, chemical shifts were reported in ppm relative to the solvent signal. Fatty acid methyl esters (FAMEs) were obtained from the extraction mixture by acid methanolysis of complex lipids (Nicolaus et al.2016) and were detected by means of a GC-MS Helwett-Packard 5890A instrument, fitted with FID detector and equipped with an HP-V column with a flow rate of 45 ml/min using the temperature program of 120 °C (1 min), from 120 °C to 250 °C at 2 °C/min. Identification of compounds was obtained with standards and by interpretation of mass spectra.

SDS-PAGE Analysis

100 mg of wet cell pellets were washed with IS solution and suspended in 300 μL of Tris HCl buffer (50 mM, pH 7.2). The cell sample was lysed in ice bath by sonication (Heat System Instrument) for 3 min and then centrifuged at 13,000 rpm for 30 min. The supernatant (crude homogenate) was assayed for protein content by means of the Bradford’s method (1976) and analysed by electrophoresis. Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis was performed at alkaline pH as described by Davis (1964), using Mini Protean II apparatus (Bio-Rad) at constant current of 150 V. Protein samples (5 μg) were analyzed by using 15% acrylamide in the resolving gel, 5% acrylamide in the stacking gel and 25 mM Tris-glycine buffer, pH 8.3 containing 1 g/l SDS as running buffer according to Laemmli method (1970). The molecular mass was determined by using a high molecular weight (HMW) protein markers (Pharmacia Biotech). HMW was composed of Myosin (212 kDa); α₂ Macroglobulin (170 kDa); β-galactosidase (116 kDa); Transferrin (76 kDa); Glutamic Dehydrogenase (53 kDa). The protein bands were stained with Coomassie brilliant blue R-250 (0.1%) for 30 min and destained with a mixture of distilled water/methanol/acetic acid (50:40:10,v/v/v). The lower limit of detection of this technique was 30 ng.

Enzyme Activities’ Assays

Ammonium sulphate (561 g L⁻¹) was added to cell free culture medium at exponential phase of growth (200 mL) under stirring at 4 °C; the resulting precipitate was recovered by centrifugation (10,000 rpm, 1 h, 4 °C), dissolved in 50 mM sodium acetate buffer pH 5.6, and finally dialyzed (cut off 12,000 to 14,000 Da) overnight against the same buffer (Finore et al.2016). For xylanase activity assay 2% birchwood xylan in 50 mM sodium acetate buffer (pH 5.6) was used as substrate and the assay was performed as previously described (Lama et al.2004). The β-xylosidase assay was based on the release ofp-nitrophenol fromp-nitrophenyl-β-D-xylopyranoside and was carried out as previously described (Lama et al.2004).

Random Amplified Polymorphic DNA- Polymerase Chain Reaction (RAPD-PCR) Assay

DNA was extracted and purified from bacterial cell cultures (about 250 mg of dry cell) using the Genomic-DNA-Buffer Set and the Genomic-tip-100/G columns (QIAGEN SpA, Milano, Italy), according to the manufacturer’s instructions (Yildiz et al.2014). DNA amplification was performed in a 50 μL PCR reaction mixture containing: 50–200 ng of genomic DNA, 1X PCR buffer (supplied as component of the DNA polymerase kit), 3 mM MgCl₂, 250 mM dNTPs, 0.5 mM of GTG5 primer (5’GTGGTGGTGGTGGTG-3′) (Versalovic et al.1994) and 2.5 units of Platinum® Taq DNA polymerase (Invitrogen) (Poli et al.2009,2011). The mixtures were amplified in a thermocycler iQ5® (Biorad). The amplification profile consisted of an initial denaturation of 2 min at 92 °C and 35 cycles of 15 s at 94 °C, annealing for 15 s at 36 °C (previously optimized by temperature gradient amplification) and elongation for 2 min at 72 °C. A final extension of 7 min was carried out at 72 °C. PCR products were analyzed by electrophoresis on microchip by using the DNA 7500 kit (Agilent) and a 2100-Bioanalyzer equipped with 2100 EXPERT software (Agilent), following the manufacturer’s instructions.

