Transcriptomic Responses of Germinating Bacillus subtilis Spores Exposed to 1.5 Years of Space and Simulated Martian Conditions on the EXPOSE-E Experiment PROTECT

Abstract

Because of their ubiquity and resistance to spacecraft decontamination, bacterial spores are considered likely potential forward contaminants on robotic missions to Mars. Thus, it is important to understand their global responses to long-term exposure to space or martian environments. As part of the PROTECT experiment, spores of B. subtilis 168 were exposed to real space conditions and to simulated martian conditions for 559 days in low-Earth orbit mounted on the EXPOSE-E exposure platform outside the European Columbus module on the International Space Station. Upon return, spores were germinated, total RNA extracted, fluorescently labeled, and used to probe a custom Bacillus subtilis microarray to identify genes preferentially activated or repressed relative to ground control spores. Increased transcript levels were detected for a number of stress-related regulons responding to DNA damage (SOS response, SPβ prophage induction), protein damage (CtsR/Clp system), oxidative stress (PerR regulon), and cell envelope stress (SigV regulon). Spores exposed to space demonstrated a much broader and more severe stress response than spores exposed to simulated martian conditions. The results are discussed in the context of planetary protection for a hypothetical journey of potential forward contaminant spores from Earth to Mars and their subsequent residence on Mars. Key Words: Bacillus—Mars—Planetary protection—Spaceflight—Spores. Astrobiology 12, 469–486.

1. Introduction

R obotic missions to potentially habitable destinations such as Mars are required to comply with international guidelines of planetary protection as established by the Committee on Space Research (COSPAR) (Bruckner et al., 2009; COSPAR, 2011) and based on Article IX of the Outer Space Treaty of the United Nations (United Nations, 1967). Critical to planetary protection guidelines are requirements for reduction or avoidance of contamination by terrestrial microorganisms on spacecraft, including measurements of bioburden and efficacy of decontamination/sterilization of spacecraft or their components before launch (Rummel, 2001; Nicholson et al., 2009; COSPAR, 2011). Monitoring of the microbial diversity of spacecraft and their assembly facilities has revealed that spore-forming Bacillus species were among the dominant isolates of cultivable microorganisms (see Moeller et al., 2012 for details). Spores of spacecraft isolates can exhibit elevated resistance to various methods for spacecraft decontamination or sterilization (Link et al., 2004; Kempf et al., 2005; Satomi et al., 2006), leading to concerns that spores could escape spacecraft decontamination/sterilization processes, survive space transit, and contaminate the target planet (reviewed in Nicholson et al., 2009).

The well-characterized spore-forming bacterium Bacillus subtilis has long been used as a model organism for astrobiological studies (reviewed in Nicholson et al., 2000, 2005, 2009; Horneck et al., 2002, 2010) and as a representative spacecraft contaminant in planetary protection studies (Nicholson et al., 2000, 2009; Fajardo-Cavazos et al., 2007). Most historical studies in which spores have been exposed to the space environment have concentrated mainly on measuring spore survival and, to a lesser extent, mutation frequency of selected markers (Horneck et al., 1984, 1994). In the PROTECT experiments presented in this volume, we attempted for the first time to elucidate some of the molecular responses of spores exposed to the conditions of outer space and simulated martian surface conditions. In addition to evaluating the spectrum of mutations induced in the rpoB gene (Moeller et al., 2012), we attempted to capture a broad range of cellular damages caused to spores by space or simulated Mars exposure, using the following rationale. It is known that spores are dormant and metabolically inactive and thus cannot repair cellular damage suffered during dormancy (Nicholson et al., 2000, 2005). Exposure to space or simulated martian conditions would therefore be expected to cause cumulative damage to cellular components. We reasoned that, upon germination, spores would detect such damages and activate the transcription of specific genes responsible for repair of each type of damage. The identification of such repair genes would thus indicate the particular type of damage inflicted on the spore by space or Mars exposure, or both. This data can enhance our understanding of how terrestrial microorganisms cope with transfer through space to an environment such as Mars and possibly become viable forward contaminants. In this communication, we present the results of these experiments.

2. Materials and Methods

2.1. Bacillus subtilis sporulation and spore purification

The laboratory strain of B. subtilis, strain 168 (trpC2) (Zeigler et al., 2008), was from the corresponding author's strain collection and was used throughout these studies. Spores were obtained by cultivation at 37°C for 5–7 days on solid agar plates of Schaeffer sporulation medium (Schaeffer et al., 1965) and were purified and stored in distilled water as described previously (Nicholson and Setlow, 1990). Spore preparations were free (>99%) of growing cells, germinated spores, and cell debris as determined by phase-contrast microscopy.

2.2. Spore sample preparation

Spacecraft-qualified, chemfilm-treated aluminum 6061 coupons (13 mm in diameter×1 mm thick; Titusville Tool & Engineering, Titusville, FL, USA) were dry-heat sterilized (132°C, 16 h) prior to use. Aliquots (20 μL) of a suspension of B. subtilis 168 spores containing ∼1×10⁸ colony-forming units were deposited aseptically onto the surface of each coupon. Spores were allowed to settle, and coupons were air-dried for 1 day under ambient laboratory conditions (temperature 20°C±2°C and relative humidity 33±5%), which resulted in layers ∼5–10 spores thick.

2.3. Spaceflight experiment

Spore samples on aluminum coupons were stacked in triplicate inside the compartments of two trays of the EXPOSE-E facility. EXPOSE-E was installed on the outside of the International Space Station (ISS), and the samples were exposed to outer space and simulated martian conditions for 559 days before being retrieved and analyzed in the laboratory. Spores in the two trays were exposed to the following parameters: (i) space vacuum (∼10⁻⁴ Pa), temperature (⁻20°C to +59°C), and galactic cosmic radiation (155 mGy); (ii) simulated martian atmospheric composition (1.6% argon, 0.15% oxygen, 2.7% nitrogen in CO₂), martian surface pressure (10³ Pa), temperature (−20°C to +59°C), and galactic cosmic radiation (134–142 mGy). Further details on the radiation dosimetry, applied UV fluences, doses of ionizing radiation, pressure values, temperature fluctuations, gas compositions, and so on are given by Horneck et al. (2012) and Rabbow et al. (2012). Only spores shielded from solar radiation (i.e., the middle and bottom coupons in triplicate stacks) were analyzed in this study. Laboratory ground controls consisted of an identical set of spore-loaded coupons stored at Earth-ambient conditions shielded from light for the duration of the experiment. Exposed spores and ground control samples were processed simultaneously.

2.4. Spore recovery from aluminum coupons

Spores were recovered from the aluminum coupons by stripping with polyvinyl alcohol as described previously (Lindberg and Horneck, 1991; Moeller et al., 2012). Spores were washed by repeated centrifugation in sterile distilled water and stored in water at 4°C. For enumeration, spores were diluted serially 10-fold in phosphate-buffered saline (PBS) (Nicholson and Setlow, 1990). Total spores recovered were enumerated by direct counts in a Petroff-Hauser cell counting chamber (Gerhardt et al., 1981), and viable spores were enumerated by spreading of appropriate dilutions on Luria-Bertani (LB) agar plates (Miller, 1972) and counting colonies after overnight incubation at 37°C.

2.5. Spore germination and total RNA isolation

To synchronize germination, spores in water (∼1×10⁹) were heat activated at 70°C for 30 min and then harvested by centrifugation. To initiate germination, spores were resuspended in 100 mL of 2× LB medium containing the germinant L-alanine (10 mM final concentration) in a 250 mL Klett flask and agitated vigorously in a rotary water bath shaker at 37°C. Germination was monitored by loss of optical density as measured in a Klett-Summerson photometer fitted with the No. 66 (660 nm; red) filter. After 60 min of germination, spores were harvested by centrifugation, and the pellets stored frozen at −20°C until further analysis. Total RNA was isolated from germinated spores by using the Ribo-Pure Bacteria Kit followed by treatment with RNase-free DNase, using the protocols supplied by the manufacturer (Ambion-Life Technologies, Grand Island, NY, USA).

