Lack of correlation of desiccation and radiation tolerance in microorganisms from diverse extreme environments tested under anoxic conditions

Lack of correlation of desiccation and radiation tolerance in microorganisms from diverse extreme... Abstract Four facultative anaerobic and two obligate anaerobic bacteria were isolated from extreme environments (deep subsurface halite mine, sulfidic anoxic spring, mineral-rich river) in the frame MASE (Mars Analogues for Space Exploration) project. The isolates were investigated under anoxic conditions for their survivability after desiccation up to 6 months and their tolerance to ionizing radiation up to 3000 Gy. The results indicated that tolerances to both stresses are strain-specific features. Yersinia intermedia MASE-LG-1 showed a high desiccation tolerance but its radiation tolerance was very low. The most radiation-tolerant strains were Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1. In both cases, cultivable cells were detectable after an exposure to 3 kGy of ionizing radiation, but cells only survived desiccation for 90 and 30 days, respectively. Although a correlation between desiccation and ionizing radiation resistance has been hypothesized for some aerobic microorganisms, our data showed that there was no correlation between tolerance to desiccation and ionizing radiation, suggesting that the physiological basis of both forms of tolerances is not necessarily linked. In addition, these results indicated that facultative and obligate anaerobic organisms living in extreme environments possess varied species-specific tolerances to extremes. correlation, desiccation, radiation, survival, anaerobes, extreme environment INTRODUCTION Anaerobic microorganisms are widely distributed in Earth's extreme environments, yet we still know little about their physiology and their capacity to adapt to extreme conditions. In particular, there is a paucity of studies whereby different anaerobic microorganisms from extreme environments are investigated to understand their diverse physiological and metabolic capabilities. In this study, the two stressors of interest were the tolerance to periods of water loss and the exposure to ionizing radiation. These stressors also occur in other extreme environments but their combination is rare. Microorganisms frequently experience periodic desiccation in subaerial environments or during dispersal. Although most natural environments do not experience ionizing radiation beyond the level of naturally occurring background radiation (Thorne 2003), this stress can be explored as a proxy for an organisms’ ability to repair general cell damage. Furthermore, there has often been a claimed correlation between desiccation and ionizing radiation resistance, which is of interest to explore further. It is suggested that the physiological basis and repair mechanisms to counteract the stress-induced damage by radiation or desiccation might be linked or might even be the same (Mattimore and Battista 1996). While there are several studies investigating the survivability of model microorganisms, such as Escherichia coli and Deinococcus radiodurans, after desiccation and after exposure to ionizing radiation tested under oxic conditions (Clavero et al.1994; Welsh and Herbert 1999; Daly 2009; Bauermeister et al.2011), there are only a few examples where facultative and strict anaerobic microorganisms were tested against these stressors under anoxic conditions. The survival capacity after exposure to one stressor in form of ionizing radiation was described for the hyperthermophilic anaerobic microorganisms, Pyrococcus furiosus and Thermococcus gammatolerans (DiRuggiero et al.1997; Jolivet et al.2003). However, there are only very few studies about the tolerance in terms of sensitivity of anaerobic microorganisms to desiccation (Fetzer, Bak and Conrad 1993; Beblo et al.2009). The application of both of these stressors gives insights into the abilities that microorganisms have evolved to survive damage to macromolecules such as proteins, membranes and nucleic acids. During desiccation, initially the free intracellular water and subsequently the hydration shell of different molecules disappear, consequently affecting cellular components in their functions. For example, protein denaturation and perturbation of the lipid membrane by phase changes, e.g. structural conversion of bilayer sheets to spherical micelles, might appear (Cox 1993; Prestrelski et al.1993; Billi and Potts 2002). The DNA is also affected through loss of water; DNA protein cross-links and strand breaks can occur (Bieger-Dose et al.1992; Dose et al.1992). Additionally, the DNA can change from the B to the A-conformation resulting in DNA single- and double-strand breaks. Dehydration for 6 weeks caused approximately 60 DNA double-strand breaks per genome in D. radiodurans (Fredrickson et al.2008). Nevertheless, the survival of the microorganisms after 6 weeks of desiccation was not even reduced by one order of magnitude (Mattimore and Battista 1996). Another form of damaging stress that occurs during desiccation is the formation of reactive oxygen species (ROS), which, in turn, leads to oxidative stress (França, Panek and Eleutherio 2007). The ROS are mainly superoxide anions (•O2−) and hydroxyl radicals (•OH) affecting all macromolecular cellular components (Cabiscol, Tamarit and Ros 2000). However, the exact origin of the radicals during rehydration is unknown, but a possible source could be the metabolism itself (e.g. respiratory chain) when the cells were growing aerobically (González-Flecha and Demple 1995; França, Panek and Eleutherio 2007).They are also formed by indirect effects through the radiation-induced radiolysis of intracellular water and surrounding water and account for approximately 80% of introduced DNA damages (Michaels and Hunt 1978; Jones et al.1994; Riley 1994). After exposure to ionizing radiation of 5 kGy, approximately 200 double-strand breaks were detected in D. radiodurans (Cox and Battista 2005). However, the survivability of D. radiodurans was not reduced (Battista, Earl and Park 1999). In addition, ionizing radiation also affects proteins through oxidation processes, lipids through lipid peroxidation and disturbance of membrane permeability in eukaryotic and prokaryotic systems (Leyko and Bartosz 1985; Daly et al.2007; Krisko and Radman 2010). MASE (Mars Analogues for Space Exploration), a project funded by the European Union, was initiated to investigate anaerobic microorganisms in terrestrial extreme environments and their physiological adaptations to these extreme environmental conditions (Cockell et al.2017). In this article, we describe the survivability after prolonged desiccation of up to 4 weeks and exposure to ionizing radiation (up to 3 kGy) of four facultative anaerobic and two strict anaerobic microorganisms isolated from different extreme environments in the frame of this project. MATERIALS AND METHODS Strains and culture conditions During the MASE project, over 30 pure cultures were obtained from various extreme environments (Cockell et al.2017). From this list, six distantly related bacterial strains were picked for further analysis. The following microorganisms, namely Acidiphilium sp. PM (DSM 24941), Buttiauxella sp. MASE-IM-9 (DSM 105071), Clostridium sp. MASE-IM-4 (DSM 105631), Halanaerobium sp. MASE-BB-1 (DSM 105537), Trichococcus sp. IM-5 (DSM 105632) and Yersinia intermedia MASE-LG-1 (DSM 102845). Media and strain-specific cultivation conditions are summarized in Table 1 and described in detail in Cockell et al. (2017). The incubation was carried out at the indicated cultivation temperature, and cultures were shaken at 50 rpm. Noteworthy, for Clostridium sp. MASE-IM-4 only vegetative cells have been observed during the applied cultivation condition. Table 1. Strains, origins and cultivation conditions.           