Bacillus Subtilis Spore Survival and Expression of Germination-Induced Bioluminescence After Prolonged Incubation Under Simulate

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ASTROBIOLOGY Volume 5, Number 4, 2005 © Mary Ann Liebert, Inc.

Research Paper

Bacillus subtilis Spore Survival and Expression of Germination-Induced Bioluminescence After Prolonged Incubation Under Simulated Mars Atmospheric Pressure and Composition: Implications for Planetary Protection and Lithopanspermia

WAYNE L. NICHOLSON1 and ANDREW C. SCHUERGER2

ABSTRACT

Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7–18 mbar) and composition (100% CO2) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes. Key Words: Bacillus subtilus spores—Germination-induced bioluminescence—Lithopanspermia—Simulated martian atmosphere. Astrobiology 5, 536–544.

INTRODUCTION 2005; Horneck et al., 2002; Schuerger, 2004, and references therein). Although a variety of mi- RISE OF INTEREST in the possibility of the sur- croorganisms have been described that could sur- Avival, growth, and metabolism of terrestrial vive the rigors of spaceflight (reviewed in Hor- bacteria in the Mars environment has resulted neck et al., 2002), spores of the gram-positive from astrobiological studies of (i) interplanetary bacterium Bacillus subtilis are considered the ex- transfer of microbes in impact ejecta and (ii) study perimental model of choice due to their hardi- of microbes as potential contaminants of Mars ness, reproducibility of their inactivation re- rovers and landers (reviewed extensively in sponse, stability, and ease of experimental Mileikowsky et al., 2000; Nicholson et al., 2000, manipulation (Nicholson et al., 2000).

Departments of 1Microbiology and Cell Science and 2Plant Pathology, University of Florida, Kennedy Space Center, Florida. This paper is Journal Series Paper R-10966 of the Florida Agriculture Experiment Station.