2.6. Microarray analyses

Total RNA samples (20 μg) were sent to the University of Florida Interdisciplinary Center for Biotechnology Research (UF-ICBR). Samples were converted into Cy3- or Cy5-labeled cDNAs, and identical quantities of cDNAs were used to probe 15,000-feature custom microarrays (Agilent) prepared from the B. subtilis strain 168 genomic sequence (Kunst et al., 1997). Background subtraction, quantile, and Loess normalization were performed by the UF-ICBR. The microarray contained 15,209 data points equivalent to 4,103 genes with an average number of 3.7 measurements per gene; the oligos were designed such that each open reading frame (ORF) was sampled at multiple locations (n=3 or 4). Log-transformed data sets for each ORF from space- or simulated Mars-exposed spores were compared to the same ORF from the ground control spores by using one-way analysis of variance (ANOVA). Data showing ≥3-fold differences with P values of ≤0.05 were considered statistically significant.

2.7. Microarray data accession number

Complete microarray data have been deposited in Gene Expression Omnibus under accession number GSE37124 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37124).

3. Results

3.1. Spore survival and germination

From the total viable versus direct spore counts, it was determined that 6.1% and 97.6% of B. subtilis 168 spores survived 559 days in outer space or under simulated martian conditions, respectively, if shielded from solar UV radiation. Note that in our samples, space-exposed spores exhibited an apparently lower survival value (6.1%) than in the samples reported by Horneck et al. (2012) (∼55%). This discrepancy is likely due to the different techniques used to assess spore viability in our two experiments (see Horneck et al., 2012 in this issue for comparison). However, these observations are in good general agreement with the results of previous flight and ground-based laboratory experiments (reviewed in Horneck et al., 2010). Making the reasonable assumption that spore inactivation is a first-order process (Nicholson, 2003), these survival rates can be converted into decimal reduction (D) values of ∼458 days (1.25 years) in space and ∼53,000 days (∼145 years) on the martian surface.

Spores were germinated in 2× LB liquid medium containing the potent germinant L-alanine (Fig. 1). It was noted that spores stored at ambient Earth conditions germinated normally, losing ∼50% of their initial optical density within the first 30 min, whereas spores exposed to space or simulated martian conditions aboard PROTECT demonstrated only ∼30% loss in optical density over the same time period (Fig. 1).

FIG. 1.

Germination of B. subtilis spores exposed to space (open squares), simulated martian conditions (open circles), or Earth conditions (open triangles) for 559 days. OD, optical density. See text for details.

3.2. Microarray data: overall trends

To visualize overall differences in the germination transcriptome of B. subtilis spores exposed to space or simulated martian conditions, microarray data were subjected to global comparisons and displayed as scatter plots (Fig. 2). As a measure of reproducibility, the complete sets of germination microarray data were compared from the two Earth control samples used in the experiment (Fig. 2A), and the two data sets demonstrated a high correlation coefficient (r=0.9781), which indicates that the methods used were inherently reproducible. When the complete sets of microarray data from space-exposed spores (Fig. 2B) or simulated Mars-exposed spores (Fig. 2C) were compared to their respective Earth controls, considerable divergence was observed (correlation coefficients of r=0.8967 and r=0.8445, respectively), which indicated that exposure to space or simulated martian conditions had resulted in alterations in the global B. subtilis germination transcriptome.

FIG. 2.

Scatter plot summaries of microarray data. Germination microarray comparisons of B. subtilis 168 spores exposed to the following conditions. (A) “Earth 1” vs. “Earth 2” controls. (B) Space-exposed spores vs. Earth-control spores. (C) Simulated Mars-exposed spores vs. Earth-control spores.

Analysis of microarray data from space- and simulated Mars-exposed spores identified genes which were significantly up- or down-regulated relative to the ground control spores. Visual inspection of the summarized data (Fig. 3) yielded the following observations. Spores exposed to full space conditions exhibited more than twice as many genes with altered expression levels (236) than did spores exposed to simulated martian conditions (112) (Fig. 3). It is particularly notable that simulated Mars exposure led to significant down-regulation of only 20 genes (Fig. 3). Furthermore, it was observed that in all spores exposed to either space or simulated martian conditions, greater than 50% of genes with altered expression belonged to Category 5, genes of unknown function (Fig. 3). The subsets of B. subtilis genes found to be significantly up-regulated or down-regulated during germination of space-exposed and simulated Mars-exposed spores are listed and compared in Tables 1 and 3, respectively. According to our hypothesis, searching through these genes should offer clues to the type(s) of cellular damage suffered by spores exposed to space or simulated martian conditions.

FIG. 3.

Summary of genes identified by microarray experiments as significantly up- or down-regulated from space- or simulated Mars-exposed spores. Categories are defined as described in Appendix 2 of Sonenshein et al. (2002). Categories are Cell envelope and cellular processes (1, dark gray bars); Intermediary metabolism (2, light gray bars); Information pathways (3, white bars); Other functions (4, black bars); and Unknown (5, hatched bars). See text for details.

Table 1.

Genes Up-Regulated during Germination of Space-Exposed (S) or Simulated Mars-Exposed (M) Spores ^a