Temperature  Strain Phylum Class  Origin  Medium  Supplements (wt/vol)  Gas phase (vol/vol)  (°C)  Acidiphilium sp. PM Proteobacteria Alphaproteiobacteria  River Rio Tinto, Spain  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30  Buttiauxella sp. MASE-IM-9, Proteobacteria Gammaproteobacteria  Islinger Mühlbach, Germany  MASE-II  0.1% Yeast extract  80% N2, 20% CO2  30  Clostridium sp. MASE-IM-4 Firmicutes Clostridia  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Dimethylamine 0.001% FeCl2  15% H2, 25% CO2, 60% N2  30  Halanaerobium sp. MASE-BB-1 Proteobacteria Gammaproteobacteria  Boulby Mine, Great Britain  HACE  0.1% Yeast extract  15% H2, 25% CO2, 60% N2  45  Trichococcus sp. MASE-IM-5 Firmicutes, Bacilli  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Na2SO4 0.01% C6H5Na3O7 × 2 H2O 0.02% KNO3  15% H2, 25% CO2, 60% N2  30  Y. intermedia MASE- LG-1 Proteobacteria Gammaproteobacteria  Lake Grænavatn, Iceland  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30            Temperature  Strain Phylum Class  Origin  Medium  Supplements (wt/vol)  Gas phase (vol/vol)  (°C)  Acidiphilium sp. PM Proteobacteria Alphaproteiobacteria  River Rio Tinto, Spain  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30  Buttiauxella sp. MASE-IM-9, Proteobacteria Gammaproteobacteria  Islinger Mühlbach, Germany  MASE-II  0.1% Yeast extract  80% N2, 20% CO2  30  Clostridium sp. MASE-IM-4 Firmicutes Clostridia  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Dimethylamine 0.001% FeCl2  15% H2, 25% CO2, 60% N2  30  Halanaerobium sp. MASE-BB-1 Proteobacteria Gammaproteobacteria  Boulby Mine, Great Britain  HACE  0.1% Yeast extract  15% H2, 25% CO2, 60% N2  45  Trichococcus sp. MASE-IM-5 Firmicutes, Bacilli  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Na2SO4 0.01% C6H5Na3O7 × 2 H2O 0.02% KNO3  15% H2, 25% CO2, 60% N2  30  Y. intermedia MASE- LG-1 Proteobacteria Gammaproteobacteria  Lake Grænavatn, Iceland  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30  View Large Desiccation and irradiation experiments For the desiccation experiments, the cells were cultivated under optimal growth conditions until stationary growth phase was reached. Desiccation experiments were performed as described by Beblo et al. (2009). Briefly, cell concentrations were determined by counting in a Thoma chamber. One milliliter of cell culture (cell densities ranged from ∼5 × 106 cells/ml to ∼5 × 107 cells/ml) was spread evenly on four glass slides and dried under anoxic conditions in an anaerobic chamber (Coy Laboratory Products Inc., [O2] < 0.0001%, relative humidity 13 ± 0.5%; both in vol/vol) in the presence of drying agent calcium chloride. Afterwards, the dried cells were stored within the anaerobic chamber. Exposure to ionizing radiation was carried out according to earlier studies (Beblo et al.2011). Stationary phase cell cultures, in liquid suspensions, were transferred anoxically into 7 ml glass HPLC vials (WICOM Germany GmbH), which were tightly sealed with rubber stoppers and aluminum caps. Irradiation was conducted with an X-ray source Gulmay RS 225A (Gulmay Medical Ltd) at 200 kV and 15 mA. Cells were irradiated at a distance of 19.5 cm below the X-ray source with 20 Gy min−1 ± 5 Gy min−1 up to 3 kGy. The dose rate was measured with a UNIDOS dosimeter (PTW Freiburg GmbH). All irradiation experiments were performed under anoxic conditions at room temperature. Determination of the survival At dedicated time points, the dried cells on glass slides or the irradiated cells as a liquid suspension were transferred under anoxic conditions into the strain-specific culture medium and incubated for up to 4 weeks (Table 1, for description of the procedure see Beblo et al.2009). Growth of the cells in all dilutions was observed visually and by phase-contrast microscopy (Zeiss® Axiolmager TM M2) with 400× or 1000× magnification. Determination of the survival and enumeration of cultivable cells was achieved by the most probable number (MPN) technique via dilution series with 10-fold dilution steps (Franson 1985). The MPN technique was applied for all six strains, since not all strains are able to grow on solid surfaces. All experiments were repeated independently at least three times, representing biological replicates. The data shown within graphs represent mean values with standard deviations. The survival (S) was calculated as relative survival after cell damaging treatment (N) compared to the non-treated control (N0) (S = N/N0). Due to the applied MPN technique and depending on the growth density of the specific strain (∼5 × 106 cells/ml to ∼5 × 107 cells/ml), the detection limit of the determination of survival was ∼1 × 10−8. RESULTS The six vegetative strains Acidiphilium sp. PM, Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Y. intermedia MASE-LG-1 showed different levels of survival after desiccation and after exposure to radiation (Fig. 1). Figure 1. View largeDownload slide Survival of the MASE isolates after anoxic desiccation (A–F) and after exposure to ionizing radiation under anoxic conditions (G–L). For desiccation experiments, the cells were applied to glass slides, dried and stored under anoxic conditions up to 184 days. For anoxic irradiation experiments, the cells were exposed to ionizing radiation up to 3 kGy in liquid culture medium under anoxic conditions. Acidiphilium sp. PM (A, G), Buttiauxella sp. MASE-IM-9 (B, H), Clostridium sp. MASE-IM-4 (C, I), Halanaerobium sp. MASE-BB-1 (D, J), Trichococcus sp. MASE-IM-5 (E, K), Y. intermedia MASE-LG-1 (F, L). Solid lines are the survival curves fitted by hand based on the survival data; N0: viable cells without desiccation or without irradiation; N: viable cells after desiccation or without irradiation (n = 3 with standard deviation); *no viable cells detected. Figure 1. View largeDownload slide Survival of the MASE isolates after anoxic desiccation (A–F) and after exposure to ionizing radiation under anoxic conditions (G–L). For desiccation experiments, the cells were applied to glass slides, dried and stored under anoxic conditions up to 184 days. For anoxic irradiation experiments, the cells were exposed to ionizing radiation up to 3 kGy in liquid culture medium under anoxic conditions. Acidiphilium sp. PM (A, G), Buttiauxella sp. MASE-IM-9 (B, H), Clostridium sp. MASE-IM-4 (C, I), Halanaerobium sp. MASE-BB-1 (D, J), Trichococcus sp. MASE-IM-5 (E, K), Y. intermedia MASE-LG-1 (F, L). Solid lines are the survival curves fitted by hand based on the survival data; N0: viable cells without desiccation or without irradiation; N: viable cells after desiccation or without irradiation (n = 3 with standard deviation); *no viable cells detected. Tolerance to desiccation The survival curves of all tested organisms showed an exponential decay as described by Chen and Alexander (1973). Thereby, the survival rate decreased substantially within the first days of desiccation and the survival decreased until it plateaued. Only Y. intermedia MASE-LG-1 was able to survive the maximum tested time period of desiccation (184 days). After 184 days, the survival of this organism was S (184 days) = 3.7 × 10−5 (Beblo-Vranesevic et al. 2017a). In contrast to this high tolerance to water loss, Clostridium sp. MASE-IM-4, Trichococcus sp. MASE-IM-5 and Halanaerobium sp. MASE-BB-1 were more sensitive to desiccation. After 4 weeks in a dry state, no living cells of Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5 were detectable (Fig. 1C and E). The survival of Halanaerobium sp. MASE-BB-1 after 4 weeks of desiccation was reduced by more than four orders of magnitude (Fig. 1D) to S (28 days) = 3.7 × 10−5 and no living cells were found after 56 days of anoxic storage in a dry state. The survival of Acidiphilium sp. PM and Buttiauxella sp. MASE-IM-9 after 4 weeks were S (28 days) = 7.0 × 10−7 and S (28 days) = 3.7 × 10−5 respectively, and these strains could outlast 84 days in a desiccated form (Fig. 1A and B). All these results summarized, the organisms can be ranked in terms of their tolerance to desiccation: Clostridium sp. MASE-IM-4 < Trichococcus sp. MASE-IM-5 < Halanaerobium sp. MASE-BB-1 < Acidiphilium sp. PM < Buttiauxella sp. MASE-IM-9 < Y. intermedia MASE-LG-1. Tolerance to ionizing radiation Survival after exposure to ionizing radiation of the tested MASE isolates varied greatly between isolates. Different types of curves were obtained and not all organisms showed the typically shouldered survival curve as it is described by Kiefer (1990). Only two organisms (Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1; Fig. 1H and J) were able to multiply after an exposure to 3 kGy (S (Buttiauxella sp. MASE-IM-9, 3 kGy) = 3.7 × 10−7; S (Halanaerobium sp. BB-1, 3 kGy) = 4 × 10−6). For Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5, the highest dose at which culturable cells were detected after reinoculation was 2.5 kGy (Fig. 1I and K). Acidiphilium sp. PM was more sensitive to ionizing radiation and only survived exposures up to 1.5 kGy (Fig. 1G). The most sensitive organism amongst this tested group of microorganisms was Y. intermedia MASE-LG-1. Multiplying cells were only observed to a dose of 0.8 kGy (Fig. 1L). Concluding these data, the tolerance to an exposure to ionizing radiation can be ranked as follows: Y. intermedia MASE-LG-1 < Acidiphilium sp. PM < Clostridium sp. MASE-IM-4 < Trichococcus sp. MASE-IM-5 < Buttiauxella sp. MASE-IM-9 < Halanaerobium sp. MASE-BB-1. Survival data for various microorganisms from