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Current interplanetary transfer models have ble to those present on the martian surface. How- calculated survival probabilities of spore popula- ever, spores buried under thin contiguous layers tions during launch, cruise phase through space, of dust (up to 500 m thick) were well protected and landing (Clark, 2001; Horneck et al., 2002; Mi- from solar UV and inactivated at a much slower liekowsky et al., 2000; Nicholson et al., 2000, 2005; rate (Schuerger et al., 2003). Schuerger, 2004). However, for successful Earth- From the above results it is clear that shielding to-Mars transport to be ecologically relevant, ter- from solar UV at the martian surface is a prereq- restrial microorganisms, once deposited on the uisite for long-term survival of spores. However, surface of Mars, would have to survive and pro- even spores shielded from UV would still be liferate in the martian environment. This possi- exposed to potentially lethal germination- or bility has received serious consideration for growth-inhibiting low temperatures, pressures, decades; the major spacefaring nations operate and desiccation. How do these Mars parameters under planetary protection protocols, originally affect the ability of spores to survive, germinate established under the auspices of COSPAR and and resume vegetative growth, even if conditions international treaties that require spacecraft des- become favorable at a later time? To answer this tined to make contact with Mars (or other poten- question, we took advantage of a B. subtilis strain tially habitable sites) to be constructed and that encodes a fusion of the luxAB genes encod- assembled in ultra-clean spacecraft assembly fa- ing luciferase to the strong sporulation-specific cilities (SAFs) under stringent hygienic condi- sspB promoter (Hill et al., 1994; Ciarciaglini et al., tions that, as nearly as possible, approach steril- 2000). During sporulation, the sspB-luxAB fusion ity (Rummel, 2001). Interestingly, the extreme strain produces luciferase in the developing fore- environments of SAFs, characterized by very low spore compartment and retains it within the dor- nutrient levels, strictly controlled humidity, and mant spores, where it is inactive as spores pos- periodic disinfection, appear to be excellent se- sess no detectable metabolism, ATP, or electron lective environments for Bacillus spp. whose transport (Hill et al., 1994). Initiation of spore me- spores exhibit ultrahigh resistance to various tabolism, and thus luciferase activity, upon ger- physical insults such as desiccation, oxidizing mination results in a burst of bioluminescence agents, ultraviolet (UV), and -radiation (La Duc that can be used as a real-time monitor of spore et al., 2003; Venkateswaran et al., 2003b; Link et germination (Hill et al., 1994; Ciarciaglini et al., al., 2004). Although the fate of such highly resis- 2000). In this communication we tested the abil- tant potential spacecraft contaminants during ity of B. subtilis spores carrying an sspB-luxAB simulated Mars missions has been considered gene fusion to survive exposure to Mars-ambient on a theoretical basis (Nicholson et al., 2005; atmospheric pressure and composition, and the Schuerger, 2004), the potential for spore survival possible effects of this exposure on subsequent and proliferation on Mars has only recently been spore germination. the subject of experimental research. Environ- mental conditions at the surface of Mars are characterized by high solar UV fluences, low atmos- MATERIALS AND METHODS pheric pressure, extreme cold temperatures, and extreme desiccation (reviewed in Schuerger, B. subtilis strain and cultivation conditions 2004; Nicholson et al., 2005). To contaminate the martian environment, spores would not only The B. subtilis strain used was PS832, a Trp have to survive these conditions, but also germi- revertant of strain 168, carrying a cassette con- nate and grow vegetatively. Using a Mars simu- sisting of an sspB-luxAB fusion and markers lation chamber (MSC), Schuerger et al. (2003) conferring resistance to macrolide-lincosamide- modeled the survival of B. subtilis spores exposed streptogramin B antibiotics and chloramphenicol to robustly simulated martian environmental (Cm), inserted in a single copy in the chromo- conditions of solar irradiation, temperature, at- some. Details of its construction have been pub- mospheric composition and pressure, and desic- lished elsewhere (Jacobs et al., 1991; Hill et al., cation, and found that solar UV was the single 1994). The strain, a generous gift from P. Setlow, most lethal environmental factor; spores were was deposited in the corresponding author’s col- rapidly inactivated (within minutes) when ex- lection as strain WN648. Spores were produced posed to a UV spectrum and fluences compara- by incubation of strain WN648 in liquid Schaef- 5765_07_p536-544 7/25/05 11:18 AM Page 538