		Fold up-regulation ^c		P value ^c
Category	Annotated function ^b	S	M	S	M
1. Cell envelope and cellular processes
1.1. Cell wall
ponA	Penicillin-binding proteins 1a/1b (PBP1) (in recUponA operon)	3.0	nsd^d	0.0137	nsd
1.2. Transport/binding proteins and lipoproteins
arsB	Extrusion of arsenite	nsd	3.6	nsd	<0.0001
arsC	Arsenate reductase	nsd	3.0	nsd	0.0008
blt	Multidrug efflux transporter	nsd	3.9	nsd	<0.0001
lmrB	Lincomycin resistance protein (in lmrAB operon)	3.1	3.6	0.0001	0.0002
zosA (ykvW)	P-type zinc-transporting ATPase, PerR regulon	nsd	3.9	nsd	0.0039
1.4. Membrane bioenergetics (electron transport chain, ATP synthase)
ndhF	NADH dehydrogenase subunit 5 (in operon with ybcCDFHIL)	5.0	3.5	<0.0001	0.0012
etfB	Electron transfer flavoprotein beta-subunit	3.9	6.0	<0.0001	<0.0001
etfA	Electron transfer flavoprotein alpha-subunit	3.1	4.8	<0.0001	<0.0001
	(both in lcfAysiABetfBA operon)
1.5. Motility and chemotaxis
fliL	Required for flagellar formation	5.1	nsd	0.0501	nsd
1.7. Cell division
gidA	glucose inhibited division protein A	3.5	nsd	<0.0001	nsd
gidB	glucose inhibited division protein B	3.1	nsd	<0.0001	nsd
1.8. Sporulation
cgeA	Maturation of outermost layer of spore	8.9	5.5	0.0035	0.0036
cgeB	Maturation of outermost layer of spore	9.7	6.7	<0.0001	<0.0001
cotG	Spore coat protein	23.4	20.4	0.0008	0.0006
cotT	Spore coat protein (inner)	7.3	6.3	0.0073	0.0045
cotV	Spore coat protein (insoluble)	12.4	10.5	0.0184	0.0224
cotW	Spore coat protein (insoluble)	8.6	7.9	0.0332	0.0870
cotX	Spore coat protein (insoluble)	14.8	13.5	<0.0001	<0.0001
cotY	Spore coat protein (insoluble)	4.3	3.6	<0.0001	<0.0001
safA	Morphogenetic protein associated with SpoVID	3.8	nsd	<0.0001	nsd
coxA	Spore cortex protein	7.5	11.2	0.0002	<0.0001
rapA	Aspartate phosphatase of Spo0F∼P	4.3	3.7	<0.0001	0.0002
phrA	RapA inhibitor peptide	7.8	6.8	0.0003	0.0007
sspE	small acid-soluble spore protein (major γ-type SASP)	nsd	5.5	nsd	0.0130
sspF	small acid-soluble spore protein (minor α/β-type SASP)	3.7	3.0	0.0076	0.0050
sspJ	small acid-soluble spore protein (minor α/β-type SASP)	4.5	4.7	0.0167	0.0061
sspM	small acid-soluble spore protein (minor α/β-type SASP)	5.4	5.1	0.0268	0.0027
sspN	small acid-soluble spore protein (minor α/β-type SASP)	13.2	14.0	0.0009	0.0017
tlp	small acid-soluble spore protein (minor α/β-type SASP)	10.7	14.8	0.0001	0.0081
sspO	Small, acid-soluble spore protein (minor)	nsd	4.3	nsd	0.0284
sspP	Small, acid-soluble spore protein (minor)	3.4	4.2	0.0020	0.0033
2. Intermediary metabolism
2.1. Metabolism of carbohydrates and related molecules
2.1.1. Specific pathways
fruR	Transcriptional repressor of fru operon (DeoR family)	24.6	29.3	<0.0001	<0.0001
fruK	Fructose-1-phosphate kinase	43.0	44.7	<0.0001	<0.0001
fruA	Fructose-specific PTS enzyme IIAB component	36.4	49.7	<0.0001	<0.0001
mtlD	Mannitol-1-phosphate 5-dehydrogenase	nsd	3.1	nsd	<0.0001
2.2. Metabolism of amino acids and related molecules
bltD	Spermine/spermidine acetyltransferase	nsd	3.0	nsd	<0.0001
2.5. Metabolism of coenzymes and prosthetic groups
hemE	Uroporphyrinogen III decarboxylase	3.6	nsd	<0.0001	nsd
hemH	Ferrochelatase	3.8	nsd	<0.0001	nsd
hemY	Protoporphyrinogen IX and coproporphyrinogen III oxidase	3.5	nsd	<0.0001	nsd
hemA	Glutamyl tRNA reductase	7.7	nsd	<0.0001	nsd
hemX	Negative regulator of the concentration of HemA	8.2	nsd	<0.0001	nsd
hemC	Porphobilinogen deaminase	6.3	nsd	<0.0001	nsd
hemD	Uroporphyrinogen III synthase	5.3	nsd	<0.0001	nsd
hemB	Porphobilinogen synthase	5.1	nsd	<0.0001	nsd
hemL	Glutamate-1-semialdehyde 2,1-aminomutase	5.2	nsd	<0.0001	nsd
3. Information pathways
3.1. DNA replication
dnaE	DNA polymerase III alpha-subunit	3.1	3.2	<0.0001	0.0002
3.2. DNA restriction/modification, repair, and recombination
addB	RecBCD homologue	3.6	nsd	<0.0001	nsd
addA	RecBCD homologue	3.4	nsd	0.0032
sbcD	Sbc ATP-dependent exonuclease D subunit	3.0	nsd	<0.0001
sbcC(yirY)	Sbc ATP-dependent exonuclease C subunit	3.1	nsd	<0.0001
yisB	Putative endonuclease associated with SbcCD	3.9	nsd	0.0100	nsd
recA	Multifunctional protein involved in homologous recombination and DNA repair	7.5	nsd	<0.0001	nsd
recU	Recombination protein; required for proper chromosome segregation	4.0	3.0	<0.0001	0.0022
umuC (yqjW)	Y-family DNA polymerase (translesion bypass DNA repair)	44.4	nsd	<0.0001	nsd
uvrB	Nucleotide excision repair excinuclease subunit B	8.4	nsd	<0.0001	nsd
uvrA	Nucleotide excision repair excinuclease subunit A	8.2	nsd	<0.0001	nsd
uvrC	Nucleotide excision repair excinuclease subunit C	3.2	nsd	<0.0001	nsd
3.5. RNA synthesis
3.5.1. Transcription initiation
sigV	RNA polymerase ECF-type sigma factor (σ^V)	4.4	5.2	0.0002	<0.0001
rsiV (yrhM)	Anti-sigma factor	5.8	5.4	<0.0001	0.0005
oatA (yrhL)	Peptidoglycan O-acetyltransferase	6.0	4.1	<0.0001	0.0001
yrhK	unknown	4.8	4.1	0.0043	0.0110
3.5.2. Transcription regulation
hrcA	Transcriptional repressor of class I heat-shock genes	4.5	4.5	<0.0001	<0.0001
lmrA	Transcriptional repressor (TetR family) of the lmrAB operon	4.1	4.5	<0.0001	<0.0001
4. Other functions
4.1. Adaptation to atypical conditions
clpE	class III stress response ATPase	56.1	53.5	<0.0001	<0.0001
clpP	ATP-dependent Clp protease (class III stress response gene)	3.3	5.2	<0.0001	<0.0001
ctsR	Transcriptional repressor of class III stress genes	3.0	nsd	0.0003	nsd
mcsA	Modulator of CtsR repression	3.6	nsd	<0.0001	nsd
mcsB	Modulator of CtsR repression	4.6	nsd	<0.0001	nsd
clpC	Class III stress-response ATPase	5.7	3.1	<0.0001	0.0007
radA	DNA repair protein, similar to ATP-dependent Lon protease	4.1	nsd	<0.0001	nsd
disA	DNA integrity scanning protein	3.6	nsd	<0.0001	nsd
mrgA	Metalloregulation DNA-binding stress protein	22.5	6.8	<0.0001	<0.0001
4.2. Detoxification
katA	Vegetative catalase 1	9.9	6.7	<0.0001	<0.0001
4.6. Miscellaneous
aprX	Intracellular alkaline serine protease	3.7	nsd	<0.0001	nsd
5. Unknown
ndhF	See Category 1.4
ybcC	Unknown	4.1	nsd	0.0127	nsd
ybcD	Unknown	7.0	3.5	<0.0001	<0.0001
ybcF	Unknown; similar to carbonic anhydrase	6.1	3.1	0.0002	0.0029
ybcH	Unknown	4.2	nsd	0.0059	nsd
ybjC	Unknown	nsd	4.2	nsd	0.0001
ydiM	Unknown	5.4	5.4	0.0485	0.0485
yebC	Unknown	nsd	3.0	nsd	<0.0001
yfhD	Unknown	13.4	18.9	0.0019	0.0041
yflS	Putative 2-oxoglutarate/malate transporter	3.3	3.3	<0.0001	<0.0001
yhaS	Unknown	3.9	5.7	nsd	nsd
yhaT	Unknown conserved protein	4.0	4.0	<0.0001	0.0001
yhaU	Similar to Na⁺/H⁺ antiporter	4.6	4.1	0.0003	<0.0001
yhbI	Putative transcription regulator (MarR family)	6.4	5.1	<0.0001	<0.0001
yhbJ	Unknown	6.3	4.7	<0.0001	<0.0001
yhcA	Similar to multidrug resistance protein	7.1	4.7	<0.0001	<0.0001
yhcB	Similar to flavodoxin	7.3	4.7	<0.0001	<0.0001
yhcC	Unknown	7.3	5.3	0.0210	0.0297
yhcQ	Unknown	7.4	11.5	0.0007	<0.0001
yhcV	Unknown; similar to IMP dehydrogenase	4.0	6.1	<0.0001	<0.0001
yhdB	Unknown	8.9	11.2	0.0062	0.0020
yhfD	Unknown	4.8	4.5	0.0056	0.0255
yhfH	Unknown	nsd	6.1	nsd	0.0195
yhjB	Probable sodium:solute symporter	3.0	nsd	0.0005	nsd
yhjN	Conserved membrane protein	3.0	nsd	0.0011	nsd
yitR	Unknown	3.0	3.1	0.0051	0.0359
yjbC	Conserved protein	3.1	nsd	0.0002	nsd
yjbE	Conserved membrane protein	3.3	nsd	0.0022	nsd
ykuA	Unknown; similar to penicillin-binding protein	nsd	3.0	nsd	0.0001
ykuF	Similar to glucose-1-dehydrogenase	11.7	8.7	<0.0001	<0.0001
ykuG	Putative cell wall binding protein	3.0	nsd	0.0130	nsd
ykvC (eag)	Putative small membrane protein	4.4	nsd	<0.0001	nsd
ykzE	Unknown	9.0	11.0	0.0031	0.0043
ymaC	Unknown; similar to phage-related protein	15.1	nsd	<0.0001	nsd
ymaD	Unknown; conserved	8.6	nsd	<0.0001	nsd
ymfJ	Unknown: conserved	4.3	4.7	0.0083	0.0038
yneA	Putative cell division suppression protein during SOS response	5.7	nsd	0.0023	nsd
yneB	Similar to resolvase	7.1	nsd	<0.0001	nsd
yoaV	Conserved membrane protein	3.2	nsd	0.0210	nsd
yobB	Unknown	3.2	nsd	0.0103	nsd
yodU	Unknown; similar to polysaccharide biosynthesis protein	4.2	nsd	0.0001	nsd
yoeB	Unknown	4.1	nsd	<0.0001	nsd
yozK-yobH	Putative Y-family DNA polymerase (translesion bypass DNA repair)	12.5	nsd	0.0260	nsd
yozN	Unknown	3.4	nsd	0.0323	nsd
yqeZ	Unknown; conserved membrane protein	3.2	nsd	<0.0001	nsd
yqfA	Unknown; conserved	2.8	nsd	0.0003	nsd
yqfB	Unknown	2.7	nsd	<0.0001	nsd
yqfX	Unknown; conserved	12.6	17.4	0.0021	0.0053
yqjL	Unknown	3.9	4.5	0.0002	0.0016
yqjW(umuC)	See Category 3.2
yqjX	Unknown; similar to SPβ phage YolD protein	48.9	nsd	<0.0001	nsd
yqjY	Unknown; putative acetyltransferase	41.0	nsd	<0.0001	nsd
yqjZ	Unknown; conserved	34.2	nsd	0.0005	nsd
yqkA	Unknown; putative acetyltransferase	33.8	nsd	<0.0001	nsd
yqkB	Unknown; conserved	7.5	nsd	0.0086	nsd
yqkC	Unknown	3.4	nsd	0.0087	nsd
yqkD	Unknown; conserved	3.4	nsd	<0.001	nsd
yqkE	Unknown (yqkD and yqkE transcribed divergently)	4.1	nsd	0.0021	nsd
yqzH	Unknown; divergent upstream from yqjW operon	3.8	nsd	0.0324	nsd
yrhH	Unknown; similar to methyltransferase	nsd	3.2	nsd	0.0013
yrkD	Unknown; conserved	nsd	3.5	nsd	0.0141
yrkE	Unknown; conserved	4.1	8.1	0.0043	0.0010
yrkF	Unknown	4.5	7.9	<0.0001	<0.0001
yrkG	Unknown; conserved	7.7	10.4	0.0020	0.0031
yrkH	Unknown; conserved	5.3	7.0	<0.0001	<0.0001
yrkI	Unknown; conserved	4.9	7.9	0.0341	0.0246
yrkO	Conserved membrane protein	3.0	nsd	0.0484	nsd
yrrD	Unknown	3.7	4.0	<0.0001	0.0002
ysiA	Putative transcriptional regulator, TetR/AcrR family (in lcfAysiABetfBA operon)	3.1	6.2	0.0005	0.0001
ysiB	Putative enoyl-CoA hydratase (in lcfAysiABetfBA operon)	4.0	6.9	0.0011	0.0002
ythC	Unknown	8.7	9.4	0.0028	0.0032
ytzC	Unknown	nsd	4.8	nsd	0.0391
yuxJ	Unknown; similar to multidrug efflux transporter	3.3	nsd	<0.0001	nsd
yvgP	Putative monovalent cation: proton antiporter	3.2	nsd	0.0003	nsd
yvqI	Unknown; conserved membrane protein	nsd	3.9	nsd	0.0323
yvqH	Unknown; similar to phage shock protein	nsd	3.0	nsd	0.0003
ywoH	Unknown; probable transcriptional regulator (MarR family)	4.1	4.1	<0.0001	<0.0001
ywoG	Unknown; similar to antibiotic resistance protein	4.1	4.1	<0.0001	<0.0001
ywtF	Unknown; similar to LytR	3.7	3.0	<0.0001	0.0015
yxaC	Unknown; conserved	nsd	3.9	nsd	<0.0001