literature data Table 2 lists organisms from other studies and their desiccation and radiation tolerance in which both parameters have been measured for a given organism. For comparability with previous studies, desiccation tolerance was defined as the tolerance to survive four weeks (28 days) in a dried state at room temperature and if applicable under anoxic conditions. Radiotolerance was defined as the ability to survive an exposure to ionizing radiation with a dose of 3 kGy. Additionally, there are many microorganisms for which only one treatment was tested and the list could be enlarged tremendously (e.g. Jolivet et al.2003; Walsh and Camilli 2011). Table 2. Overview of microbial survival after desiccation (28 days) and ionizing radiation (3 kGy) from literature data. Organism  Oxidative state of the experimental setup  Desiccation (28 days)  Radiation (3 kGy)  Reference  Acidiphilium sp. PM  Anoxic  +  –  In this study  Buttiauxella sp. MASE-IM-10  Anoxic  +  +  In this study  Clostridium sp. MASE-IM-4  Anoxic  –  –  In this study  Halanaerobium sp. MASE-BB-1  Anoxic  +  +  In this study  Trichococcus sp. MASE-IM-5  Anoxic  –  –  In this study  Yersinia intermedia MASE-LG-1  Anoxic  +  –  In this study  Archaeoglobus fulgidus  Anoxic  +  +  Beblo et al.2009; Beblo et al.2011  Ignicoccus hospitalis  Anoxic  –  +  Beblo et al.2009; Koschnitzki 2016  Methanocaldococcus jannaschii  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Methanosarcina barkeri  Anoxic  +  +b  Morozova and Wagner 2007; Anderson, Apolinario and Sowers 2012  Methanothermobacter thermoautotrophicus  Anoxic  +  –c  Beblo et al.2009; Beblo et al.2011  Nanoarchaeum equitans  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Pyrococcus furiosus  Anoxic  –  +  DiRuggiero et al.1997; Beblo et al.2009  Thermoproteus tenax  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Thermofilum pendens  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Aquifex pyrophilus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Hydrogenothermus marinus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Metallosphaera sedula  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Sulfolobus metallicus  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Acinetobacter radioresistens  Oxic  +  +  Nishimura, Ino and Iizuka 1988; Jawad et al.1998  Brevundimonas sp.  Oxic  +  +  Dartnell et al.2010; Musilova et al.2015  Chroococcidiopsis sp.  Oxic  +  +  Caiola et al.1996; Billi et al.2000  Deinococcus radiodurans  Oxic  +  +  Daly 2009; Bauermeister et al.2011  Deinococcus geothermalis  Oxic  +  +  Ferreira et al.1997; Frösler et al.2017  Escherichia coli  Oxic  –  –  Clavero et al.1994; Welsh and Herbert 1999  Geodermatophilus poikilotrophi  Oxic  +  +  Montero-Calasanz et al.2014  Halobacterium salinarum  Oxic  +a  +  Kottemann et al.2005; Leuko and Rettberg 2017  Halococcus hamelinensis  Oxic  +  +  Leuko and Rettberg 2017  Halococcus morrhuae  Oxic  +  +  Leuko and Rettberg 2017  Halomonas sp.  Oxic  +  +  Musilova et al.2015  Kocuria polaris  Oxic  –  +  Shirsalimian et al.2016  Listeria monocytogenes  Oxic  +a  –  Niemira et al.2003; Hingston et al.2013  Methylobacterium extorquens  Oxic  +  +  Romanovskaya et al.2002  Rhodococcus sp.  Oxic  –  +  Dartnell et al.2010; Musilova et al.2015  Salmonella typhimurium  Oxic  +a  +  Thayer and Boyd 1991; Li et al.2012  Yersinia pestis  Oxic  –  –  Rose et al.2003; Sommers and Cooke 2009  Organism  Oxidative state of the experimental setup  Desiccation (28 days)  Radiation (3 kGy)  Reference  Acidiphilium sp. PM  Anoxic  +  –  In this study  Buttiauxella sp. MASE-IM-10  Anoxic  +  +  In this study  Clostridium sp. MASE-IM-4  Anoxic  –  –  In this study  Halanaerobium sp. MASE-BB-1  Anoxic  +  +  In this study  Trichococcus sp. MASE-IM-5  Anoxic  –  –  In this study  Yersinia intermedia MASE-LG-1  Anoxic  +  –  In this study  Archaeoglobus fulgidus  Anoxic  +  +  Beblo et al.2009; Beblo et al.2011  Ignicoccus hospitalis  Anoxic  –  +  Beblo et al.2009; Koschnitzki 2016  Methanocaldococcus jannaschii  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Methanosarcina barkeri  Anoxic  +  +b  Morozova and Wagner 2007; Anderson, Apolinario and Sowers 2012  Methanothermobacter thermoautotrophicus  Anoxic  +  –c  Beblo et al.2009; Beblo et al.2011  Nanoarchaeum equitans  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Pyrococcus furiosus  Anoxic  –  +  DiRuggiero et al.1997; Beblo et al.2009  Thermoproteus tenax  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Thermofilum pendens  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Aquifex pyrophilus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Hydrogenothermus marinus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Metallosphaera sedula  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Sulfolobus metallicus  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Acinetobacter radioresistens  Oxic  +  +  Nishimura, Ino and Iizuka 1988; Jawad et al.1998  Brevundimonas sp.  Oxic  +  +  Dartnell et al.2010; Musilova et al.2015  Chroococcidiopsis sp.  Oxic  +  +  Caiola et al.1996; Billi et al.2000  Deinococcus radiodurans  Oxic  +  +  Daly 2009; Bauermeister et al.2011  Deinococcus geothermalis  Oxic  +  +  Ferreira et al.1997; Frösler et al.2017  Escherichia coli  Oxic  –  –  Clavero et al.1994; Welsh and Herbert 1999  Geodermatophilus poikilotrophi  Oxic  +  +  Montero-Calasanz et al.2014  Halobacterium salinarum  Oxic  +a  +  Kottemann et al.2005; Leuko and Rettberg 2017  Halococcus hamelinensis  Oxic  +  +  Leuko and Rettberg 2017  Halococcus morrhuae  Oxic  +  +  Leuko and Rettberg 2017  Halomonas sp.  Oxic  +  +  Musilova et al.2015  Kocuria polaris  Oxic  –  +  Shirsalimian et al.2016  Listeria monocytogenes  Oxic  +a  –  Niemira et al.2003; Hingston et al.2013  Methylobacterium extorquens  Oxic  +  +  Romanovskaya et al.2002  Rhodococcus sp.  Oxic  –  +  Dartnell et al.2010; Musilova et al.2015  Salmonella typhimurium  Oxic  +a  +  Thayer and Boyd 1991; Li et al.2012  Yersinia pestis  Oxic  –  –  Rose et al.2003; Sommers and Cooke 2009  a Survival was tested at a maximum of 20 days of desiccation. bSurvival was tested after exposure to 2.5 kGy. cSurvival was tested after exposure to 5 kGy. View Large All combinations of tolerance to desiccation and to ionizing radiation were prevalent within the MASE strains (Tables 2 and 3). Yersinia intermedia MASE-LG-1 and Acidiphilium sp. MASE-PM showed tolerance to desiccation, but not to ionizing radiation. Halanaerobium sp. MASE-BB-1 and Buttiauxella sp. MASE-IM-9 showed tolerances to both treatments (28 days desiccation; 3 kGy); Trichococcus sp. MASE-IM-5 and Clostridium sp. MASE-IM-4 did not survive either 4 weeks of desiccation, or exposure to 3 kGy of ionizing radiation. In general, there is a trend of correlation between tolerance to desiccation and to ionizing radiation; 50% of the organisms possessed the ability to survive both stresses. However, 33% of organisms showed no correlation and can outlast only one of the applied stresses. Additionally, 17% could not survive even one stress (Table 3). Most importantly, as visible in the rankings of the investigated organisms, the idea that ionizing radiation resistance is caused by desiccation resistance is not supported since some strains have high ionizing radiation resistance, but not desiccation resistance and vice versa. Table 3. Distribution of resistance to desiccation and ionizing radiation amongst the investigated organisms from Table 2. Desiccation (28 days)  Radiation (3 kGy)  Percentage  –  –  17%  –  +  22%  +  –  11%  +  +  50%  Desiccation (28 days)  Radiation (3 kGy)  Percentage  –  –  17%  –  +  22%  +  –  11%  +  +  50%  View Large DISCUSSION Tolerance of vegetative cells to desiccation and to ionizing radiation seems to be a common phenomenon present in all domains in the tree of life including Bacteria and Archaea (Potts 1994; DiRuggiero et al.1997; Beblo et al.2009, 2011; Confalonieri and Sommer 2011). However, the response of anaerobic microorganisms to different extremes, such as drought and radiation, is still poorly understood and a systematic comparison of survival cannot be made due to the different experimental setups. In this study, the survival of six facultative or obligate anaerobic microorganisms after exposure to desiccation and to ionizing radiation under anoxic conditions was examined and compared to data in the literature. The tolerance to desiccation and to radiation of the tested microorganisms was found to vary substantially. These variations were found within genera as in the case of the genus Yersinia, indicating that both tolerances are a species-specific feature. Several