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fer Sporulation Medium (SSM) (Schaeffer et al., aliquot of 10 l was removed from each sample 1965) containing 5 g of Cm/ml (SSMCm) at for viability assay, and the remaining 90 l was 37°C for 48 h in a rotary shaker with vigorous used for germination bioluminescence assay. aeration. Cultures were harvested by centrifuga- tion (10,000 g, 10 min, 25°C), and spores were pu- Viability assay rified using the lysozyme and buffer washing Spore samples were diluted serially 1:10 in method (Nicholson and Setlow, 1990), heat- PBS, aliquots were plated on SSMCm and in- shocked (80°C, 10 min), and stored in deionized cubated for 18–24 h at 37°C, colonies were water at 4°C. All spore preparations were deter- counted, and spore titer was calculated. Further mined by phase-contrast microscopy to be essen- incubation of plates for 2 days yielded no further tially free of vegetative cells and consist of increase in colony-forming units (cfu). 99.9% phase-bright spores. The viable titer of all spore preparations was determined by serial 10-fold dilution in phosphate-buffered saline Germination bioluminescence assay (PBS) (10 mM potassium phosphate and 150 mM To each well of a 96-well microtiter plate con- NaCl, pH 7.4) and plating on SSMCm solidified taining 90 l of spores was added 100 l of liq- with 1.7% agar. uid SSM containing a final concentration of 10 mM L-alanine and 0.003% (vol/vol) n-decanal, Exposure of spores to Earth and simulated freshly prepared for each experiment. Microtiter Mars atmosphere plates were placed in a luminometer (Harta In- struments, Gaithersburg, MD), and at the indi- Spores (1 108 in 125 l of water) were cated times readings were taken in “glow” mode pipetted into several wells of 96-well sterile poly- with a dwell time of 1 s. Data are expressed in styrene microtiter plates and air-dried overnight terms of arbitrary relative light units (RLU). Cu- at 37°C. One microtiter plate was covered loosely mulative bioluminescence was determined by and placed in the laboratory at Earth-normal at- computing a running total of RLU measurements mosphere, ambient temperature (20°C), relative during the course of germination of each sample. humidity (60%), and pressure. Daily barometric pressure was taken from the weather data col- Statistical treatment of data lected at the nearby Cape Canaveral Weather Sta- tion. The other plate was covered loosely, placed All viability and germination bioluminescence inside the MSC, and equilibrated to simulated assays were performed on triplicate samples. Basic statistical data and analysis of variance Mars atmosphere (100% CO2) at 20°C and Mars-normal pressure (average 12.5 mbar; range (ANOVA) were computed using the statistical 7–18 mbar; see below). Temperature and pressure package supplied with KaleidaGraph version within the MSC were measured using an auto- 3.6.2 (Synergy Software, Reading, PA). matic data logging system (model SNAP Ulti- mate I/O, Opto 22, Temecula, CA). Although a pure CO atmosphere is not exactly like Mars 2 RESULTS [95.3% (Owen, 1992)], work by Schuerger et al. (2003) indicated that gas composition had no ef- Survival and germination of B. subtilis spores fect on the survival and subsequent germination of endospores of B. subtilis, and, thus, the pure exposed to simulated Mars atmosphere CO2 atmosphere was selected to simplify the ex- B. subtilis strain WN648 spores were exposed perimental procedures. At 7, 12, and 19 days, mi- as dried films to either Earth-ambient or simu- crotiter plates were removed for viability assays lated Mars-ambient atmospheric composition and luciferase assays. Triplicate wells were used and pressure. Over the 19-day course of the ex- for each determination. Spores were rehydrated periment, from 11 August 2004 to 30 August 2004, by addition of 100 l of sterile deionized water the barometric pressure measured from the and incubation at room temperature for 60 min. nearby Cape Canaveral Weather Station aver- Rehydrated spores were resuspended thoroughly aged 1,017.5 mbar and varied between 1,014 and by pipetting and examined under the phase-con- 1,021 mbar. In the MSC, the simulated Mars at- trast microscope before further processing. An mospheric pressure averaged 12.5 mbar, and var- 5765_07_p536-544 7/25/05 11:18 AM Page 539