Survival to Space Simulated Conditions

As previously described (Mastascusa et al.2014),G. thermantarcticus possess a high resistance to a variety of conditions simulating spatial travelling like the exposition to extreme temperature’s variations and UVC rays irradiation. After all the above mentioned treatments, cells preserved their ability to proliferate since they were able to restart growth in the standard conditions. Cells that were exposed for 3 months to a wide range of temperatures (−196 °C, −80 °C, −20 °C, 0 °C, 4 °C, 10 °C, 25 °C, 50 °C, 70 °C, 85 °C) showed a high resistance since they were able to afford microbial growth in most cases higher than 90% with respect to the standard conditions. The lower values of percent cells proliferation i.e. 30% and 68.3% with respect to the control, were found for cells stored at −196 °C and 85 °C, respectively (Mastascusa et al.2014). On the other hand, after the exposition of cells in a complex medium, the microbial growth’s percentage was on average about 80–90% with respect to the control; the most significant loss of cell growth was observed after 60 min of exposition, when a decrease of about 50% of cells production was observed (Mastascusa et al.2014). In order to complete our knowledge ofG. thermantarcticus’s ability to retain the growing capability after exposition to the extreme conditions mimicking the travelling in space environment, in this paper the bacterium’s resistance to long term desiccation, to X-rays irradiation and to UVC radiation in isotonic solution was also investigated. To assess the resistance in the absence of water, cells were desiccated and stored for time intervals ranging from one day up to one year, either at room temperature or at −20 °C. The resistance ofG. thermantarcticus was assessed by verifying the ability of treated cells to grow after the stressing conditions. As shown in Fig.1a (white bars) all the desiccated cells’ samples stored at room temperature were able to grow as much as in the standard conditions for all the desiccation time intervals tested; similar results were also obtained when desiccated cells were stored at −20 °C (Fig.1a, black bars).G. thermantarcticus was also investigated for its resistance to X-rays irradiation: cells seeded as a uniform layer in a sterile Petri dish were exposed to radiation doses ranging from 50 Gy to 1000 Gy. As shown by Fig.1b, after irradiation with all the X-rays doses applied, the cells were able to grow when re-suspended in the standard growth medium, thus confirming that this bacterial species is highly resistant to simulated space conditions. Finally, the UVC irradiation was performed by suspending cells in a sterile isotonic solution, in order to avoid the radiations’ shielding effect of nutrients present in the complex medium that was previously observed (Mastascusa et al.2014). Cells were exposed to UV radiation at increasing intensities and, as shown in Fig.1c, they were able to restart growth as much as the control un-treated cells, after irradiation up to 250 J/m². On the other hand, irradiation with higher intensity caused the cells’ death, indeed after irradiation at 1000 J/m² cells were no longer able to restart growth. SinceG. thermantarcticus is a sporulating species, the UVC irradiation experiment was performed on a purified sample of bacterial spores to assess the fraction of dormant cells that survived to UVC radiations. As shown in Fig.2 after irradiation at 125 J/m² the colony’s forming ability decreased of one order of magnitude, instead after irradiation at 250 J/m² a decrease of four orders of magnitude was found. Irradiation at higher doses caused the total loss of colony forming ability ofG. thermantarcticus’ spores. As previously observed for other space simulation experiments (Mastascusa et al.2014), X-rays irradiation and desiccation did not cause any significant delay in the lag phase of growth was observed. Indeed, as shown in Fig.3, the curve of microbial growth of cells either after exposition to X-rays at 1000 Gy (■) or after exposition to desiccation for 1 year at room temperature (▲), was closely comparable to that observed for not stressed cells (●).

