JOURNAL OF BACTERIOLOGY, Apr. 2007, p. 3306–3311 Vol. 189, No.

8
0021-9193/07/$08.00⫹0 doi:10.1128/JB.00018-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis

Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV,
and Ionizing Radiation䌤
Ralf Moeller,1,2 Erko Stackebrandt,2 Günther Reitz,1 Thomas Berger,1 Petra Rettberg,1
Aidan J. Doherty,3 Gerda Horneck,1 and Wayne L. Nicholson4*
German Aerospace Center, Institute of Aerospace Medicine, Radiation Biology Division, Research Group Photo- and Exobiology,
Cologne, Germany1; German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany2;
Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, United Kingdom3; and

Downloaded from http://jb.asm.org/ on March 9, 2015 by ST ANDREWS UNIV

Department of Microbiology and Cell Science, University of Florida,
Kennedy Space Center, Florida 328994
Received 4 January 2007/Accepted 5 February 2007

The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation,
and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV
mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were
significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating
that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.

It has been shown that endospores of gram-positive bacteria
can remain viable for at least thousands of years (5, 44, 54;
reviewed in reference 31). Bacterial spores persist in a meta-
bolically inactive state, and environmental damage to spore
cellular components accumulates unrepaired until germination
and outgrowth (32). However, Bacillus subtilis spores are
highly resistant to different environmental stresses, such as
toxic chemicals and biocidal agents, desiccation, pressure and
temperature extremes, and ionizing and UV radiation. The
reason for this high resistance to environmental extremes lies
partly in the spore structure itself: spores possess thick layers
of highly cross-linked coat proteins (13), a modified pepti-
doglycan spore cortex, and abundant intracellular constituents
such as the calcium chelate of dipicolinic acid and small, acid-
soluble spore proteins (␣/␤-type SASP), as protectants of
spore DNA (46, 50). Binding of ␣/␤-type SASP to spore DNA,
coupled with spore core dehydration, appears to change the
helical conformation of spore DNA from the B form to an
A-like form (34, 48), which in turn alters its UV photochem-
istry to favor the production of 5-thyminyl-5,6-dihydrothymine,
the unique spore-specific spore photoproduct (SP) (8, 32, 35,
50). For the removal of the SP, spores possess an SP-specific
repair enzyme called SP lyase, encoded by the splB gene, that
monomerizes the SP dimer back to two thymine residues in an
adenosyl-radical-dependent reaction (4, 28, 42).
While the UV photochemistry of spore DNA and the repair
of UV damage to DNA during germination are well described
(12, 32, 33, 47, 50), there has been relatively little work on the
nature of DNA damage in spores caused by ionizing radiation
or extreme dryness and on the occurrence of a specific DNA
* Corresponding author. Mailing address: Rm. 201-B, Space Life
Sciences Laboratory, Building M6-1025/SLSL, Kennedy Space Center,
FL 32953. Phone: (321) 861-3487. Fax: (321) 861-2925. E-mail: WLN
@ufl.edu.
䌤
Published ahead of print on 9 February 2007.
repair system(s) for repair of this damage. It is assumed that
DNA double-strand breaks (DSB), which are the most critical
damage caused by ionizing radiation (57) and desiccation (9,
10, 39) in vegetative cells, are also induced in bacterial spores.
Spores of B. subtilis contain a single chromosome arranged in
a toroidal shape (16, 41); therefore, the homologous recombi-
nation pathway, which requires at least two homologous chro-
mosomes, cannot operate on DSB during spore germination
(55). An alternative repair pathway for DSB induced in spore
DNA, nonhomologous-end joining (NHEJ) (3, 56), is consid-
ered here. This pathway as it occurs in eukaryotic cells requires
a DNA end-binding component called Ku (Ku70 and Ku80)
(58). The first step in the NHEJ DNA repair mechanism in-
volves the binding of the Ku complex to the two DSB ends. The
next proteins to be recruited to the complex are those that lead
to resection of the ends of a DSB. The final set of steps in
NHEJ leads to direct joining of the two ends by a specific DNA
ligase that restores the integrity of the DNA. The ligation of
DNA strands is an energy-dependent process, and ATP is
required for the catalysis of the formation of a phosphodiester
at the site of a single-strand break (SSB) in a duplex DNA (58).
Recently, Weller et al. (57) identified in B. subtilis a Ku ho-
molog (encoded by the ykoV gene), which retains the biochem-
ical characteristics of the eukaryotic Ku heterodimer. The bac-
terial Ku specifically recruits a DNA ligase (encoded by ykoU)
to DNA ends and thereby stimulates DNA ligation. Loss of
these proteins leads to hypersensitivity of stationary-phase B.
subtilis cells to ionizing radiation (57). From the observation
that the Ku system is conserved in spore-forming bacterial
species (e.g., Bacillus and Streptomyces spp.), Weller et al. (57)
speculated that NHEJ via the prokaryotic Ku system might
function during subsequent spore germination to repair DSB
induced in dormant bacterial spores. Recently, Wang et al.
(55) showed by microarray analyses that the ykoU and ykoV
genes of the ykoWVU operon were under the control of sigma-G
RNA polymerase during forespore development. They showed