Microarrays were probed with Cy3- or Cy5-labeled cDNA isolated from 60 min germinated space-exposed or simulated Mars-exposed spores and compared to 60 min germinated ground control spores.

Annotated functions assigned using the Bacillus subtilis Genome Database BSORF (http://bacillus.genome.ad.jp).

Genes showing an average≥3-fold up-regulation (n=4) with a P≤0.05 were considered significant.

nsd, not significantly different.

3.3. Up-regulated genes in space- and/or simulated Mars exposed-spores versus ground control spores

All bacteria, including B. subtilis, encode a large number of cross-regulated and interlocking regulons devoted to coping with environmental stresses (reviewed in Storz and Hengge, 2011). In B. subtilis, these stress regulons are controlled by a number of “master regulator” proteins such as RecA/LexA, sigma-B, CtsR, Fur, Spx, PerR, HrcA, ECF sigma factors, and so on (Tam et al., 2006; Elsholz et al., 2010). We found elevated transcript levels of several of these master regulators, including recA, sigV, hrcA, and ctsR; in most (but not all) cases, induction of these master stress-responsive genes was higher in space-exposed spores than in simulated Mars-exposed spores (Table 1).

3.3.1. RecA and the SOS regulon

Visual inspection of the data immediately revealed a striking up-regulation of genes involved in DNA repair and the SOS response in space-exposed spores but not in simulated Mars-exposed spores (Table 1). This result confirmed several earlier observations that exposure of spores to space conditions damages DNA (see Moeller et al., 2012 for details). The SOS regulon has been well characterized at the molecular level in vegetative cells of B. subtilis, including its response to DNA-damaging agents such as UV radiation, mitomycin-C, and mild ionizing radiation (Au et al., 2005; Goranov et al., 2006). These previous studies enabled us to compare transcriptional activation of SOS genes in space-exposed or simulated Mars-exposed spores to known genes involved in the SOS response.

The classic SOS regulon has been defined by genes whose expression was induced significantly by exposure of vegetatively growing B. subtilis cells to the DNA-damaging agents mitomycin-C (which causes mainly interstrand crosslinks) and 254 nm UV (which mainly causes pyrimidine dimers) (Au et al., 2005; Friedberg et al., 2006). We therefore compared the DNA repair and SOS regulon genes identified from germinated space- or simulated Mars-exposed spores (Table 1) to the set of genes previously identified by microarray experiments as belonging to the classic B. subtilis SOS regulon (Au et al., 2005) (Fig. 4). We observed that out of a total of 38 SOS-regulon genes defined by Au et al. (2005), 23 SOS genes were not significantly up-regulated during germination of either space- or simulated Mars-exposed spores (Fig. 4, white arrows). Fifteen SOS regulon genes were identified as induced significantly during germination of spores exposed to space but not in spores exposed to simulated martian conditions (Fig. 4, black arrows). A single SOS regulon gene (dnaE) was significantly up-regulated during germination of both space- and simulated Mars-exposed spores (Fig. 4, gray arrow with black outline). The yqjWXYZ operon was identified as belonging to the SOS regulon (Au et al., 2005) and was significantly up-regulated in space-exposed, but not simulated Mars-exposed, spores (Fig. 4). The first gene of the operon, yqjW, has been identified as encoding the homologue of the Y-family translesion bypass DNA polymerase UmuC (Sung et al., 2003; Rivas-Castillo et al., 2010), but the functions of the downstream genes yqjX, yqjY, or yqjZ are currently unknown (Table 1). Upstream from the yqjWXYZ operon is located the divergently transcribed yqzH gene, and downstream from yqjWXYZ are located the yqkABC operon and the yqkED divergon (Fig. 4). The functions of these flanking genes are currently not known, nor were they previously identified as SOS regulon genes. It is interesting to note, however, that expression of all genes in this region was specifically up-regulated during germination of space-exposed, but not simulated Mars-exposed, spores (Table 1; Fig. 4, gray arrows).

FIG. 4.

Map of the B. subtilis genome summarizing the locations of SOS regulon genes (modified from Au et al., 2005). Scale is in kilobase pairs. Shown are SOS regulon genes whose expression was not induced by exposure of spores to space or simulated martian conditions (white arrows); SOS regulon genes that were up-regulated in spores exposed to space, but not simulated martian, conditions (black arrows); genes whose expression was up-regulated in space-exposed spores, but not in Mars-exposed spores or defined in the SOS regulon (gray arrows); and the dnaE gene, which was identified as an SOS regulon gene and whose expression was up-regulated in both space- and simulated Mars-exposed spores (gray arrow with black outline). Overlapping arrows denote genes that belong to putative operons. The gray bars denote the locations of prophages PBSX and SPβ.

A novel set of SOS genes induced by treatments that introduce double-strand (ds) breaks in DNA was recently uncovered, including yneAB, msm, dinB, aprX, ymaC, yhaZ, lexA, and recA (Simmons et al., 2009). It is of interest to note that we observed significant up-regulation of a subset of these genes (yneAB, aprX, ymaCD, and recA) during germination of space-exposed, but not simulated Mars-exposed, spores (Table 1), which supports earlier findings that space exposure causes ds DNA breaks in spores (reviewed in Nicholson et al., 2000).

A dramatic consequence of the classic SOS response in B. subtilis is prophage induction, which results from activated RecA cleavage of prophage repressors (Yasbin et al., 1993). Bacillus subtilis strain 168 carries two resident prophages, SPβ and the defective prophage PBSX (Zahler, 1993). No up-regulated PBSX genes were identified in germinating space-exposed or simulated Mars-exposed spores; however, a large number of SPβ genes were up-regulated (Table 2). Consistent with an SOS-like response, out of a total of 186 SPβ genes (Sonenshein et al., 2002), 68 were up-regulated in space-exposed spores, and a subset of 38 were also up-regulated in simulated Mars-exposed spores (Table 2).