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ied from a minimum of 7 mbar to a maximum of tion by expression of sspB-luxAB and retained 18 mbar. Spores were assayed for viability at 0, within the dormant spore. After 90 min, lumi- 7, 12, and 19 days (Table 1). Spores survived well nescence declined, presumably resulting from under both Earth or simulated Mars atmospheres; proteolytic degradation of luciferase in the ger- indeed, a slight increase in viable titer was noted minating spores and subsequent vegetatively on Days 12 and 19 under both atmospheres (Table growing cells. Because expression of luxAB is dri- 1). Because of this slight day-to-day variation ven by the sporulation-specific sspB promoter, noted, comparisons were only made between which is inactive during germination and vege- spore titers measured on the same day of expo- tative growth, luciferase was not replaced during sure (Table 1). Differences in spore numbers mea- these developmental periods, hence the decline sured after exposure to Earth- or simulated Mars in activity. There was no apparent difference in atmospheric conditions were not statistically sig- either the kinetics or absolute level of germina- nificant at the P 0.05 confidence level, as mea- tion bioluminescence between the two sets of sured by ANOVA (Table 1). Therefore it was spores at Day 0; both spore populations exhibited concluded that exposure to simulated martian at- maximum bioluminescence at 90 min, and the ki- mosphere and low pressure in the MSC for 19 netic curves were superimposable (Fig. 1A). At 7 days did not significantly affect viability (i.e., abil- days (Fig. 1B), 12 days (Fig. 1C), and 19 days (Fig. ity of spores to form colonies) upon return to 1D) of exposure to Earth- or simulated Mars at- Earth atmosphere and nutrient conditions. Be- mosphere, triplicate spore samples were again re- cause colony formation is necessarily dependent moved and assayed for germination-induced upon spore germination, it can be inferred that bioluminescence. Spores exposed to simulated spores exposed in the MSC for 19 days retained Mars atmosphere in the MSC for 7, 12, and 19 their ability to sense the return of a favorable days exhibited lower levels of germination bio- (Earth) environment and respond by germination luminescence than did the parallel spore samples and subsequent growth. exposed to Earth-normal conditions for the same number of days (Fig. 1B–D). In addition, spores exposed in the MSC for 7 and 12 days exhibited Kinetics of germination luminescence of a slight delay in maximum bioluminescence, B. subtilis spores exposed to simulated from 90 to 120 min (Fig. 1B and C). Mars atmosphere We observed differences in levels of germina- Before placement in the MSC at Day 0, tripli- tion-induced bioluminescence between Earth- cate spore samples from both sets of plates were and simulated Mars atmosphere exposures, and assayed for germination bioluminescence (Fig. documented considerable day-to-day variation in 1A). As previously reported (Hill et al., 1994; Ciar- the absolute values of germination biolumines- ciaglini et al., 2000), germinating spores of strain cence (note the changes in y-axis scales in each WN648 produced a burst of luminescence, which panel of Fig. 1). These differences were attributed peaked at 90 min. The initial rise in luminescence to variations in the preparation of the germina- was due to the activity of luciferase, which had tion solution for each day. We thus concluded been produced in the forespore during sporula- that valid statistical comparisons could only be

TABLE 1. SURVIVAL OF B. SUBTILUS STRAIN WN648 SPORES AFTER EXPOSURE TO EARTH OR SIMULATED MARS ATMOSPHERE

Days of exposure Condition Mean SD P value (ANOVA)

0 Earth 1.08 108 3.5 107 0.977 (NSD) MSC 1.09 108 1.3 107 7 Earth 1.14 108 3.0 107 0.729 (NSD) MSC 1.27 108 5.3 107 12 Earth 4.4 108 5.3 107 0.222 (NSD) MSC 5.2 108 8.0 107 19 Earth 4.0 108 7.2 107 1 (NSD) MSC 4.0 108 2.0 107

Data are averages standard deviation (SD) of triplicate determinations. NSD, not significantly different. 5765_07_p536-544 7/25/05 11:18 AM Page 540

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FIG. 1. Kinetics of bioluminescence during germination of B. subtilus strain WN648 spores at 0 days (A), 7 days (B), 12 days (C), and 19 days (D) of exposure to Earth atmosphere (open circles) and simulated Mars atmosphere in the MSC (filled circles) compared with background luminescence (dashed lines). Data points represent averages standard deviations of triplicate determinations.

made between parallel sets of samples assayed tions. Examination of plots of total cumulative bi- on the same day. oluminescence versus time (Fig. 2) reinforced this In addition, pairwise statistical analysis by notion. As judged visually by overlap in error ANOVA of particular time points gave mixed re- bars, there was no significant difference in cu- sults. For example, exposure of spores for 7 days mulative germination-induced bioluminescence in the MSC (Fig. 1B) resulted in significantly between spore populations at 0 days (Fig. 2A). lower germination bioluminescence at 60 min However, after 19 days in the MSC, cumulative (P 0.0048) and 90 min (P 0.0026), but not at germination-induced bioluminescence was sig- 120 min (P 0.257). We reasoned that the total nificantly lower than the control kept at Earth at- cumulative bioluminescence generated during mospheric conditions (Fig. 2B). Guided by the the time course of germination could be used as above results (Fig. 2), we calculated the total cu- a basis of comparison for parallel spore popula- mulative germination bioluminescence generated 5765_07_p536-544 7/25/05 11:18 AM Page 541