previous works reported on the distribution of desiccation and radiation tolerance within phylogenetically diverse microorganisms (Thomas et al.2006; LaDuc et al.2007; Musilova et al.2015). Especially, La Duc and colleagues (2007) showed that there is no general correlation between short desiccation periods up to 7 days and radiation tolerance: 34 strains withstood desiccation, but surprisingly none of these strains tested strains survived a treatment of 5 kGy. Yersinia intermedia MASE-LG-1 tested here was highly desiccation tolerant which is in contrast with the desiccation-sensitive strain Y. pestis which is not able to survive desiccation on glass for 24 h (Rose et al.2003). Similar species-specific specificities have been shown for the aerobic deinococcal radiation-sensitive representatives (Callegan et al.2008) It has been postulated that tolerance to desiccation is correlated with tolerance to radiation, since desiccation would select for repair capabilities that serendipitously allow for radiation tolerance, even though these organisms do not grow in naturally high-radiation environments (e.g. Mattimore and Battista 1996). Our data allow us to compare the desiccation and radiation tolerance of our selected anoxically grown isolates, and thus to investigate whether the postulated correlation is true for all organisms. There are different theories which try to explain the presumed correlation or relationship between tolerance to desiccation and radiotolerance. There are some reasons which are associated with the natural environment and with the cell as a whole like (i) habitat and (ii) cell aggregates and biofilms. Additionally, there are some intracellular factors which play a role inside the cells like (iii) specific enzymes and (iv) compatible solutes. Nevertheless, if other factors like a toroidal genome structure (Levin-Zaidman et al.2003; Cox and Battista 2005), or the intracellular ion content (Daly et al.2004; Daly 2009) as it is described for D. radiodurans play a role in the investigated strains remains speculative. Habitat One explanation is based on the assumption that the organisms’ original habitat influences their tolerance against stressful conditions. This has been observed for microorganisms that grow in dry habitats like deserts or highly saline areas, such as various deinococci, Chroococcidiopsis and some haloarchaea (Caiola, Billi and Friedmann 1996; Billi et al.2000; Stan-Lotter and Fendrihan 2015). Additionally, different microbial strains were isolated around Chernobyl. Those strains are able to tolerate better the exposure to different doses of radiation than bacterial communities from other sites with lower background radiation levels (Ruiz-González et al.2016). However, some deep-sea organisms such as Archaeoglobus fulgidus and Aquifex pyrophilus also show correlation between radiation and desiccation resistance (Stetter 1988; Huber et al.1992; Beblo et al.2009, 2011). The same correlation was shown in this work for Buttiauxella sp. MASE-IM-9 and Halanerobium sp. MASE-BB-1. For both organisms, it is unlikely that they experience desiccation or high levels of radiation in their natural (aqueous) environment (Cockell et al.2017). Furthermore, in the Boulby mine located 1100 m below ground the habitat of Halanaerobium sp. MASE-BB-1, the background radiation level was determined to be lower than on the surface (Malczewski, Kisiel and Dorda 2013). Cell aggregates and biofilm The capability of cells to form aggregates or to live in biofilms may also enable them to survive desiccation and radiation. Filament-forming cyanobacteria and tetrad-forming strains such as D. radiodurans and Chroococcidiopsis sp. are tolerant to desiccation (de Winder, Matthijs and Mur 1989; Jena et al.2006; Thomas et al.2006; Baqué et al.2013). It was hypothesized that cells attached to each other in a biofilm help each other during repair processes, for example with the exchange of genetic material (Cvitkovitch 2004). For D. geothermalis, Chroococcidiopsis sp. and two Streptococcus strains, it was shown that these microorganisms are able to form biofilms. As a part of a biofilm, they are more tolerant to cell damaging treatment compared to planktonic cells (Baqué et al.2013; Marks, Reddinger and Hakansson 2014; Frösler et al.2017). In our case, all MASE isolates grow as single cells under applied optimal growth conditions. At suboptimal growth conditions Halanaerobium sp. MASE-BB-1, and at high-sulfate concentrations Y. intermedia MASE-LG-1 grow in chains (Schwendner et al. 2018). The first, Halanaerobium sp. MASE-BB-1, is able to survive desiccation and ionizing radiation. In contrast, Y. intermedia MASE-LG-1 was the only strain to show a tolerance to long-term desiccation being able to survive up to half a year while being sensitive to radiation. Specific enzymes There are additional factors to expect a correlation between desiccation and radiation tolerance especially in (facultative) anaerobic microorganisms. The strains tested here were facultative and obligate anaerobes, and some of the other listed representatives are strictly anaerobes and consequently oxygen-sensitive. During and after desiccation and exposure to ionizing radiation, ROS production has been demonstrated (Jones et al.1994; França, Panek and Eleutherio 2007). The capacity of these anaerobic strains to effectively protect their intracellular components and to eliminate ROS is of crucial importance for their survival. One strategy is the elimination of ROS by the superoxide dismutase or the superoxide reductase (Cannio et al.2000). This enzymatic system produces H2O2 which is later eliminated by peroxidases, catalases or hydroperoxide reductases (Seaver and Imlay 2001). Nevertheless, not all facultative or obligate anaerobic strains were tolerant to desiccation and ionizing irradiation and a general protection by superoxide dismutase/reductase system can be neglected. Compatible solutes Water loss and high salinity have similar effects on a microbial cell. To counteract osmotic stress, several microorganisms take up or produce intracellular compatible solutes or follow the salt-in strategy which is most commonly detected in halophiles (Galinski 1995; Kempf and Bremer 1998). It has been shown that compatible solutes, due to their radical scavenging capacity, their ability to stabilize proteins and membranes, positively influence the desiccation tolerance of microorganisms but the desiccation itself is not inducing compatible solute accumulation (Smirnoff and Cumbes 1989; Lippert and Galinski 1992; Hincha and Hagemann 2004). Recently, it has been reported that the response of Y. intermedia MASE-LG-1 to salt stress (e.g. NaCl) involves an accumulation of L-asparagine and sucrose which might be one explanation for its tolerance to desiccation (Schwendner et al. 2017). For Halanaerobium praevalens, a close relative to the MASE strain Halanaerobium sp. MASE-BB-1, it was shown that the organisms is using the salt-in strategy and KCl is accumulated to respond to changes in the osmotic balance (Oren, Heldal and Norland 1997). A possible link between intracellular osmoadaptation compounds and microbial tolerance to ionizing radiation has also been discussed (Kish et al.2009; Webb and DiRuggiero 2012). Additionally, for the compatible solute ectoine, a protective influence on isolated DNA was shown (Hahn et al.2017). However, in Hydrogenothermus marinus and A. fulgidus only an enhanced desiccation tolerance but no improvement of the radiation tolerance due to cultivation at hyper optimal salinity (NaCl) has been observed (Beblo-Vranesevic et al.2017b). In Y. intermedia MASE-LG-1, L-asparagine and sucrose are produced due to high osmolality, but the radiation sensitivity was not altered. CONCLUSION Our data demonstrated that (facultative) anaerobes from extreme environments showed different response to desiccation and ionizing radiation. We did not observe an obvious correlation between desiccation and radiation tolerance, suggesting that although some of the biochemical basis behind desiccation and radiation tolerance, such as in the quenching of ROS, may be similar, the pathways determining desiccation and radiation tolerance in microorganisms are likely different to involve distinct biochemical pathways. Indeed, the diversity of possible responses that microorganisms can deploy to cope with these extremes may explain why high tolerance to one stress does not imply high tolerance to the other. Although the matter remains open as to whether desiccation stress can select for high radiation stress in some organisms, our data showed that microorganisms can possess tolerance to ionizing radiation and yet be sensitive to desiccation stress. Further work to elucidate the pathways of ionizing and radiation stress in microorganisms is merited. FUNDING This work was supported by European Community's Seventh Framework Program (FP7/2007-2013) [grant agreement no. 607297]. Conflict of interest. None declared. REFERENCES Anderson KL, Apolinario EE, Sowers KR. Desiccation as a long-term survival mechanism for the archaeon Methanosarcina barkeri . Appl Environ Microb  2012; 78: 1473– 9. Google Scholar CrossRef Search ADS   Battista JR, Earl AM, Park MJ. 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Abstract