Table 2.

Prophage SPβ Genes Up-Regulated during Germination of Space-Exposed (S) or Simulated Mars-Exposed (M) Spores ^a

		Fold up-regulation ^c		P value ^c
SPβ gene	Annotated function ^b	S	M	S	M
bnrdE (yosN, yosO)	Putative ribonucleoside-diphosphate reductase alpha chain	4.8	nsd^d	0.0044	nsd
bnrdF (yosP)	Putative ribonucleoside-diphosphate reductase beta chain	6.0	nsd	<0.0001	nsd
bnrdI (yosM)	Putative ribonucleotide reductase associated protein	6.6	nsd	0.0288	nsd
ligB (yoqV)	ATP-dependent DNA ligase	6.2	6.0	0.0002	0.0003
mtbP	modification methylase Bsu	3.6	nsd	0.0012	nsd
sspC	Minor small, acid-soluble spore protein (SASP)	6.3	3.3	0.0318	0.0349
uvrX (yolE)	Translesion bypass DNA repair polymerase	10.2	nsd	0.0500	nsd
yolD	Unknown; probably cotranscribed with uvrX	10.2	nsd	0.0359	nsd
yomQ	Unknown	3.9	nsd	0.0358	nsd
yomZ	Unknown	3.2	nsd	0.0224	nsd
yonF	Unknown	3.1	nsd	0.0004	nsd
yonJ	Unknown	3.4	nsd	<0.0001	nsd
yonT	Unknown	78.9	9.0	0.0006	0.0036
yonU	Unknown	38.2	8.3	0.0012	0.0004
yonV	Unknown	17.3	nsd	0.0006	nsd
yonX	Unknown	119.3	42.8	<0.0001	<0.0001
yopA	Unknown	15.7	8.3	<0.0001	<0.0001
yopP	Putative integrase/recombinase	3.8	4.1	<0.0001	<0.0001
yopX	Unknown	8.9	7.2	<0.0001	0.0001
yopY	Unknown	7.0	6.0	0.0089	0.0046
yopZ	Unknown	7.1	7.4	0.0005	0.0500
yoqA	Unknown	6.5	6.6	0.0268	0.0093
yoqB	Unknown	3.2	nsd	0.0422	nsd
yoqD	Putative DNA-binding protein	3.3	3.4	0.0021	0.0064
yoqE	Unknown	18.4	13.2	0.0047	0.0151
yoqG	Unknown	10.8	8.5	0.0062	0.0038
yoqH	Unknown	12.0	6.9	<0.0001	0.0003
yoqI	Unknown	5.5	4.6	0.0078	0.0098
yoqJ	Unknown	6.1	3.5	<0.0001	<0.0001
yoqX	Unknown	3.8	nsd	0.0039	nsd
yoqY	Unknown	4.0	nsd	0.0131	nsd
yoqZ	Unknown	3.0	nsd	0.0008	nsd
yorE	Unknown	4.4	nsd	0.0348	nsd
yorF	Unknown	7.1	nsd	<0.0001	nsd
yorG	Unknown	10.9	4.0	<0.0001	0.0054
yorH	Unknown	10.0	nsd	0.0003	nsd
yorI	Putative replicative DNA helicase	14.8	3.6	<0.0001	0.0333
yorJ	Unknown	4.0	nsd	0.0248	nsd
yorK	Putative single-strand DNA-specific exonuclease	3.6	nsd	0.0023	nsd
yorM	Unknown	3.8	nsd	<0.0001	nsd
yorR	Unknown	3.5	nsd	<0.0001	nsd
yorS	Unknown	4.7	nsd	0.0011	nsd
yorT	Unknown	3.4	nsd	0.0062	nsd
yorZ	Unknown	5.3	4.0	0.0153	0.0082
yosA	Unknown	22.9	12.5	0.0055	0.0091
yosB	Unknown	15.6	8.2	0.0122	0.0290
yosC	Unknown	16.4	6.3	<0.0001	<0.0001
yosD	Unknown	10.1	3.7	0.0345	0.0251
yosF	Unknown	32.7	12.2	<0.0001	0.0034
yosG	Unknown	34.0	11.9	0.0246	0.0298
yosH	Unknown	24.0	7.6	<0.0001	0.0004
yosJ	Unknown	25.3	7.1	0.0188	0.0222
yosL	Unknown	14.0	3.2	0.0050	nsd
yosQ	Putative homing endonuclease	4.5	nsd	0.0033	nsd
yosS	Putative dUTP nucleotidohydrolase	5.7	nsd	<0.0001	nsd
yosT	Putative DNA gyrase inhibitory protein	4.0	nsd	0.0221	nsd
yosV	Unknown	5.5	nsd	0.0017	nsd
yosX	Unknown	5.6	3.4	0.0192	0.0051
yosZ	Unknown	7.9	4.8	0.0002	0.0053
yotB	Unknown	6.3	nsd	0.0002	nsd
yotC	Unknown	16.0	4.6	0.0419	nsd
yotD	Unknown	8.7	4.1	0.0194	nsd
yotE	Unknown	12.2	5.9	0.0446	nsd
yotF	Unknown	9.0	3.8	0.0196	0.0166
yotG	Unknown	24.0	7.0	0.0007	nsd
yotH	Unknown	10.6	5.3	0.0041	0.0221
yotI	Unknown	16.9	3.8	0.0493	nsd
yotM	Unknown	4.9	nsd	0.0038	nsd

a,b,c,d

Footnotes are as described in Table 1.

3.3.2. Non-SOS regulon DNA repair genes

Further inspection of the data in Table 1 revealed a number of genes involved in DNA repair and recombination, not previously identified as part of the SOS regulon, that were significantly up-regulated in space-exposed but not simulated Mars-exposed spores (Table 1). The addABsbcDCyisB operon encodes the AddAB helicase/nuclease, the homologue of the E. coli RecBCD recombination repair protein (Kooistra et al., 1993; Yeeles et al., 2011), and sbcDC encode the SbcCD complex important in repair of strand breaks and crosslinks in B. subtilis (Sharples and Lloyd, 1993; Mascarenhas et al., 2006). The recUponA operon encodes RecU, a Holliday junction resolvase important for both double-strand break repair (Sanchez et al., 2005) and chromosome segregation (Pedersen and Setlow, 2000; Carrasco et al., 2004), and PonA, a penicillin binding protein (PBP-1) important in forming the cell division septum (Pedersen et al., 1999). The addABsbcDCyisB operon was only significantly up-regulated during germination of space-exposed spores, while the recU gene (but not the ponA gene) was up-regulated in both space- and simulated Mars-exposed spores (Table 1). In addition, the radA and disA DNA repair and integrity-scanning genes embedded in the ctsR operon (see Section 3.3.3 below) were induced only in space-exposed spores. Germination induction of these genes further supports the notion that DNA is a major target of damage by space exposure and, to a much lesser extent, by simulated Mars exposure.

3.3.3. Other stress response regulators

Various environmental stresses provoke protein damage and misfolding, and B. subtilis possesses a system for monitoring protein quality within the cell. A number of ATPases (ClpC, ClpE, ClpX, ClpY) bind to misfolded proteins and either act as molecular chaperones or direct the misfolded protein to the associated proteases ClpP or ClpQ for degradation (Gerth et al., 2004). The Clp protease system is regulated by the CtsR repressor encoded in the ctsRmcsABclpCradAdisA operon, which was found to be significantly up-regulated in space-exposed, but not simulated Mars-exposed, spores (Table 1). However, the Clp ATPase-encoding clpC and clpE genes, and the Clp protease-encoding clpP gene, were all significantly up-regulated in both space- and simulated Mars-exposed spores (Table 1). Particularly notable was the dramatic (>50-fold) up-regulation of clpE, perhaps due to the dual role of ClpE in protein quality monitoring and proteolytic regulation of CtsR activity itself (Miethke et al., 2006). The data suggest that protein damage/denaturation is also a likely consequence of exposure to space and, to a lesser extent, simulated martian conditions.

The HrcA repressor controls expression of the class I heat shock regulon, which consists of only two operons coding mostly for protein chaperones for refolding denatured proteins, hemNhrcAgrpEdnaK and groESgroEL (Schumann, 2003). Interestingly, only the hrcA transcript itself, not its target operons, was significantly up-regulated in both space- and simulated Mars-exposed spores (Table 1).