B. SUBTILIS SPORE GERMINATION: MARS ATMOSPHERE 541

FIG. 2. Cumulative bioluminescence during germination of B. subtilus strain WN648 spores after 0 days (A) and 19 days (B) of exposure to Earth atmosphere (open circles) and simulated Mars atmosphere (filled circles) compared with background luminescence (dashed lines). Data points represent averages standard deviation of three independent determinations.

during the entire germination time course of each aldehyde, oxygen, and reduced FMN (Hastings sample, normalized the resulting cumulative val- et al., 1969; Nealson et al., 1970). Because decanal ues to the spore titer of each suspension, and com- and oxygen were supplied exogenously in the pared the normalized values by ANOVA (Table assay, the observed decrease in germination-in- 2). The data clearly showed that 7, 12, or 19 days duced bioluminescence after prolonged exposure of spore exposure to simulated Mars atmospheric to simulated Mars atmosphere in the MSC could conditions in the MSC resulted in a highly statis- be due to inactivation of LuxAB luciferase activ- tically significant decrease in germination-in- ity, to impairment in FMN reduction, or to both. duced bioluminescence, up to a 32% decrease by It should be noted that the Mars atmosphere sim- Day 19 (Table 2). ulation used here was quite rudimentary, in that it lacked trace gases, low temperatures, and UV spectrum and flux characteristic of the true mar- DISCUSSION tian atmosphere (Schuerger et al., 2003; Cockell et al., 2005; Nicholson et al., 2005). However, the Germination-induced bioluminescence has data are consistent with the hypothesis that days been considered to be an effective real-time mon- of exposure to the low pressure and high CO2 itor of the germination and outgrowth process, components of the Mars atmosphere alone, in the and a measurement of how this process is af- absence of the full suite of harsh martian envi- fected by inhibitory or lethal treatments to spores ronmental conditions, slowly degrade the ability (Hill et al., 1994; Ciarciaglini et al., 2000). The data of spores to germinate upon return to favorable presented here indicate that exposure to simu- environmental conditions. lated Mars atmosphere (100% CO2, average of To date, interplanetary transfer models have 12.5 mbar) for 19 days significantly reduced ger- only considered the probability of survival of en- mination-induced bioluminescence of B. subtilis dolithic bacteria to forces generated during the WN648 spores (Figs. 1 and 2 and Table 2), but transfer process itself (i.e., impact-mediated ejec- was insufficient to inactivate spores or com- tion, transit through space, and entry and depo- pletely prevent spore germination, as measured sition on the target planet) (Clark, 2001; by the viability assay (Table 1). Light production Mileikowsky et al., 2000; Nicholson et al., 2000; in the bacterial luciferase reaction used in this Horneck et al., 2002). In these models, transfer in study depends on the LuxAB protein, long-chain either direction between Earth and Mars has been 5765_07_p536-544 7/25/05 11:18 AM Page 542

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TABLE 2. COMPARISON OF NORMALIZED CUMULATIVE GERMINATION-INDUCED BIOLUMINESCENCE OF SPORES EXPOSED TO EARTH OR SIMULATED MARS ATMOSPHERE

Day, condition Replicate C-RLU S C-RLU/S Average SD P value (ANOVA)

0 Earth 1 20,147 8.03 2,509 2 21,953 7.83 2,804 2,435 412 3 16,091 8.08 1,991 0.747 (NSD) MSC 1 22,790 8.05 2,831 2 17,919 7.97 2,248 2,306 499 3 14,852 8.08 1,838 7 Earth 1 10,403 8.03 1,296 2 9,949 7.94 1,253 1,300 50 3 11,045 8.17 1,352 0.0026 MSC 1 8,482 8.06 1,052 2 8,419 8.27 1,018 1,002 60 3 7,408 7.91 936 12 Earth 1 33,063 8.66 3,818 2 30,715 8.68 3,539 3,703 146 3 32,049 8.58 3,753 0.041 MSC 1 28,591 8.64 3,309 2 30,870 8.78 3,516 3,380 118 3 28,919 8.72 3,316 19 Earth 1 37,577 8.53 4,405 2 32,751 8.58 3,817 4,059 308 3 34,318 8.68 3,954 MSC 1 25,711 8.56 3,004 0.0043 2 23,472 8.62 2,723 2,755 235 3 21,879 8.62 2,538