Ability ofG. thermantarcticus’s cells to restart growth after exposition to space simulated conditions. Panel (a): growth ability of cells after exposition to desiccation and storage at room temperature (white bars) and at −20 °C (black bars). Panel (b): growth ability of cells after exposition to X-rays exposition. Panel (c): growth ability of cells after exposition to UVC irradiation at 254 nm. (O.D.: optical density at 540 nm after 18 h incubation in the standard conditions)

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Resistance ofG. thermantarcticus’s spores after UVC irradiation at 254 nm. (CFU/mL: colony forming units)

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Growth’s curves ofG. thermantarcticus cells after exposition to space simulated conditions. ● control cell growth; ▲ cell growth after desiccation; ■ cell growth after X-rays irradiation. (O.D. optical density at 540 nm)

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Effects of Exposition to Space Simulated Conditions on Cell Morphology

The analysis of morphology ofG. thermantarcticus was carried out by means of electron scanning microscopy for the cells that were able to restart growth after the exposition to the desiccation stress for 40 days either at room temperature or at −20 °C, and for 1 year at room temperature (Fig.4). In standard conditions the cells are regular rods, 0.5–2.0 μm in diameter and 2.0 to 5.0 μm long, as shown in Fig.4 (panels a and b). The cells grown after desiccation and storage at room temperature for 40 days (Fig.4, panels c and d) still retained the same shape and dimensions. The more significant variations occurred when cells were desiccated and stored for 40 days at −20 °C. As shown in Fig.4 (panels e and f),G. thermantarcticus’s morphology changed significantly. In this condition, the average length increased up to 7–9 μm. A similar effect was induced by desiccation and storage for 1 year at room temperature (Fig.4, panels g and h), indeed also in this condition the length of a significant fraction of cells raised up to 10–11 μm, about 2–3 fold longer then the control sample. The observed elongation could be ascribed to a delay in the cell division process, this phenomenon can indeed be associated to exposition to stressing conditions as previously described (Mathis and Ackermann2016).

G. thermantarcticus’s morphology after desiccation experiments. Panels (a) and (b), control sample; (c) and (d), desiccation and storage at room temperature for 40 days; (e) and (f), desiccation and storage at −20 °C for 40 days; (g) and (h), desiccation and storage at room temperature for 1 year

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Effects of Exposition to Space Simulated Conditions on membrane’s Lipids

The effect of the exposition to the space simulated conditions on the membranes’ composition was evaluated by analysis of total lipid extract obtained by treating cells in organic solvents, as described in the experimental section. The¹H NMR spectrum (Fig.5a) of the total lipid extract from control cells showed a very complex pattern of signals due to the presence of different lipid species, i.e. membrane soluble quinones and long chain fatty acids that are the main components of membrane’s lipids. With regard to the quinones, as previously described, one major group of isoprenoid quinones that can be found in bacteria is represented by the respiratory quinone species like the menaquinones (Fig.5, panel c) and their derivatives (Collins and Jones1981). The signals typical of these quinones can be found in three main regions of the spectrum i.e. the region from 8.10 to 6.75 ppm, relative to the naphtoquinone moiety (Fig.5c); the region from 5.10 to 4.90 ppm relative to the olefinic hydrogens of the polyisoprenoid chain (Fig.5b) and finally the region from 2.11 to 1.40 ppm relative to the methyl groups of the isoprenoid chain. The analysis of¹H–NMR spectrum of control cells showed, in the naphtoquinone ring’s region, the presence of two main spin systems and of a doublet at 7.079 ppm. The first system includes the two multiplet signals at 8.081 ppm and 7.685 ppm (Fig.5, panel c) that, based on literature’s data, are attributable respectively to H-5/H-8 and to H-6/H-7 aromatic hydrogens of a menaquinone species (MK-n) (Das et al.1989). The second spin system includes the three broad singlets at 7.111 ppm. 7.046 ppm and 6.897 ppm (Fig.5, panel c). Based on the data reported in literature, these signals can be ascribed to a reduced demethylated menaquinone (DMK-n) (Das et al.1989): the signals at δ 7.111 and 7.046 are attributable to the hydrogens linked to the C-5/C-8 and at C-6/C-7 respectively, while the broad singlet at δ 6.897 is indicative of the quinonoide hydrogen at C-2. Finally, the doublet at 7.079 ppm is indicative of another spin system whose counterpart couldn’t be identified in the spectrum. The presence of three different quinone species was confirmed by the analysis of the isoprenoid chain’s hydrogens that indeed showed three main multiplets at 5.422, 5.437 and 5.109 ppm (panel b in Fig.5). The identification in the region from 1.5 to 0.5 ppm of the signals of the methyl groups of the polyisoprenoid chain was hampered by the presence of the intense signals of the ω-methyl groups of the fatty acids of the other membrane’s lipids.