3306
VOL. 189, 2007 NOTES 3307

TABLE 1. B. subtilis strains used in this study ykoV and the double ykoU ykoV mutant (57) were used (Table
Strain Genotype and/or phenotype b
Source (reference)
1). All the strains are isogenic with the 168 wild-type strain.
Spores of each strain were obtained by cultivation under vig-
a
168 trpC2 DSM 402, DSMZ (53) orous aeration in liquid Schaeffer sporulation medium (43),
WN463 trpC2 recA::ermC MLSr R. E. Yasbin (6)
TKJ6324 trpC2 splB1 N. Munakata (14) purified, and stored as described previously (2, 25, 36). The
BFS1845 trpC2 ykoU::pMUTIN4 Cmr A. J. Doherty (57) mutations did not significantly affect sporulation efficiency.
BFS1846 trpC2 ykoV::pMUTIN4 Cmr A. J. Doherty (57) Spore preparations were free (⬎98%) of growing cells, germi-
BFS1846 trpC2 ykoU ykoV::pMUTIN4 Cmr A. J. Doherty (57) nating spores, and cell debris, as seen in the phase-contrast
a
Obtained from the German Collection of Microorganisms and Cell Cultures microscope. Spore samples consisted of air-dried spore mono-
(DSMZ) GmbH, Braunschweig, Germany. layers (2 ⫻ 107 spores) immobilized on 7-mm-diameter quartz
b
MLSr, resistant to lincomycin (25 ␮g/ml) and erythromycin (1 ␮g/ml); Cmr,
resistant to chloramphenicol (5 ␮g/ml). splB1 carries two point mutations causing discs (Heraeus Quarzglas GmbH & Co. Kg, Hanau, Germany),
changes in the amino acids G168R and G242D in SP lyase (14). as described previously (40). For studying the effects of UV

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that ykoV and ykoU ykoV mutant spores were significantly
more sensitive than wild-type spores to dry heat, a treatment
known to cause DNA strand breaks, but no difference in hy-
drogen peroxide resistance between mutant and wild-type
spores was observed (55). These observations led to the as-
sumption that SP lyase, also expressed by sigma-G RNA poly-
merase during sporulation (32, 37), and NHEJ are both spore-
specific DNA repair pathways, components of which are
synthesized in the forespore during sporulation, packed in the
dormant spore, and activated during germination.
We have been conducting ongoing studies on the role of
DNA repair in the resistance of bacterial spores to the space
environment, which is characterized by extreme vacuum, high
solar UV flux, and ionizing radiation (reviewed in references
15, 18, 20, 32, and 33), all of which are implicated in the
formation of DSB and SSB in DNA. Therefore, in this com-
munication we report results from experiments examining the
role of NHEJ in spore resistance to UV, ionizing radiation,
and extreme dryness induced by high vacuum. To investigate
the capability for DNA repair via the NHEJ pathway during
spore germination, spores of B. subtilis 168 wild type (53) and
of repair-deficient mutants with mutations in the homologous
recombination gene recA (6), the SP lyase gene splB (11, 14),
the NHEJ ligase-like gene ykoU, and the NHEJ Ku-like gene
and ionizing-radiation-induced DNA damage on spore sur-
vival, samples were exposed to monochromatic UV-C radia-
tion from a low-pressure mercury lamp (NN 8/15; Heraeus,
Berlin, Germany) with a major emission line at 254 nm, to
defined spectral ranges of UV-A plus UV-B (UV-A⫹B) (290
to 400 nm) or UV-A (320 to 400 nm) radiation obtained with
a 1,000-W xenon arc lamp (Polytech, Waldbronn, Karlsruhe,
Germany) and optical filter combinations (Schott AG, Mainz,
Germany) (24), or to ionizing radiation provided in the form of
X rays (150 keV/19 mA) generated by an X-ray tube (Mueller
type MG 150, MCN 165; Phillips, Hamburg, Germany). Do-
simetry and dose calculations were performed as described
previously (23). For studying the effect of vacuum-induced
extreme desiccation, samples were exposed to ultrahigh vac-
uum produced by an ion-getter pumping system (400 liter/s;
Varian SpA, Torino, Italy) reaching a final pressure of 10⫺7 Pa
(18, 29).
To recover the spores from the quartz discs after treatments,
the spore monolayer was covered by a 10% aqueous polyvinyl
alcohol solution; after air drying, the spore-polyvinyl alcohol
layer was stripped off as described previously (19). Spores were
resuspended in 1 ml sterile distilled water, resulting in ⬎95%
recovery of the spores. This procedure has no geno- or cyto-
toxic effect on the viability of the spores (21). Spore survival
was determined from serial dilutions in distilled water as CFU
after growth overnight on nutrient broth agar (Difco, Detroit,