C-RLU, cumulative RLU; NSD, not significantly different; S, log10 cfu of spores; SD, standard deviation.

considered; natural Earth-to-Mars transfers were threefold. First, during spacecraft assembly, the considered less probable because of Earth’s use of bioluminescent or fluorescent biomarkers denser atmosphere and higher escape velocity to monitor viable bioloads must be confirmed to (Mileikowsky et al., 2000). For the interplanetary directly correlate with cell viability before any transfer process to be ecologically relevant, the one assay is relied upon for microbial detection habitabilities of the donor and recipient planets, and quantification. As sterilizing protocols are which constrain both the initial populations sub- applied to spacecraft materials during assembly jected to launch and the fate of survivors upon (i.e., heat, chemical, and gaseous treatments), it arrival, must be considered. The present results might be possible that the different sterilizing combined with the data and considerations pre- protocols may degrade viability, biolumines- viously discussed (Schuerger et al., 2003; Nichol- cence, and fluorescent biomarkers to different de- son et al., 2004) suggest that, at least for Bacillus grees, thus obscuring the evaluation of the level spp. spores and currently prevailing environ- of cleanliness achieved. Second, the use of biolu- mental conditions, the environment of the Mars minescence in future life-detection experiments surface, characterized by high solar UV, low pres- on Mars has been proposed for the search for an sure, low temperature, and predominance of at- extant Mars microbiota, as well as any terrestrial mospheric CO2, may present a formidable barrier contamination on Mars (Venkateswaran et al., to the establishment of viable populations result- 2003a; Steele et al., 2004). The results presented ing from natural Earth-to-Mars interplanetary here indicate that spore viability and biolumi- transfer events. nescence of terrestrial bacteria might not always The implications of these results for planetary match, and that caution should be used when in- protection issues related to Mars exploration are terpreting the intensity of bioluminescence as a 5765_07_p536-544 7/25/05 11:18 AM Page 543

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biomarker for cell viability. A similar effect was REFERENCES reported by Cockell et al. (2005), who found that the viability of cells of the cyanobacterium Ciarciaglini, G., Hill, P.J., Davies, K., McClure, P.J., Kilsby, Chroococcidiopsis sp. 029 did not match several flu- D., Brown M.H., and Coote, P.J. (2000) Germination-induced bioluminescence, a route to determine the in- orescent biomarkers used to assay cell viability hibitory effect of a combination preservation treatment following exposure to martian conditions. This on bacterial spores. Appl. Environ. Microbiol. 66, study reported that viability of Chroococcidiopsis 3735–3742. sp. 029 decreased rapidly under martian condi- Clark, B. (2001) Planetary interchange of bioactive mate- tions, while several fluorescent biomarkers re- rial: Probability factors and implications. Orig. Life Evol. tained their intensity over much longer time Biosph. 31, 185–197. frames. Although the effects in the study of Cock- Cockell, C., Schuerger, A.C., Billi, D., Friedmann, E.I., and Panitz, C. (2005) Effects of a simulated martian UV flux ell et al. (2005) were opposite those reported here, on the cyanobacterium Chroococcidiopsis sp. 029. Astro- both studies indicate that bioluminescence or biology 5, 127–140. fluorescent biomarkers might not always match Hastings, J.W., Weber, K., Friedland, J., Eberhard, A., the viability of terrestrial microorganisms ex- Mitchell, G.W., and Gunsalus, A. (1969) Structurally- posed to martian conditions. Third, the use of distinct bacterial luciferases. Biochemistry 8, 4681–4689. bioluminescence or fluorescent biomarkers as a Hill, P.J., Hall, L., Vinicombe, D.A., Soper, C.J., Setlow, P., single check for terrestrial back-contamination Waites, W.M., Denyer, S., and Stewart, G.S.A.B. (1994) Bioluminescence and spores as biological indicators of of returned samples from Mars may not be ad- inimical processes. J. Appl. Bacteriol. Symp. Suppl. 76, equate for confirming either the presence or ab- 129S–134S. sence of terrestrial microorganisms. As the re- Horneck, G., Miliekowsky, C., Melosh, H.J., Wilson, J.W., sults of the current study and the report by Cucinotta, F.A., and Gladman, B. (2002) Viable transfer Cockell et al. (2005) clearly demonstrate, detec- of microorganisms in the solar system and beyond. In tion of bioluminescence or fluorescent biomark- Astrobiology: The Quest for the Conditions of Life, edited ers may not always correlate to the viability of by G. Horneck and C. Baumstark-Khan, Springer, the bioload of a sample. Berlin, pp. 55–76. Jacobs, M., Hill, P.J., and Stewart, G.S.A.B. (1991) Highly