¹H–NMR spectrum of the total lipid extract from not treated cells ofG. thermantarcticus. Panel (a), full range; panel (b), detail of the polyisoprenoid chain protons’ range from 4.0 to 5.5; panel (c), detail of aromatic protons’ region from 8.3 to 4.7 ppm. Chemical shifts are reported in ppm relative to CHCl₃ (7.26 ppm). Legend: MK-n, menaquinone; DMK-n, demethylated menaquinone

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The effect of the different stressing condition tested resulted in most cases in the modification of the aromatic region of the quinone species as confirmed by the detailed analysis of the region from 8.10 to 6.75 ppm (Fig.6). For the majority of conditions explored, the menaquinone species was not affected by exposition to the extreme parameters of irradiation (Fig.6, panel a), desiccation (Fig.6, panel a) and temperature (Fig.6, panels b, c and d) that were tested. The most significant effect was the disappearance of the spin system relative to the probable demethylated menaquinone, that was observed in almost all the conditions, with the only exceptions of X-rays irradiation at 1000Gy and storage at −80 °C and 0 °C.

Effect of exposition to the space simulated parameters on the quinone species ofG. thermantarcticus: details of quinone ring’s region in the¹H–NMR spectra. Panel (a): after UVC at 125 J/m² and X-rays irradiation at 100Gy, desiccation and storage at −20 °C and room temperature. Panel (b): after storage for 3 months at T from −196 °C to −20 °C. Panel (c): after storage for 3 months at T from 0 °C to 25 °C (panelc). Panel (d): after storage for 3 months at T from 50 °C to 85 °C (paneld). Chemical shifts are reported in ppm relative to CHCl₃ (7.26 ppm)

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As previously stated, the analysis of¹H–NMR spectrum (Fig.5) of the total lipid extract showed very intense signals in the upfield region that can be attributed to the aliphatic protons of long chain fatty acids from the cells membranes. The fatty acids’ composition ofG. thermantarcticus’s membrane after the exposition to the space simulated parameters was determined by means of GC-MS analysis of fatty acids’ methyl esters (FAME). The results obtained after all the tested stressing condition were reported in Table1 as percent composition, in comparison with the control conditions.

The control sample i.e.G. thermantarcticus cells grown in the standard conditions and harvested at the beginning of growth’s stationary phase, showed as main fatty acid methyl esters theiso C15:0,anteiso C16:0 andnormal C16:0, besides theiso C17:0 andanteiso C:17:0. The main variation of lipid’s profile was represented, for all the stressing conditions examined, by the appearance of short chain C14 and long chain C19 fatty acids; moreover for all the stressed cells an increase of theiso:anteiso ratio was observed. Other remarkable effects were represented by the differences in lipid’s profiles of sample stored at −196 °C and for the cells that were desiccated and stored for 1 year at room temperature. As reported in Table1, for the cells stored at −196 °C the levels ofiso C15:0 andanteiso C18:0 were about 38.8% and 35.9% of the control values, respectively. Main differences were found for the cells subjected to desiccation then stored at room temperature (RT) for 1 year: in this case the percentage values ofiso C15:0,anteiso C15:0,iso C17:0,anteiso C17:0 were lower compared to control (50.7%, 42.1%, 62.3%, and 68.4%, respectively) while the percentage ofnormal C15:0,normal C16:0 andnormal C17:0 were 2.5-, 3,5- and 3-fold with respect to the control sample.