FIG. 1. Survival curves of B. subtilis strains in response to 254-nm UV-C (A), 290- to 400-nm UV-A⫹B (B) and 320- to 400-nm UV-A
(C) radiation. Strains: 168 (wild type) (F), recA mutant (E), ykoU mutant (Œ), ykoV mutant (‚), ykoU ykoV mutant (f), and splB mutant (ƒ). Data
are averages and standard deviations (n ⫽ 3).
3308 NOTES J. BACTERIOL.

TABLE 2. Survival characteristics of treated B. subtilis sporesa

F10c
b % S after vacuum
Strain genotype D10 (Gy) of X rays
UV-C (J/m2)
UV-A⫹B
UV-A (kJ/m2) desiccationd
(kJ/m2)

Wild type 838.1 ⫾ 98.0 273.1 ⫾ 52.2 11.4 ⫾ 1.4 364.5 ⫾ 12.3 33.4 ⫾ 10.1
recA 378.9 ⫾ 57.7* 189.2 ⫾ 29.5* 8.3 ⫾ 2.1* 136.3 ⫾ 23.0* (43.1 ⫾ 17.4) ⫻ 10⫺2*
splB 694.4 ⫾ 86.9 91.7 ⫾ 26.1* 2.2 ⫾ 0.6* 43.9 ⫾ 8.3* 19.8 ⫾ 7.6
ykoU 280.3 ⫾ 46.1* 183.2 ⫾ 30.3* 6.9 ⫾ 1.2* 110.2 ⫾ 19.7* (7.6 ⫾ 2.3) ⫻ 10⫺2*
ykoV 188.4 ⫾ 40.1* 164.7 ⫾ 25.0* 5.7 ⫾ 0.9* 86.7 ⫾ 15.5* (0.4 ⫾ 0.1) ⫻ 10⫺2*
ykoU ykoV 146.4 ⫾ 35.4* 138.8 ⫾ 17.6* 4.1 ⫾ 0.6* 63.2 ⫾ 9.5* (0.2 ⫾ 0.1) ⫻ 10⫺2*
a
Data are averages and standard deviations (n ⫽ 3). Asterisks indicate survival values that were significantly different (P ⱕ 0.05) from the survival value for wild-type
spores of Bacillus subtilis 168.
b
Dose reducing the survival of the spore population to 10%.

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c
Fluence of UV-C (254 nm), UV-A⫹B (290 to 400 nm), and UV-A (320 to 400 nm) irradiation reducing the survival of the spore population to 10%.
d
% S, survival after 30 days of exposure to high vacuum (10⫺7 Pa).