bioluminescent Bacillus subtilis obtained through high- level expression of a luxAB fusion gene. Mol. Gen. Genet. ACKNOWLEDGMENTS 230, 251–256. La Duc, M.T., Nicholson, W.L., Kern, R., and Venkateswaran, K. (2003) Microbial characterization of The authors thank Peter Setlow for the gener- the Mars Odyssey spacecraft and its encapsulation fa- ous gift of the B. subtilis strain used. This work cility. Environ. Microbiol. 5, 977–985. was supported by a grant (20020023/21988) from Link, L., Sawyer, J., Venkateswaran, K., and Nicholson, the University of Florida/University of Central W.L. (2004) Extreme spore UV resistance of Bacillus Florida Space Research Initiative to A.C.S. and pumilus isolates obtained from an ultra-clean Spacecraft W.L.N. and NASA Exobiology grant NNA04- Assembly Facility. Microb. Ecol. 47, 159–163. Mileikowsky, C., Cucinotta, F.A., Wilson, J.W., Gladman, CI35A to W.L.N. The MSC used in this study was B., Horneck, G., Lindegren, L., Melosh, H.J., Rickman, built with support from the University of Florida, H., Valtonen, M., and Zheng, J.Q. (2000) Natural trans- NASA’s Planetary Protection Office (NRA grant fer of viable microbes in space. Part 1: From Mars to NAS2-00087), and the University of Florida/Uni- Earth and Earth to Mars. Icarus 145, 391–427. versity of Central Florida Space Research Initia- Nealson, K.H., Platt, T., and Hastings, J.W. (1970) Cellu- tive mentioned above. lar control of the synthesis and activity of the bacterial luminescent system. J. Bacteriol. 104, 313–322. Nicholson, W.L. and Setlow, P. (1990) Sporulation, germination, and outgrowth. In Molecular Biological Meth- ABBREVIATIONS ods for Bacillus, edited by C.R. Harwood and S.M. Cut- ting, John Wiley & Sons, Chichester, UK, pp. 391–450. ANOVA, analysis of variance; cfu, colony- Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., forming units; Cm, chloramphenicol; MSC, Mars and Setlow, P. (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. simulation chamber; PBS, phosphate-buffered Microbiol. Mol. Biol. Rev. 64, 548–572. saline; RLU, relative light units; SAF, spacecraft Nicholson, W.L., Schuerger, A.C., and Setlow, P. (2005) assembly facility; SSM, Schaeffer Sporulation The solar UV environment and bacterial spore UV re- Medium; UV, ultraviolet. sistance: Considerations for Earth-to-Mars transport by 5765_07_p536-544 7/25/05 11:18 AM Page 544

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