Effects of Exposition to Space Simulated Conditions on Protein Pattern

The effect on the endocellular proteins’ pattern ofG. thermantarcticus was investigated by means of SDS-PAGE analysis of cell’s lysates (Fig.7). In all cases the most significant variations in the protein pattern were found in the molecular weight’s range from 76 kDa to 53 kDa. Indeed, in the case of cells exposed to UVC radiation (at 125 J/m²) and to X-rays (at 1000 Gy) the comparison of two apparently new protein bands around 60 kDa was found (Fig.7, panel a). A similar result was found for the cells underwent to the desiccation and storage at room temperature for 40 days and for 1 year, and for desiccated cells stored at −20 °C (Fig.7, panel b): moreover, for the desiccated cells stored for 1 year, a depletion in the intensity of the protein band located at about 65 kDa was registered. Finally, the protein pattern of cells stored for 3 months at temperatures ranging from −196 °C up to 10 °C (Fig.7, panels c and d) also showed the appearance of two new protein bands around 60 kDa; in particular, after storage at temperatures below 10 °C the comparison of the new band was accompanied by the depletion in the intensity of the protein band located at about 65 kDa.

Effect of exposition to the space simulated parameters on the endocellular protein pattern ofG. thermantarcticus. After stress experiments cells were grown for 18 h in the standard conditions and endocellular proteins were recovered by cell lysis. Legend: proteins’ SDS-PAGE analysis after UVC at 125 J/m² and X-rays irradiation at 1000Gy (panela); after desiccation and storage at −20 °C and room temperature (panelb), after storage for 3 months at temperature from −196 °C to 85 °C (panelsc andd)

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G. thermantarcticus has been reported to be able to produce extracellular glycoside hydrolases activities, namely xylanase and β-xylosidase. The effect of the space simulation on extracellular proteins was assessed by measuring the production of these enzymes that are expressed in a late exponential phase of cellular growth (Lama et al.2012). The activity of both the enzymes was mainly affected by desiccation and storage for 1 year at room temperature and UVC irradiation at 125 J/m². As shown in Table2, where the comparison between the specific activity in control cells and in stressed cells has been reported, the β-xylosidase activity’s expression decreased of 68.4% for desiccation and storage for 1 year at room temperature and of 76.9% after UVC irradiation stress, respectively. On the contrary, the xylanase activity increased of 50% after UVC irradiation and of 72.3% for the desiccated sample stored for 1 year at room temperature.

Effects of Exposition to Space Simulated Conditions on DNA

The effect of the different stressing conditions tested onG. thermantarcticus’s DNA was investigated by means of RAPD-PCR. Therefore, fingerprint patterns were produced by using GTG5 primer on DNA samples extracted form cells exposed to storage for three months at 10 °C, to desiccation and storage for 1 year at room temperature, to UVC at 125 J/m² and to X-rays irradiation at 1000Gy (Fig.8). The use of GTG5 primer underlined that, overall,G. thermantarcticus DNA was not significantly affected by the different exposition treatments, indeed the most evident mutations were found mainly for UVC irradiated cells (Fig.8, line 3): in this case a new band at about 700 kbp, that was absent in the control (Fig.8, line 5), appeared after the irradiation at λ = 254 nm.

RAPD-PCR-fingerprint ofG. thermantarcticus DNA. Legend: L, molecular weight standard; 1, storage for 3 months at 10 °C; 2, desiccation and storage for 1 year at room temperature; 3, UV irradiation at 125 J/m²; 4, X-rays irradiation at 100Gy; 5, control sample (untretaed cells); 6, blank (GTG5 primer). (Nicolaus et al.2016)