Abstract Four facultative anaerobic and two obligate anaerobic bacteria were isolated from extreme environments (deep subsurface halite mine, sulfidic anoxic spring, mineral-rich river) in the frame MASE (Mars Analogues for Space Exploration) project. The isolates were investigated under anoxic conditions for their survivability after desiccation up to 6 months and their tolerance to ionizing radiation up to 3000 Gy. The results indicated that tolerances to both stresses are strain-specific features. Yersinia intermedia MASE-LG-1 showed a high desiccation tolerance but its radiation tolerance was very low. The most radiation-tolerant strains were Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1. In both cases, cultivable cells were detectable after an exposure to 3 kGy of ionizing radiation, but cells only survived desiccation for 90 and 30 days, respectively. Although a correlation between desiccation and ionizing radiation resistance has been hypothesized for some aerobic microorganisms, our data showed that there was no correlation between tolerance to desiccation and ionizing radiation, suggesting that the physiological basis of both forms of tolerances is not necessarily linked. In addition, these results indicated that facultative and obligate anaerobic organisms living in extreme environments possess varied species-specific tolerances to extremes. correlation, desiccation, radiation, survival, anaerobes, extreme environment INTRODUCTION Anaerobic microorganisms are widely distributed in Earth's extreme environments, yet we still know little about their physiology and their capacity to adapt to extreme conditions. In particular, there is a paucity of studies whereby different anaerobic microorganisms from extreme environments are investigated to understand their diverse physiological and metabolic capabilities. In this study, the two stressors of interest were the tolerance to periods of water loss and the exposure to ionizing radiation. These stressors also occur in other extreme environments but their combination is rare. Microorganisms frequently experience periodic desiccation in subaerial environments or during dispersal. Although most natural environments do not experience ionizing radiation beyond the level of naturally occurring background radiation (Thorne 2003), this stress can be explored as a proxy for an organisms’ ability to repair general cell damage. Furthermore, there has often been a claimed correlation between desiccation and ionizing radiation resistance, which is of interest to explore further. It is suggested that the physiological basis and repair mechanisms to counteract the stress-induced damage by radiation or desiccation might be linked or might even be the same (Mattimore and Battista 1996). While there are several studies investigating the survivability of model microorganisms, such as Escherichia coli and Deinococcus radiodurans, after desiccation and after exposure to ionizing radiation tested under oxic conditions (Clavero et al.1994; Welsh and Herbert 1999; Daly 2009; Bauermeister et al.2011), there are only a few examples where facultative and strict anaerobic microorganisms were tested against these stressors under anoxic conditions. The survival capacity after exposure to one stressor in form of ionizing radiation was described for the hyperthermophilic anaerobic microorganisms, Pyrococcus furiosus and Thermococcus gammatolerans (DiRuggiero et al.1997; Jolivet et al.2003). However, there are only very few studies about the tolerance in terms of sensitivity of anaerobic microorganisms to desiccation (Fetzer, Bak and Conrad 1993; Beblo et al.2009). The application of both of these stressors gives insights into the abilities that microorganisms have evolved to survive damage to macromolecules such as proteins, membranes and nucleic acids. During desiccation, initially the free intracellular water and subsequently the hydration shell of different molecules disappear, consequently affecting cellular components in their functions. For example, protein denaturation and perturbation of the lipid membrane by phase changes, e.g. structural conversion of bilayer sheets to spherical micelles, might appear (Cox 1993; Prestrelski et al.1993; Billi and Potts 2002). The DNA is also affected through loss of water; DNA protein cross-links and strand breaks can occur (Bieger-Dose et al.1992; Dose et al.1992). Additionally, the DNA can change from the B to the A-conformation resulting in DNA single- and double-strand breaks. Dehydration for 6 weeks caused approximately 60 DNA double-strand breaks per genome in D. radiodurans (Fredrickson et al.2008). Nevertheless, the survival of the microorganisms after 6 weeks of desiccation was not even reduced by one order of magnitude (Mattimore and Battista 1996). Another form of damaging stress that occurs during desiccation is the formation of reactive oxygen species (ROS), which, in turn, leads to oxidative stress (França, Panek and Eleutherio 2007). The ROS are mainly superoxide anions (•O2−) and hydroxyl radicals (•OH) affecting all macromolecular cellular components (Cabiscol, Tamarit and Ros 2000). However, the exact origin of the radicals during rehydration is unknown, but a possible source could be the metabolism itself (e.g. respiratory chain) when the cells were growing aerobically (González-Flecha and Demple 1995; França, Panek and Eleutherio 2007).They are also formed by indirect effects through the radiation-induced radiolysis of intracellular water and surrounding water and account for approximately 80% of introduced DNA damages (Michaels and Hunt 1978; Jones et al.1994; Riley 1994). After exposure to ionizing radiation of 5 kGy, approximately 200 double-strand breaks were detected in D. radiodurans (Cox and Battista 2005). However, the survivability of D. radiodurans was not reduced (Battista, Earl and Park 1999). In addition, ionizing radiation also affects proteins through oxidation processes, lipids through lipid peroxidation and disturbance of membrane permeability in eukaryotic and prokaryotic systems (Leyko and Bartosz 1985; Daly et al.2007; Krisko and Radman 2010). MASE (Mars Analogues for Space Exploration), a project funded by the European Union, was initiated to investigate anaerobic microorganisms in terrestrial extreme environments and their physiological adaptations to these extreme environmental conditions (Cockell et al.2017). In this article, we describe the survivability after prolonged desiccation of up to 4 weeks and exposure to ionizing radiation (up to 3 kGy) of four facultative anaerobic and two strict anaerobic microorganisms isolated from different extreme environments in the frame of this project. MATERIALS AND METHODS Strains and culture conditions During the MASE project, over 30 pure cultures were obtained from various extreme environments (Cockell et al.2017). From this list, six distantly related bacterial strains were picked for further analysis. The following microorganisms, namely Acidiphilium sp. PM (DSM 24941), Buttiauxella sp. MASE-IM-9 (DSM 105071), Clostridium sp. MASE-IM-4 (DSM 105631), Halanaerobium sp. MASE-BB-1 (DSM 105537), Trichococcus sp. IM-5 (DSM 105632) and Yersinia intermedia MASE-LG-1 (DSM 102845). Media and strain-specific cultivation conditions are summarized in Table 1 and described in detail in Cockell et al. (2017). The incubation was carried out at the indicated cultivation temperature, and cultures were shaken at 50 rpm. Noteworthy, for Clostridium sp. MASE-IM-4 only vegetative cells have been observed during the applied cultivation condition. Table 1. Strains, origins and cultivation conditions.           