The PerR regulon consists of a group of genes induced in response to oxidative stress, including mrgA, katA, ahpCF, a heme biosynthetic operon (hemAXCDBL), a zinc uptake system (zosA), fur, and perR itself (Herbig and Helmann, 2002). Although perR, ahpCF, and fur transcripts were not significantly elevated, we did observe significant up-regulation of mrgA and katA during germination of both space- and simulated Mars-exposed spores. Also, significant up-regulation of hemAXCDBL was observed in space-exposed, but not simulated Mars-exposed, spores (Table 1). Interestingly, we also noted significant up-regulation of the hemEHY operon during germination of space-exposed, but not simulated Mars-exposed, spores (Table 1). The prior observation that heme biosynthesis requires all nine products from both hemAXCDBL and hemEHY operons (Hansson and Hederstedt, 1992) and that both hem operons were induced during germination of space-exposed spores (Table 1) suggests that the hemEHY operon may also be part of the PerR regulon.

The sigV gene encodes SigV, one of seven so-called extracytoplasmic function (ECF) sigma factors involved in response to antibiotics and other cell envelope-stressing stimuli (Guariglia-Oropeza and Helmann, 2011). A major operon induced by SigV is the autoregulated sigVrsiVoatAyrhK operon encoding SigV itself, which we found up-regulated by 4- to 6-fold during germination of both space- and simulated Mars-exposed spores (Table 1). However, another major SigV-dependent operon, dltABCDE (Guariglia-Oropeza and Helmann, 2011), was not significantly up-regulated in either set of spores.

3.3.4. Sporulation genes

It has previously been reported that a number of transcripts encoded by sporulation genes were found in the dormant spore, including the unknown genes ykzE, ymfJ, yhcV, yqfX, ythC, ythD, and coxA; genes encoding the minor small, acid-soluble spore proteins (SASPs) (sspN, tlp, and sspO); and the major SASP-γ gene sspE (Keijser et al., 2007). It was postulated that these represented abundant late sporulation transcripts that persisted into the dormant spore, consistent with the observation that these transcripts were rapidly degraded and disappeared within the first 30 min of spore germination (Keijser et al., 2007). We also found a number of sporulation genes apparently preferentially up-regulated during germination of both space- or simulated Mars-exposed spores. As reported by Keijser et al. (2007), we also noted increased transcript levels of the unknown genes yhcV, ykzE, ymfJ, yqfX, and ythC (but not ythD); genes encoding minor SASPs of the α/β-type (sspF, sspJ, sspM, sspNtlp, and sspOP) (Setlow, 2007); and the major SASP-γ gene (sspE) (Vyas et al., 2011) (Table 1). In addition, however, we noted significant up-regulation of the cgeAB operon, products of which are involved in maturation of the outermost spore layer (Roels and Losick, 1995); cotG, cotT, and cotVWXY, products of which are components of the spore coat (Driks, 1999; Kim et al., 2006); the safAcoxA operon, products of which are involved in spore morphogenesis and spore cortex formation (Ozin et al., 2000); and rapAphrA, products of which are involved in initiation of sporulation (Pottathil and Lazazzera, 2003) (Table 1).

3.4. Down-regulated genes in space- and/or simulated Mars-exposed spores versus ground control spores

In addition to genes up-regulated during germination, a smaller subset of significantly down-regulated genes was found (Table 3). Only 22 and 9 genes of known function were significantly down-regulated during germination of space- and simulated Mars-exposed spores, respectively (Fig. 2). Many of the down-regulated genes encoded products involved in the following: the cell envelope, such as transport proteins, lipid biosynthetic enzymes, and membrane-associated nuclease; the translational apparatus, such as a few ribosomal proteins and tRNA synthetases; and cold-shock proteins. Large-scale expression patterns of known regulons could not be readily discerned from the data without heavy speculation; thus the data are presented but not discussed in full detail.

Table 3.

Genes Down-Regulated during Germination of Space-Exposed (S) or Simulated Mars-exposed (M) Spores ^a