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As previously reported for some extremophilic microorganisms (Leuko et al.2015; Mancinelli et al.1998) the viable cells of the thermophilic speciesG. thermantarcticus have been shown to resist to space simulated conditions. The results here reported suggested that this species could be able to repair the possible damages induced by the exposition to temperatures values far from the bacterium’s optimum, by the absence of water (that is usually a consequence of the absence of gravity in the space), and by irradiation with the full solar radiation spectrum. The ability ofG. thermantarcticus to restart cellular growth after exposition to the space simulated conditions could also be ascribed to the fact that is a spore-forming species. Indeed, spores of other species belonging to the genusBacillus have been widely investigated as biological models to assess the response to terrestrial and extraterrestrial radiation in outer space or in space simulation facilities (Nicholson et al.2005; Horneck et al.2001,2010). Like other species belonging to the same genus,G. thermantarcticus’ spores also displayed a good resistance to the exposition to UVC radiations. In order to assess the survival ofG. thermantarcticus at a molecular level, we investigated the morphology, the membrane’s lipids profile, the protein pattern and the DNA patterns of the cells that were produced from those ones that were exposed to the above mentioned simulated space conditions. The adaptive response ofG. thermantarcticus was elicited by changes in the morphology that, after desiccation and storage at room temperature or at −20 °C, resulted in the elongation of cells with respect to the standard growth conditions. In parallel the cell membranes’ composition changed either for the lipids profile or the quinones pattern. With regard to the lipids profile, the response to the stressing conditions ofG. thermantarcticus was implemented by increasing of membrane fluidity: indeed, after all the stressing experiments, the newborn cells showed an higher ratio of levels ofiso vsanteiso forms of branched fatty methyl esters, a variation that typically causes the increase of membrane’s fluidity. On the other hand, after the exposition to all the space simulated parameters, the redox quinones’ pattern showed as main modification the loss of the minor component i.e. DMK-n, since the main species MK-n was still produced in the second generation’s cells. Membrane soluble quinones like MK-n play a crucial role in the energy transduction since they are among the key components of the respiratory chain of virtually all the bacterial cells. Our results showed that cells ofG. thermantarcticus are able to protect their respiratory chain machinery thus being able to produce energy also after the exposition to the space simulated conditions. The survival of this species may also due by its ability to change the proteins pattern: as a matter of fact, after all the stressing treatments, the expression of total endocellular proteins did not dramatically changed and the main differences were represented by a higher production of low molecular proteins whose involvement in the resistance response could be assessed only by means of a proteomic approach. Finally, the probable mutagenic effects of the exposition to the extreme conditions tested in this work, was in almost all experiments counteracted in the second generation cells since apparently no significant variation in their DNA patterns was evidenced. The only appreciable modifications were found in the cells that were irradiated by UVC for which a slightly different pattern of DNA’s amplification was found. Nevertheless, a complete genomic analysis, that was out of the scopes of this work, will be needed to confirm this result.

The identification of bacterial species that are able to survive and to adapt to space environments is of particular concern for astrobiology, either for the search of the possible origin of life on earth or for finding the most probable candidates for possible future colonization of exoplantes (Moissl-Eichinger et al.2016). To approach to this issue it is necessary to identify those living organisms that are able to survive to space travelling and to proliferate after the transfer from or to other planets. In this frame, the study of the extremophiles is a key point since these species have evolved on Earth to be able to survive into a wide variety of extreme environments. Extremophiles have developed several strategies to react to different kinds of stressing conditions by producing biomolecules (proteins, lipids, osmolytes, et cetera) or reorganizing cellular organelles. Therefore, their ability to survive and adapt to different extreme conditions suggests that they could also adapt to the harsh space environments. In this work we verified the ability of the thermophilic speciesG. thermantarcticus to survive to space simulated conditions by being able to restart cellular growth with the same viability of non-stressed cells.G. thermantarcticus cells showed similar responses to the different stressing conditions by being able to efficiently protect the membrane’s, DNA’s and proteins’ integrity. These results suggested thatG. thermantarcticus could be a good biological model for astrobiology studies since it is able to repair the possible modifications induced by stressing space conditions thus counteracting the harmful effects of exposition to space conditions.