Temperature  Strain Phylum Class  Origin  Medium  Supplements (wt/vol)  Gas phase (vol/vol)  (°C)  Acidiphilium sp. PM Proteobacteria Alphaproteiobacteria  River Rio Tinto, Spain  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30  Buttiauxella sp. MASE-IM-9, Proteobacteria Gammaproteobacteria  Islinger Mühlbach, Germany  MASE-II  0.1% Yeast extract  80% N2, 20% CO2  30  Clostridium sp. MASE-IM-4 Firmicutes Clostridia  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Dimethylamine 0.001% FeCl2  15% H2, 25% CO2, 60% N2  30  Halanaerobium sp. MASE-BB-1 Proteobacteria Gammaproteobacteria  Boulby Mine, Great Britain  HACE  0.1% Yeast extract  15% H2, 25% CO2, 60% N2  45  Trichococcus sp. MASE-IM-5 Firmicutes, Bacilli  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Na2SO4 0.01% C6H5Na3O7 × 2 H2O 0.02% KNO3  15% H2, 25% CO2, 60% N2  30  Y. intermedia MASE- LG-1 Proteobacteria Gammaproteobacteria  Lake Grænavatn, Iceland  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30            Temperature  Strain Phylum Class  Origin  Medium  Supplements (wt/vol)  Gas phase (vol/vol)  (°C)  Acidiphilium sp. PM Proteobacteria Alphaproteiobacteria  River Rio Tinto, Spain  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30  Buttiauxella sp. MASE-IM-9, Proteobacteria Gammaproteobacteria  Islinger Mühlbach, Germany  MASE-II  0.1% Yeast extract  80% N2, 20% CO2  30  Clostridium sp. MASE-IM-4 Firmicutes Clostridia  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Dimethylamine 0.001% FeCl2  15% H2, 25% CO2, 60% N2  30  Halanaerobium sp. MASE-BB-1 Proteobacteria Gammaproteobacteria  Boulby Mine, Great Britain  HACE  0.1% Yeast extract  15% H2, 25% CO2, 60% N2  45  Trichococcus sp. MASE-IM-5 Firmicutes, Bacilli  Islinger Mühlbach, Germany  MASE-II—FeCl2  0.01% Na2SO4 0.01% C6H5Na3O7 × 2 H2O 0.02% KNO3  15% H2, 25% CO2, 60% N2  30  Y. intermedia MASE- LG-1 Proteobacteria Gammaproteobacteria  Lake Grænavatn, Iceland  MASE-I  0.01% KNO3 0.01% C-Org-Mix  80% N2, 20% CO2  30  View Large Desiccation and irradiation experiments For the desiccation experiments, the cells were cultivated under optimal growth conditions until stationary growth phase was reached. Desiccation experiments were performed as described by Beblo et al. (2009). Briefly, cell concentrations were determined by counting in a Thoma chamber. One milliliter of cell culture (cell densities ranged from ∼5 × 106 cells/ml to ∼5 × 107 cells/ml) was spread evenly on four glass slides and dried under anoxic conditions in an anaerobic chamber (Coy Laboratory Products Inc., [O2] < 0.0001%, relative humidity 13 ± 0.5%; both in vol/vol) in the presence of drying agent calcium chloride. Afterwards, the dried cells were stored within the anaerobic chamber. Exposure to ionizing radiation was carried out according to earlier studies (Beblo et al.2011). Stationary phase cell cultures, in liquid suspensions, were transferred anoxically into 7 ml glass HPLC vials (WICOM Germany GmbH), which were tightly sealed with rubber stoppers and aluminum caps. Irradiation was conducted with an X-ray source Gulmay RS 225A (Gulmay Medical Ltd) at 200 kV and 15 mA. Cells were irradiated at a distance of 19.5 cm below the X-ray source with 20 Gy min−1 ± 5 Gy min−1 up to 3 kGy. The dose rate was measured with a UNIDOS dosimeter (PTW Freiburg GmbH). All irradiation experiments were performed under anoxic conditions at room temperature. Determination of the survival At dedicated time points, the dried cells on glass slides or the irradiated cells as a liquid suspension were transferred under anoxic conditions into the strain-specific culture medium and incubated for up to 4 weeks (Table 1, for description of the procedure see Beblo et al.2009). Growth of the cells in all dilutions was observed visually and by phase-contrast microscopy (Zeiss® Axiolmager TM M2) with 400× or 1000× magnification. Determination of the survival and enumeration of cultivable cells was achieved by the most probable number (MPN) technique via dilution series with 10-fold dilution steps (Franson 1985). The MPN technique was applied for all six strains, since not all strains are able to grow on solid surfaces. All experiments were repeated independently at least three times, representing biological replicates. The data shown within graphs represent mean values with standard deviations. The survival (S) was calculated as relative survival after cell damaging treatment (N) compared to the non-treated control (N0) (S = N/N0). Due to the applied MPN technique and depending on the growth density of the specific strain (∼5 × 106 cells/ml to ∼5 × 107 cells/ml), the detection limit of the determination of survival was ∼1 × 10−8. RESULTS The six vegetative strains Acidiphilium sp. PM, Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Y. intermedia MASE-LG-1 showed different levels of survival after desiccation and after exposure to radiation (Fig. 1). Figure 1. View largeDownload slide Survival of the MASE isolates after anoxic desiccation (A–F) and after exposure to ionizing radiation under anoxic conditions (G–L). For desiccation experiments, the cells were applied to glass slides, dried and stored under anoxic conditions up to 184 days. For anoxic irradiation experiments, the cells were exposed to ionizing radiation up to 3 kGy in liquid culture medium under anoxic conditions. Acidiphilium sp. PM (A, G), Buttiauxella sp. MASE-IM-9 (B, H), Clostridium sp. MASE-IM-4 (C, I), Halanaerobium sp. MASE-BB-1 (D, J), Trichococcus sp. MASE-IM-5 (E, K), Y. intermedia MASE-LG-1 (F, L). Solid lines are the survival curves fitted by hand based on the survival data; N0: viable cells without desiccation or without irradiation; N: viable cells after desiccation or without irradiation (n = 3 with standard deviation); *no viable cells detected. Figure 1. View largeDownload slide Survival of the MASE isolates after anoxic desiccation (A–F) and after exposure to ionizing radiation under anoxic conditions (G–L). For desiccation experiments, the cells were applied to glass slides, dried and stored under anoxic conditions up to 184 days. For anoxic irradiation experiments, the cells were exposed to ionizing radiation up to 3 kGy in liquid culture medium under anoxic conditions. Acidiphilium sp. PM (A, G), Buttiauxella sp. MASE-IM-9 (B, H), Clostridium sp. MASE-IM-4 (C, I), Halanaerobium sp. MASE-BB-1 (D, J), Trichococcus sp. MASE-IM-5 (E, K), Y. intermedia MASE-LG-1 (F, L). Solid lines are the survival curves fitted by hand based on the survival data; N0: viable cells without desiccation or without irradiation; N: viable cells after desiccation or without irradiation (n = 3 with standard deviation); *no viable cells detected. Tolerance to desiccation The survival curves of all tested organisms showed an exponential decay as described by Chen and Alexander (1973). Thereby, the survival rate decreased substantially within the first days of desiccation and the survival decreased until it plateaued. Only Y. intermedia MASE-LG-1 was able to survive the maximum tested time period of desiccation (184 days). After 184 days, the survival of this organism was S (184 days) = 3.7 × 10−5 (Beblo-Vranesevic et al. 2017a). In contrast to this high tolerance to water loss, Clostridium sp. MASE-IM-4, Trichococcus sp. MASE-IM-5 and Halanaerobium sp. MASE-BB-1 were more sensitive to desiccation. After 4 weeks in a dry state, no living cells of Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5 were detectable (Fig. 1C and E). The survival of Halanaerobium sp. MASE-BB-1 after 4 weeks of desiccation was reduced by more than four orders of magnitude (Fig. 1D) to S (28 days) = 3.7 × 10−5 and no living cells were found after 56 days of anoxic storage in a dry state. The survival of Acidiphilium sp. PM and Buttiauxella sp. MASE-IM-9 after 4 weeks were S (28 days) = 7.0 × 10−7 and S (28 days) = 3.7 × 10−5 respectively, and these strains could outlast 84 days in a desiccated form (Fig. 1A and B). All these results summarized, the organisms can be ranked in terms of their tolerance to desiccation: Clostridium sp. MASE-IM-4 < Trichococcus sp. MASE-IM-5 < Halanaerobium sp. MASE-BB-1 < Acidiphilium sp. PM < Buttiauxella sp. MASE-IM-9 < Y. intermedia MASE-LG-1. Tolerance to ionizing radiation Survival after exposure to ionizing radiation of the tested MASE isolates varied greatly between isolates. Different types of curves were obtained and not all organisms showed the typically shouldered survival curve as it is described by Kiefer (1990). Only two organisms (Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1; Fig. 1H and J) were able to multiply after an exposure to 3 kGy (S (Buttiauxella sp. MASE-IM-9, 3 kGy) = 3.7 × 10−7; S (Halanaerobium sp. BB-1, 3 kGy) = 4 × 10−6). For Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5, the highest dose at which culturable cells were detected after reinoculation was 2.5 kGy (Fig. 1I and K). Acidiphilium sp. PM was more sensitive to ionizing radiation and only survived exposures up to 1.5 kGy (Fig. 1G). The most sensitive organism amongst this tested group of microorganisms was Y. intermedia MASE-LG-1. Multiplying cells were