		Fold down-regulation ^c		P value ^c
Category	Annotated function ^b	S	M	S	M
1. Cell envelope and cellular processes
1.2. Transport/binding proteins and lipoproteins
fhuD	Ferrichrome transport, substrate-binding protein	4.6	nsd^d	<0.0001	nsd
glpT	Glycerol-3-phosphate permease (in glpTQ operon)	4.5	4.0	0.0053	0.0049
manP	PTS mannose-specific enzyme IIBCA component	3.0	3.0	0.0003	0.0003
opuAA	Glycine betaine transport system, ATP-binding protein	4.4	nsd	<0.0001	nsd
opuAB	Glycine betaine transport system, permease	3.5	nsd	0.0010	nsd
opuAC	Glycine betaine transport system, substrate-binding protein	3.8	nsd	<0.0001	nsd
1.8. Sporulation
rapI	Response regulator aspartate phosphatase	3.5	nsd	0.0500	nsd
phrI	Regulator of RapI activity (both in rapIphrIyddM operon)	3.3	nsd	0.0266	nsd
1.10. Transformation/competence
comI (nucA)	Membrane-associated nuclease	3.2	nsd	0.0001	nsd
2. Intermediary metabolism
2.1. Metabolism of carbohydrates and related molecules
2.1.1. Specific pathways
manA	Mannose-6-phosphate isomerase	8.0	8.0	0.0009	0.0009
pta	Phosphotransacetylase	5.3	3.5	0.0042	0.0097
2.1.2. Main glycolytic pathways
fbaA	Fructose-bisphosphate aldolase	3.3	nsd	0.0003	nsd
2.2. Metabolism of amino acids and related molecules
speE	Spermidine synthase	3.4	nsd	<0.0001	nsd
2.3. Metabolism of nucleotides and nucleic acids
purA	Adenylosuccinate synthetase	3.6	nsd	0.0003	nsd
2.4. Metabolism of lipids
acpA	Acyl carrier protein	3.1	nsd	0.0020	nsd
glpQ	Glycerophosphoryl diester phosphodiesterase (in glpTQ operon)	4.3	3.5	<0.0001	<0.0001
plsC	1-acylglycerol-3-phosphate O-acyltransferase	3.6	nsd	<0.0001	nsd
fabI	Enoyl-acyl-carrier protein reductase	4.3	nsd	<0.0001	nsd
2.5. Metabolism of coenzymes and prosthetic groups
nadE	NH₃-dependent NAD⁺ synthetase	3.5	nsd	<0.0001	nsd
3. Information pathways
3.4. DNA packaging and segregation
hbs	Non-specific DNA-binding protein HBsu	nsd	3.8	nsd	0.0068
3.5. RNA synthesis
3.5.2. Transcription regulation
abrB	Pleiotropic transcription regulator of transition state genes	3.6	nsd	0.0009	nsd
degU	Two-component response regulator, involved in degradative enzyme and competence regulation	3.4	nsd	<0.0001	nsd
lrpA	Transcriptional regulator involved in glyA transcription and KinB-dependent sporulation	5.3	nsd	<0.0001	nsd
manR	Transcriptional antiterminator (BglG family), regulates manPAyjdF operon	7.9	7.9	0.0001	0.0001
pyrR	Transcriptional attenuator of the pyrimidine operon	3.4	nsd	<0.0001	nsd
3.5.3. Transcription elongation
rpoE	RNA polymerase delta subunit	3.7	nsd	<0.0001	nsd
3.7. Protein synthesis
3.7.1. Ribosomal proteins
rpsT	30S ribosomal protein S20	3.4	nsd	0.0014	nsd
rpsO	30S ribosomal protein S15	3.3	nsd	0.0024	nsd
3.7.2. Amino acyl-tRNA synthetases
serS	Seryl-tRNA synthetase	3.9	nsd	<0.0001	nsd
3.8. Protein folding
ppiB	Peptidyl-prolyl isomerase	4.3	nsd	0.0002	nsd
4. Other functions
4.1. Adaptation to atypical conditions
cspB	Major cold-shock protein; RNA chaperones facilitating translation initiation at low temperatures	6.0	3.2	0.0009	0.0033
cspD	Cold-shock protein; RNA chaperone facilitating translation initiation at low temperatures	3.9	nsd	0.0068	nsd
dps	Stress- and starvation-induced gene controlled by sigma-B	3.8	3.8	0.0004	<0.0001
4.2. Detoxification
sodA	Superoxide dismutase	4.6	nsd	<0.0001	nsd
5. Unknown
ybcP	Cell killing factor production; similar to coenzyme PQQ synthesis protein	5.0	nsd	<0.0001	nsd
ybfF	Unknown	4.3	3.2	0.0001	0.0001
ycdA	Unknown	4.2	4.1	<0.0001	0.0024
yclQ	Probable ferrichrome transport system substrate-binding protein	3.2	nsd	0.0142	nsd
ycsE	Unknown	3.2	nsd	0.0007	nsd
yddM	Unknown (in rapIphrIyddM operon)	3.2	nsd	<0.0001	nsd
yddK	Unknown	6.9	nsd	0.0055	nsd
yddQ	Unknown	4.6	nsd	<0.0001	nsd
yddR	Unknown	6.8	nsd	<0.0001	nsd
ydeA	Unknown	4.0	nsd	<0.0001	nsd
ydeS	Putative transcriptional regulator (TetR/AcrR family)	3.3	nsd	<0.0001	nsd
ydhO	Putative PTS oligo-beta-mannoside-specific enzyme IIC component	3.6	nsd	<0.0001	nsd
ydiR	DNA restriction protein	3.7	nsd	0.0003	nsd
ydiS	DNA restriction protein	3.8	nsd	0.0028	nsd
yfhQ	Unknown; putative iron(III) dicitrate transport system substrate-binding protein	4.8	nsd	<0.0001	nsd
yfiY	Unknown; similar to iron(III) dicitrate transport permease	6.4	3.3	<0.0001	<0.0001
yfmC	Unknown; putative ferrichrome transport system substrate-binding protein	4.1	nsd	<0.0001	nsd
yjcS	Unknown; probable acetyltransferase	4.0	nsd	0.0035	nsd
yjdF	Unknown (in manPAyjdF operon)	4.6	3.1	<0.0001	<0.0001
ykuJ	Unknown	3.3	nsd	0.0324	nsd
ykuK	Unknown	3.2	nsd	<0.0001	nsd
ykuN	Similar to flavodoxin	5.0	nsd	0.0007	nsd
ykuO	Unknown	4.8	nsd	<0.0001	nsd
ykuP	Similar to flavodoxin	4.5	nsd	<0.0001	nsd
ykyA	Unknown	3.2	nsd	0.0023	nsd
ylbN	Unknown	3.3	nsd	0.0002	nsd
ynaB	Unknown	4.7	nsd	0.0038	nsd
ynaE	Unknown	6.7	nsd	<0.0001	nsd
ynaF	Unknown	3.7	nsd	0.0007	nsd
yneF	Unknown	4.4	nsd	0.0307	nsd
yobL	Unknown	3.3	nsd	0.0002	nsd
yobI	Unknown	3.7	nsd	0.0002	nsd
yocH	Putative peptidoglycan hydrolase	5.1	nsd	0.0005	nsd
yqgA	Unknown	4.8	nsd	<0.0001	nsd
yqzC	Unknown	3.4	nsd	0.0013	nsd
yqzI	SKIN protein	3.0	nsd	0.0015	nsd
ytrE	Possible acetoin transport system ATP-binding protein	3.2	nsd	<0.0001	nsd
yufK	Unknown	3.3	nsd	0.0002	nsd
yukE	Unknown	5.4	4.0	0.0382	0.0399
yvaW	Production of extracellular factor able to induce yvbA transcription	3.5	nsd	0.0103	nsd
yviA	Unknown	3.6	nsd	0.0014	nsd
yvyC	Similar to flagellar protein	3.1	nsd	0.0092	nsd
ywiB	Unknown	3.6	nsd	<0.0001	nsd
ywpB	Putative hydroxymyristoyl-(acyl carrier protein) dehydratase	5.0	nsd	0.0232	nsd
yxaI	Unknown; putative membrane protein	3.2	nsd	<0.0001	nsd
yxeB	Putative ferrichrome transport system substrate-binding protein	9.1	3.8	<0.0001	<0.0001
yxiD	Unknown	6.5	nsd	<0.0001	nsd
yxxD	Unknown	11	3.7	0.0005	0.0205
yxxE	Unknown	5.5	nsd	0.0225	nsd
yybN	Unknown	3.0	nsd	<0.0001	nsd
yybL	Unknown	3.9	3.8	<0.0001	0.0002
yybK	Unknown	3.5	3.5	0.0081	0.0061
yybJ	Similar to ABC transporter ATP-binding protein	3.0	3.0	<0.0001	<0.0001
yydA	Conserved protein	3.8	nsd	<0.0001	nsd
yydB	Unknown	6.4	nsd	<0.0001	nsd
yydD	Conserved protein	7.5	nsd	<0.0001	nsd
yydF	Unknown	2.1	nsd	0.1227	nsd
yydG	Unknown	3.1	nsd	<0.0001	nsd
yydH	Membrane metalloprotease	8.6	6.2	<0.0001	0.0001
yydI	Similar to ABC transporter (ATP-binding protein)	4.2	nsd	0.0127	nsd

a,b,c,d

Footnotes as in Table 1.

4. Discussion

4.1. Microarray analysis of PROTECT spore samples

Dormant B. subtilis spores were exposed to the space environment in low-Earth orbit and to a simulation of martian surface conditions for 559 days aboard the PROTECT experiment on the ISS. These environmental parameters were designed to simulate a hypothetical journey to, and residence on, Mars of spacecraft-contaminant spores (see Horneck et al., 2012). It has been known from previous experiments spanning the past 40 years that spore exposure to the space environment causes both lethal and mutagenic DNA damage to spores (reviewed in Nicholson et al., 2000; Horneck et al., 2012; Moeller et al., 2012). More recent research has also shown significant lethal and mutagenic DNA damage to spores exposed to simulated martian surface conditions (Schuerger et al., 2003, 2006; Perkins et al., 2008; Fajardo-Cavazos et al., 2010; Moeller et al., 2011). However, very little attention has been devoted to studying damage to other cellular constituents by space or Mars exposure. We knew that dormant spores lack metabolism and thus cannot repair cellular damage until metabolism is re-activated during germination (Nicholson et al., 2000). We reasoned that germinating spores would induce the expression of specific cellular repair pathways in response to the specific type of cellular component damaged; we could thus infer the type(s) of damage spores suffered by identifying which genes were specifically induced. To accomplish this goal, we performed transcription microarray analyses on RNA isolated from 60 min germinated space- or simulated Mars-exposed spores, versus RNA isolated from 60 min germinated spores from unexposed ground control samples. We found that germinating spores exhibited a strong stress response by inducing the expression of several known stress-related regulators including recA, sigV, hrcA, and ctsR. In general, two interesting observations were made. (i) Induction of these master stress-responsive genes was generally stronger and more extensive in space-exposed spores than in simulated Mars-exposed spores, which indicates that spore exposure to the space environment caused a wider spectrum and more severe cellular damages than spore exposure to simulated martian conditions. (ii) Induced expression of a particular master regulator usually caused induction of only a subset of that regulator's putative target genes, which implies that space or Mars exposure exerted a unique set of stresses upon dormant spores. It can be imagined that some of these stresses (e.g., ultrahigh vacuum or low-pressure CO₂ atmosphere) are outside the range of conditions normally encountered by spores on Earth and thus provoke novel combinations of both cellular damages and stress responses. Specific responses will be discussed below.

4.2. SOS response

By far the major stress regulon induced in space-exposed (but not simulated Mars-exposed) spores was the SOS regulon. The recA and lexA gene products serve as the master regulators of the classic SOS regulon in B. subtilis (Au et al., 2005). The LexA protein is a transcriptional repressor of SOS-inducible genes. DNA damage provokes production of single-stranded DNA, which binds to RecA and converts it into a protease specific for cleavage of repressors of the LexA family. Cleavage of LexA leads to derepression of ∼50 LexA-repressed SOS target genes (including recA and lexA themselves).