only observed to a dose of 0.8 kGy (Fig. 1L). Concluding these data, the tolerance to an exposure to ionizing radiation can be ranked as follows: Y. intermedia MASE-LG-1 < Acidiphilium sp. PM < Clostridium sp. MASE-IM-4 < Trichococcus sp. MASE-IM-5 < Buttiauxella sp. MASE-IM-9 < Halanaerobium sp. MASE-BB-1. Survival data for various microorganisms from literature data Table 2 lists organisms from other studies and their desiccation and radiation tolerance in which both parameters have been measured for a given organism. For comparability with previous studies, desiccation tolerance was defined as the tolerance to survive four weeks (28 days) in a dried state at room temperature and if applicable under anoxic conditions. Radiotolerance was defined as the ability to survive an exposure to ionizing radiation with a dose of 3 kGy. Additionally, there are many microorganisms for which only one treatment was tested and the list could be enlarged tremendously (e.g. Jolivet et al.2003; Walsh and Camilli 2011). Table 2. Overview of microbial survival after desiccation (28 days) and ionizing radiation (3 kGy) from literature data. Organism  Oxidative state of the experimental setup  Desiccation (28 days)  Radiation (3 kGy)  Reference  Acidiphilium sp. PM  Anoxic  +  –  In this study  Buttiauxella sp. MASE-IM-10  Anoxic  +  +  In this study  Clostridium sp. MASE-IM-4  Anoxic  –  –  In this study  Halanaerobium sp. MASE-BB-1  Anoxic  +  +  In this study  Trichococcus sp. MASE-IM-5  Anoxic  –  –  In this study  Yersinia intermedia MASE-LG-1  Anoxic  +  –  In this study  Archaeoglobus fulgidus  Anoxic  +  +  Beblo et al.2009; Beblo et al.2011  Ignicoccus hospitalis  Anoxic  –  +  Beblo et al.2009; Koschnitzki 2016  Methanocaldococcus jannaschii  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Methanosarcina barkeri  Anoxic  +  +b  Morozova and Wagner 2007; Anderson, Apolinario and Sowers 2012  Methanothermobacter thermoautotrophicus  Anoxic  +  –c  Beblo et al.2009; Beblo et al.2011  Nanoarchaeum equitans  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Pyrococcus furiosus  Anoxic  –  +  DiRuggiero et al.1997; Beblo et al.2009  Thermoproteus tenax  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Thermofilum pendens  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Aquifex pyrophilus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Hydrogenothermus marinus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Metallosphaera sedula  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Sulfolobus metallicus  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Acinetobacter radioresistens  Oxic  +  +  Nishimura, Ino and Iizuka 1988; Jawad et al.1998  Brevundimonas sp.  Oxic  +  +  Dartnell et al.2010; Musilova et al.2015  Chroococcidiopsis sp.  Oxic  +  +  Caiola et al.1996; Billi et al.2000  Deinococcus radiodurans  Oxic  +  +  Daly 2009; Bauermeister et al.2011  Deinococcus geothermalis  Oxic  +  +  Ferreira et al.1997; Frösler et al.2017  Escherichia coli  Oxic  –  –  Clavero et al.1994; Welsh and Herbert 1999  Geodermatophilus poikilotrophi  Oxic  +  +  Montero-Calasanz et al.2014  Halobacterium salinarum  Oxic  +a  +  Kottemann et al.2005; Leuko and Rettberg 2017  Halococcus hamelinensis  Oxic  +  +  Leuko and Rettberg 2017  Halococcus morrhuae  Oxic  +  +  Leuko and Rettberg 2017  Halomonas sp.  Oxic  +  +  Musilova et al.2015  Kocuria polaris  Oxic  –  +  Shirsalimian et al.2016  Listeria monocytogenes  Oxic  +a  –  Niemira et al.2003; Hingston et al.2013  Methylobacterium extorquens  Oxic  +  +  Romanovskaya et al.2002  Rhodococcus sp.  Oxic  –  +  Dartnell et al.2010; Musilova et al.2015  Salmonella typhimurium  Oxic  +a  +  Thayer and Boyd 1991; Li et al.2012  Yersinia pestis  Oxic  –  –  Rose et al.2003; Sommers and Cooke 2009  Organism  Oxidative state of the experimental setup  Desiccation (28 days)  Radiation (3 kGy)  Reference  Acidiphilium sp. PM  Anoxic  +  –  In this study  Buttiauxella sp. MASE-IM-10  Anoxic  +  +  In this study  Clostridium sp. MASE-IM-4  Anoxic  –  –  In this study  Halanaerobium sp. MASE-BB-1  Anoxic  +  +  In this study  Trichococcus sp. MASE-IM-5  Anoxic  –  –  In this study  Yersinia intermedia MASE-LG-1  Anoxic  +  –  In this study  Archaeoglobus fulgidus  Anoxic  +  +  Beblo et al.2009; Beblo et al.2011  Ignicoccus hospitalis  Anoxic  –  +  Beblo et al.2009; Koschnitzki 2016  Methanocaldococcus jannaschii  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Methanosarcina barkeri  Anoxic  +  +b  Morozova and Wagner 2007; Anderson, Apolinario and Sowers 2012  Methanothermobacter thermoautotrophicus  Anoxic  +  –c  Beblo et al.2009; Beblo et al.2011  Nanoarchaeum equitans  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Pyrococcus furiosus  Anoxic  –  +  DiRuggiero et al.1997; Beblo et al.2009  Thermoproteus tenax  Anoxic  –  +  Beblo et al.2009; Beblo et al.2011  Thermofilum pendens  Anoxic  –  –c  Beblo et al.2009; Beblo et al.2011  Aquifex pyrophilus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Hydrogenothermus marinus  Microoxic  +  +  Beblo et al.2009; Beblo et al.2011  Metallosphaera sedula  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Sulfolobus metallicus  Microoxic/oxic  –  +  Beblo et al.2009; Beblo et al.2011  Acinetobacter radioresistens  Oxic  +  +  Nishimura, Ino and Iizuka 1988; Jawad et al.1998  Brevundimonas sp.  Oxic  +  +  Dartnell et al.2010; Musilova et al.2015  Chroococcidiopsis sp.  Oxic  +  +  Caiola et al.1996; Billi et al.2000  Deinococcus radiodurans  Oxic  +  +  Daly 2009; Bauermeister et al.2011  Deinococcus geothermalis  Oxic  +  +  Ferreira et al.1997; Frösler et al.2017  Escherichia coli  Oxic  –  –  Clavero et al.1994; Welsh and Herbert 1999  Geodermatophilus poikilotrophi  Oxic  +  +  Montero-Calasanz et al.2014  Halobacterium salinarum  Oxic  +a  +  Kottemann et al.2005; Leuko and Rettberg 2017  Halococcus hamelinensis  Oxic  +  +  Leuko and Rettberg 2017  Halococcus morrhuae  Oxic  +  +  Leuko and Rettberg 2017  Halomonas sp.  Oxic  +  +  Musilova et al.2015  Kocuria polaris  Oxic  –  +  Shirsalimian et al.2016  Listeria monocytogenes  Oxic  +a  –  Niemira et al.2003; Hingston et al.2013  Methylobacterium extorquens  Oxic  +  +  Romanovskaya et al.2002  Rhodococcus sp.  Oxic  –  +  Dartnell et al.2010; Musilova et al.2015  Salmonella typhimurium  Oxic  +a  +  Thayer and Boyd 1991; Li et al.2012  Yersinia pestis  Oxic  –  –  Rose et al.2003; Sommers and Cooke 2009  a Survival was tested at a maximum of 20 days of desiccation. bSurvival was tested after exposure to 2.5 kGy. cSurvival was tested after exposure to 5 kGy. View Large All combinations of tolerance to desiccation and to ionizing radiation were prevalent within the MASE strains (Tables 2 and 3). Yersinia intermedia MASE-LG-1 and Acidiphilium sp. MASE-PM showed tolerance to desiccation, but not to ionizing radiation. Halanaerobium sp. MASE-BB-1 and Buttiauxella sp. MASE-IM-9 showed tolerances to both treatments (28 days desiccation; 3 kGy); Trichococcus sp. MASE-IM-5 and Clostridium sp. MASE-IM-4 did not survive either 4 weeks of desiccation, or exposure to 3 kGy of ionizing radiation. In general, there is a trend of correlation between tolerance to desiccation and to ionizing radiation; 50% of the organisms possessed the ability to survive both stresses. However, 33% of organisms showed no correlation and can outlast only one of the applied stresses. Additionally, 17% could not survive even one stress (Table 3). Most importantly, as visible in the rankings of the investigated organisms, the idea that ionizing radiation resistance is caused by desiccation resistance is not supported since some strains have high ionizing radiation resistance, but not desiccation resistance and vice versa. Table 3. Distribution of resistance to desiccation and ionizing radiation amongst the investigated organisms from Table 2. Desiccation (28 days)  Radiation (3 kGy)  Percentage  –  –  17%  –  +  22%  +  –  11%  +  +  50%  Desiccation (28 days)  Radiation (3 kGy)  Percentage  –  –  17%  –  +  22%  +  –  11%  +  +  50%  View Large DISCUSSION Tolerance of vegetative cells to desiccation and to ionizing radiation seems to be a common phenomenon present in all domains in the tree of life including Bacteria and Archaea (Potts 1994; DiRuggiero et al.1997; Beblo et al.2009, 2011; Confalonieri and Sommer 2011). However, the response of anaerobic microorganisms to different extremes, such as drought and radiation, is still poorly understood and a systematic comparison of survival cannot be made due to the different experimental setups. In this study, the survival of six facultative or obligate anaerobic microorganisms after exposure to desiccation and to ionizing radiation under anoxic conditions was examined and compared to data in the literature. The tolerance to desiccation and to radiation of the tested microorganisms was found to vary substantially. These variations were found within genera as in the case of the genus Yersinia, indicating that both tolerances are a species-specific feature. Several previous works reported on the distribution of desiccation and radiation tolerance within phylogenetically diverse microorganisms (Thomas et al.2006; LaDuc et al.2007; Musilova et al.2015). Especially, La Duc and colleagues (2007) showed that there is no general correlation between short desiccation periods up to 7 days and radiation tolerance: 34 strains withstood desiccation, but surprisingly none of these strains tested strains survived a treatment of 5 kGy. Yersinia intermedia MASE-LG-1 tested here was highly desiccation tolerant which is in contrast with the desiccation-sensitive strain Y. pestis which is not able to survive desiccation on glass for 24 h (Rose et al.2003). Similar species-specific specificities have been shown for the aerobic deinococcal radiation-sensitive representatives (Callegan et al.2008) It has been postulated that tolerance to desiccation is correlated with tolerance to radiation, since desiccation would select for repair capabilities that serendipitously allow for radiation tolerance, even though these organisms do not grow in naturally high-radiation environments (e.g. Mattimore and Battista 1996). Our data allow us to compare the desiccation and radiation tolerance of our selected anoxically grown isolates, and thus to investigate whether the postulated correlation is true for all organisms. There are different theories which try to explain the presumed correlation or relationship between tolerance to desiccation and radiotolerance. There are some reasons which are associated with the natural environment and with the cell as a whole like (i) habitat and (ii) cell aggregates and biofilms. Additionally, there are some intracellular factors which play a role inside the cells like (iii) specific enzymes and (iv) compatible solutes. Nevertheless, if other factors like a toroidal genome structure (Levin-Zaidman et al.2003; Cox and Battista 2005), or the intracellular ion content (Daly et al.2004; Daly 2009) as it is described for D. radiodurans play a role in the investigated strains remains speculative. Habitat One explanation is based on the assumption that the organisms’ original habitat influences their tolerance against stressful conditions. This has been observed for microorganisms that grow in dry habitats like deserts or highly saline areas, such as various deinococci, Chroococcidiopsis and some haloarchaea (Caiola, Billi and Friedmann 1996; Billi et al.2000; Stan-Lotter and Fendrihan 2015). Additionally, different microbial strains were isolated around Chernobyl. Those strains are able to tolerate better the exposure to different doses of radiation than bacterial communities from other sites with lower background radiation levels (Ruiz-González et al.2016). However, some deep-sea organisms such as Archaeoglobus fulgidus and Aquifex pyrophilus also show correlation between radiation and desiccation resistance (Stetter 1988; Huber et al.1992; Beblo et al.2009, 2011). The same correlation was shown in this work for Buttiauxella sp. MASE-IM-9 and Halanerobium sp. MASE-BB-1. For both organisms, it is unlikely that they experience desiccation or high levels of radiation in their natural (aqueous) environment (Cockell et al.2017). Furthermore, in the Boulby mine located 1100 m below ground the habitat of Halanaerobium sp. MASE-BB-1, the background radiation level was determined to be lower than on the surface (Malczewski, Kisiel and Dorda 2013). Cell aggregates and biofilm The capability of cells to form aggregates or to live in biofilms may also enable them to survive desiccation and radiation. Filament-forming cyanobacteria and tetrad-forming strains such as D. radiodurans and Chroococcidiopsis sp. are tolerant to desiccation (de Winder, Matthijs and Mur 1989; Jena et al.2006; Thomas et al.2006; Baqué et al.2013). It was hypothesized that cells attached to each other in a biofilm help each other during repair processes, for example with the exchange of genetic material (Cvitkovitch 2004). For D. geothermalis, Chroococcidiopsis sp. and two Streptococcus strains, it was shown that these microorganisms are able to form biofilms. As a part of a biofilm, they are more tolerant to cell damaging treatment compared to planktonic cells (Baqué et al.2013; Marks, Reddinger and Hakansson 2014; Frösler et al.2017). In our case, all MASE isolates grow as single cells under applied optimal growth conditions. At suboptimal growth conditions Halanaerobium sp. MASE-BB-1, and at high-sulfate concentrations Y. intermedia MASE-LG-1 grow in chains (Schwendner et al. 2018). The first, Halanaerobium sp. MASE-BB-1, is able to survive desiccation and ionizing radiation. In contrast, Y. intermedia MASE-LG-1 was the only strain to show a tolerance to long-term desiccation being able to survive up to half a year while being sensitive to radiation. Specific enzymes There are additional factors to expect a correlation between desiccation and radiation tolerance especially in (facultative) anaerobic microorganisms. The strains tested here were facultative and obligate anaerobes, and some of the other listed representatives are strictly anaerobes and consequently oxygen-sensitive. During and after desiccation and exposure to ionizing radiation, ROS production has been demonstrated (Jones et al.1994; França, Panek and Eleutherio 2007). The capacity of these anaerobic strains to effectively protect their intracellular components and to eliminate ROS is of crucial importance for their survival. One strategy is the elimination of ROS by the superoxide dismutase or the superoxide reductase (Cannio et al.2000). This enzymatic system produces H2O2 which is later eliminated by peroxidases, catalases or hydroperoxide reductases (Seaver and Imlay 2001). Nevertheless, not all facultative or obligate anaerobic strains were tolerant to desiccation and ionizing irradiation and a general protection by superoxide dismutase/reductase system can be neglected. Compatible solutes Water loss and high salinity have similar effects on a microbial cell. To counteract osmotic stress, several microorganisms take up or produce intracellular compatible solutes or follow the salt-in strategy which is most commonly detected in halophiles (Galinski 1995; Kempf and Bremer 1998). It has been shown that compatible solutes, due to their radical scavenging capacity, their ability to stabilize proteins and membranes, positively influence the desiccation tolerance of microorganisms but the desiccation itself is not inducing compatible solute accumulation (Smirnoff and Cumbes 1989; Lippert and Galinski 1992; Hincha and Hagemann 2004). Recently, it has been reported that the response of Y. intermedia MASE-LG-1 to salt stress (e.g. NaCl) involves an accumulation of L-asparagine and sucrose which might be one explanation for its tolerance to desiccation (Schwendner et al. 2017). For Halanaerobium praevalens, a close relative to the MASE strain Halanaerobium sp. MASE-BB-1, it was shown that the organisms is using the salt-in strategy and KCl is accumulated to respond to changes in the osmotic balance (Oren, Heldal and Norland 1997). A possible link between intracellular osmoadaptation compounds and microbial tolerance to ionizing radiation has also been discussed (Kish et al.2009; Webb and DiRuggiero 2012). Additionally, for the compatible solute ectoine, a protective influence on isolated DNA was shown (Hahn et al.2017). However, in Hydrogenothermus marinus and A. fulgidus only an enhanced desiccation tolerance but no improvement of the radiation tolerance due to cultivation at hyper optimal salinity (NaCl) has been observed (Beblo-Vranesevic et al.2017b). In Y. intermedia MASE-LG-1, L-asparagine and sucrose are produced due to high osmolality, but the radiation sensitivity was not altered. CONCLUSION Our data demonstrated that (facultative) anaerobes from extreme environments showed different response to desiccation and ionizing radiation. We did not observe an obvious correlation between desiccation and radiation tolerance, suggesting that although some of the biochemical basis behind desiccation and radiation tolerance, such as in the quenching of ROS, may be similar, the pathways determining desiccation and radiation tolerance in microorganisms are likely different to involve distinct biochemical pathways. 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