We observed that expression of the recA gene was induced 7.5-fold in space-exposed spores but was not significantly induced in simulated Mars-exposed spores. Surprisingly, however, the microarray data failed to detect a statistically significant elevation in lexA expression in either space- or simulated Mars-exposed spores. This observation was puzzling because in previous spore germination microarray experiments we observed significant induction of both recA (5-fold) and lexA (3-fold) transcription. Bacillus subtilis 168 spores germinated for 60 min after irradiation with 254 nm UV (Moeller et al., unpublished), as would be expected for a classic SOS response. The data suggest that space exposure of spores may have induced a partial SOS response that was apparently independent of LexA cleavage. It is instructive to recall that the PROTECT spores analyzed in these experiments were shielded from solar UV (an inducer of the classic SOS response) but were subjected to space vacuum, extreme desiccation, and ionizing radiation, each of which are known to cause ds DNA breaks (reviewed in Nicholson et al., 2000). We know that the SOS response in B. subtilis is considerably more complex than its counterpart in E. coli (Yasbin et al., 1993) and that the two systems have been demonstrated to respond differently to ds DNA breaks induced in vegetative B. subtilis cells either artificially or by mild doses of ionizing radiation (Simmons et al., 2009). Furthermore, it has been shown that the DNA repair responses to ds DNA breaks in B. subtilis are substantially different in vegetative cells versus sporulating cells and spores (Moeller et al., 2007; Simmons et al., 2009). Therefore, it is not unreasonable to suppose that the circuitry controlling the SOS response during germination of spores exposed to space conditions could exhibit substantial differences from the classic SOS response. This might also explain the induction of non-SOS DNA repair genes such as addABsbcDC or recU. Regardless of the molecular details, the results strongly indicate that DNA is a major target of damage in spores exposed to the space environment and furthermore suggest that ds DNA breaks are involved.

4.3. Interlocking stress responses

From the PROTECT microarray experiments it became apparent that spores exposed to the space or simulated martian environment accumulated damage to cellular components in addition to DNA. Certainly spore proteins also sustained damage, as evidenced by partial induction of the CtsR/Clp chaperone/protease systems in both space- and simulated Mars-exposed spores. What environmental parameter(s) may have caused protein damage? Proteins can be denatured by heat or by extreme desiccation resulting from ultrahigh vacuum (Jaenicke, 1981), or they can become inactivated by ionizing radiation and reactive oxygen species (Berlett and Stadtman, 1997). In exposure to space on PROTECT, spores encountered cycles of heating and cooling, ultrahigh vacuum, and ionizing radiation, any of which could damage or denature proteins. In addition, contact with water and oxygen during subsequent germination of space-exposed spores would be expected to generate reactive oxygen species in levels higher than those experienced during the normal oxidative burst in germinating spores (Ibarra et al., 2008). Many individual stress regulons are controlled by partially overlapping control circuitries that respond to diverse environmental stimuli (reviewed in Herbig and Helmann, 2002; for recent examples, see Chaturongakul et al., 2011; Faulkner and Helmann, 2011; Hesketh et al., 2011). Therefore, it is perhaps not surprising to observe partial induction of overlapping stress regulons such as PerR, HrcA, CtsR, and SigV during germination of space- or simulated Mars-exposed spores.

4.4. Sporulation genes

As noted above (Section 3.3.3), a number of genes usually associated with sporulation were found to be up-regulated during germination of spores exposed to space or simulated martian conditions. Transcripts for some of these genes (ykzE, ymfJ, yhcV, yqfX, ythC, ythD, coxA, sspN tlp, sspO, and sspE) were previously found in dormant spores and disappeared rapidly within 30 min of initiation of germination (Keijser et al., 2007). In the PROTECT samples, were these sporulation transcripts simply left over from the previous round of sporulation, or were they synthesized de novo during germination? It is difficult to envision the former possibility for the following reasons. (i) Our RNA samples were harvested at 60 min of germination, by which time these same transcripts had disappeared in the germination experiments of Keijser et al. (2007). (ii) The induction levels we report here were calculated relative to RNA from non-treated ground control spores also harvested at 60 min of germination, thus controlling for any elevated transcript levels in non-treated spores. (iii) We found up-regulation of transcripts not found in the dormant spore, such as: sporulation genes expressed early in sporulation (rapAphrA); the additional SASP genes sspF, sspJ and sspM; and genes normally expressed from sigma-K promoters only in the mother cell (cgeAB, cotG, cotT, cotVWXY). Why space- or simulated Mars-exposed spores would up-regulate transcription of some sporulation genes is a mystery at present and can be resolved only by closer analysis of the expression of individual genes during germination with an independent method such as lacZ gene fusions.

4.5. Down-regulated genes

Although several apparently down-regulated genes were identified during germination of space- or simulated Mars-exposed spores, closer examination failed to find unifying regulons to which these genes belonged. There are likely a number of reasons for this. The majority of down-regulated genes belonged to the Unknown function category. Few genes of known function were found to be significantly down-regulated in space-exposed (22 genes) or simulated Mars-exposed (9 genes) spores, and most of these known genes were only modestly (3- to 5-fold) down-regulated. Previous investigations in the literature included measurement of the mRNA levels expressed by the same gene with two complementary techniques, microarray and quantitative reverse transcriptase-polymerase chain reaction, and concluded that the correlation between the two techniques was much greater for up-regulated genes (0.700; Spearman's Rho, P<0.0001, n=169) than for down-regulated genes (0.356; Spearman's Rho, P=0.0002, n=108); furthermore, the differences between the two groups were highly statistically significant (ANOVA, P=0.0042, n=10) (Morey et al., 2006). In other words, the authors concluded that microarray analysis is much better at identifying up-regulated than down-regulated genes (Miron et al., 2006; Morey et al., 2006).

4.6. Conclusions

Regarding planetary protection, the collective spore survival data (Horneck et al., 2012; Moeller et al., 2012; and Section 3.1 in this communication) indicate that spores shielded from solar UV radiation would likely suffer ∼50–90% reduction in viability during the cruise phase to Mars but could potentially survive for decades once deposited on the martian surface. The data presented in this communication demonstrate that microarray profiling can yield a substantial amount of insight into the types of cellular damages suffered by spores during long-term exposure to space or to a simulated martian environment. The results (i) confirm earlier investigations that indicated that spore DNA is a major molecular target for damage, (ii) demonstrate that the UV-shielded martian surface environment is much less harsh than is the UV-shielded space environment, and (iii) indicate that germinating spores are faced with repair of oxidative damage likely resulting from long-term exposure to ionizing radiation, vacuum, extreme desiccation, or a combination of these parameters. From a planetary protection perspective, the results indicate that a fraction of forward-contaminant spores on robotic spacecraft could survive an Earth-to-Mars transfer but would suffer substantial cellular damage during the journey.

(Other) Members of the PROTECT Team and Their Affiliations

Jean Cadet: Laboratoire “Lésions des Acides Nucléiques,” SCIB-UMR-E n°3 (CEA/UJF) Institut Nanosciences et Cryogénie CEA/Grenoble, Grenoble, France.

Thierry Douki: Laboratoire “Lésions des Acides Nucléiques,” SCIB-UMR-E n°3 (CEA/UJF) Institut Nanosciences et Cryogénie CEA/Grenoble, Grenoble, France.

Rocco L. Mancinelli: Bay Area Environmental Research Institute, NASA Ames Research Center, Moffett Field, California, USA.

Corinna Panitz: Institut für Flugmedizin, Technical University RWTH, Aachen, Germany.

Elke Rabbow: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology Department, Cologne, Germany.

Petra Rettberg: German Aerospace Center (DLR), Institute of Aerospace Medicine, Radiation Biology Department, Cologne, Germany.

Andrew Spry: Jet Propulsion Laboratory, Pasadena, California, USA.

Erko Stackebrandt: German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany.

Parag Vaishampayan: Jet Propulsion Laboratory, Pasadena, California, USA.

Kasthuri J. Venkateswaran: Jet Propulsion Laboratory, Pasadena, California, USA.

Footnotes

Acknowledgments

We thank Patricia Fajardo-Cavazos for insightful discussions, Samantha Waters for careful examination of the microarray analyses, and the UF-ICBR for performing the microarray experiments described in this work. This study was supported in part by grants from the NASA Planetary Protection Office (NNA06CB58G and NNX10AV22G) to W.L.N. and by the DLR grant DLR-FuE Projekt ISS-Nutzung in der Biodiagnostik, Programm RF-FuW, Teilprogramm 475 to R.M.

Author Disclosure Statement

No competing financial interests exist.

Abbreviations

ANOVA, analysis of variance; COSPAR, the Committee on Space Research; ds, double strand; ECF, extracytoplasmic function; ISS, International Space Station; LB, Luria-Bertani; ORF, open reading frame; PBS, phosphate-buffered saline; SASP, small, acid-soluble spore protein; UF-ICBR, University of Florida Interdisciplinary Center for Biotechnology Research.

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