MI) at 37°C. The surviving fraction was determined from the
ratio of N/N0, with N being the number of CFU of the treated
sample and N0 the CFU of the nontreated controls. Plotting
the logarithm of N/N0 as a function of each treatment, survival
curves were obtained. Each experiment was repeated at least
three times, and the data shown are expressed as averages ⫾
standard deviations. The results were compared statistically
using Student’s t test (21, 24, 25). Values were analyzed in
multigroup pairwise combinations, and differences with P val-
ues of ⱕ0.05 were considered statistically significant.
Inactivation kinetics of wild-type and mutant B. subtilis
spores were determined in response to mono- and polychro-
matic UV radiation spanning wavelengths from 254 to 400 nm
(Fig. 1). Nearly exponential UV survival curves were obtained
for spores of all strains of B. subtilis tested (Fig. 1), and the
best-fit curves were used to calculate F10 values (i.e., fluences
reducing survival to 10%) for statistical comparison (Table 2).
With UV-C, the recA, ykoU, ykoV, and ykoU ykoV strains pro-
duced spores with slightly (1.5- to 2-fold) but significantly
lower F10 values than wild-type spores (Table 2). In contrast,
spores carrying the splB mutation were threefold more UV-C
sensitive than wild-type spores (Table 2). The slight but signif-
icant UV sensitivity of ykoU and ykoV mutant spores likely
reflects the fact that the majority of the 254-nm UV photo-
products in spore DNA are SP, whereas strand breaks such as
SSB and DSB are only minor photoproducts (52). Relative to
wild-type spores, in response to all UV wavelengths (UV-C,
UV-A⫹B, and UV-A), splB spores showed the highest rate
of inactivation (ranging from three- to ninefold), followed
by the ykoU ykoV double mutant, ykoV and ykoU single
mutants, and the recA-deficient strain (Fig. 1; Tables 2 and
3). Interestingly, both DNA strand break-repairing mecha-
nisms, homologous recombination mediated by recA and
ykoUV-mediated NHEJ, became progressively more impor-
tant to spore resistance in response to longer UV wave-
lengths (Tables 2 and 3). These observations are in good
agreement with the previous demonstration that exposure of
spores to longer artificial or solar UV-B and UV-A wave-
lengths produced a higher proportion of SSB and DSB in
spore DNA (52). The results suggest that NHEJ provides a
major DNA strand break repair strategy for spores irradi-
ated with UV-B and UV-A wavelengths of ⬎290 nm, such as
encountered on Earth’s surface (32).
Ionizing radiation induces less specific damage to DNA than
does UV radiation. There are two alternative routes of radia-
tion damage to biological components such as proteins, RNA,
and DNA: direct energy absorption (direct radiation effect)
and interactions with radicals, e.g., those produced by radiol-
ysis of cellular water molecules (indirect radiation effect) (22).
Both processes mainly cause SSB or DSB in DNA (9–11, 22,
30). The decreased water content of the spore core, which may
reduce the indirect effect, has been suggested to be responsible
for increased spore resistance to ionizing radiation (32, 49).
However, to date no DNA repair system has been identified
that acts specifically on spore DNA damage caused by ionizing
radiation. Therefore, wild-type and mutant spores were as-
sayed for their resistance to ionizing radiation delivered in the
form of X rays (Fig. 2). After exposure to X rays, strictly

TABLE 3. Repair capacity of spores of B. subtilis 168 and mutants after irradiation with mono- and polychromatic UV, X-ray irradiation, and
high-vacuum-induced extreme desiccationa,b
F10REP/F10WT D10REP/D10WT SREP/SWT
Strain genotype
UV-C UV-A⫹B UV-A (X rays) (desiccation)

Wild type 1 1 1 1 1
recA 0.7 ⫾ 0.1* 0.7 ⫾ 0.2* 0.4 ⫾ 0.1* 0.5 ⫾ 0.1* (1.3 ⫾ 0.3) ⫻ 10⫺2*
splB 0.3 ⫾ 0.1* 0.2 ⫾ 0.1* 0.2 ⫾ 0.1* 0.8 ⫾ 0.2 0.7 ⫾ 0.2
ykoU 0.7 ⫾ 0.2* 0.6 ⫾ 0.1* 0.3 ⫾ 0.1* 0.3 ⫾ 0.1* (22.9 ⫾ 6.3) ⫻ 10⫺4*
ykoV 0.6 ⫾ 0.1* 0.5 ⫾ 0.1* 0.2 ⫾ 0.1* 0.2 ⫾ 0.1* (1.2 ⫾ 0.3) ⫻ 10⫺4*
ykoU ykoV 0.5 ⫾ 0.1* 0.4 ⫾ 0.1* 0.2 ⫾ 0.1* 0.2 ⫾ 0.1* (0.6 ⫾ 0.2) ⫻ 10⫺4*
a
REP, repair-deficient spores, WT, wild type.
b
S, spore survival after 30 days of vacuum-induced desiccation. Asterisks indicate values that are significantly different (P ⱕ 0.05) from the wild-type value, as
determined by multigroup pairwise combinations (Student’s t test). Data are averages and standard deviations (n ⫽ 3).
VOL. 189, 2007
FIG. 2. Survival curves of B. subtilis strains in response to X-ray
irradiation. Strains: 168 (wild type) (F), recA mutant (E), ykoU mutant
(Œ), ykoV mutant (‚), ykoU ykoV mutant (■), and splB mutant (ƒ).
Data are averages and standard deviations (n ⫽ 3).
exponential survival curves were obtained for spores of all
repair-deficient strains of B. subtilis tested, while the wild-type
strain showed a slight shoulder in its survival curve (Fig. 2).
More dramatic differences were observed in the spore survival
kinetics of the various strains exposed to X rays (Fig. 2) than
the same strains exposed to UV-C (Fig. 1A). Indeed, the X-ray
data closely mimicked the response of the recA, ykoU, ykoV,
and ykoU ykoV strains to UV-A (Fig. 1C; Tables 2 and 3),
further supporting the notion that both UV-A and X rays
produce substantial SSB and DSB in spore DNA. This conten-
tion is further strengthened by the observation that spores of
the splB strain, which is defective in repair of the UV-induced
photoproduct SP, were not significantly more sensitive to X
rays than wild-type spores (Tables 2 and 3).
In the case of X-ray resistance, the spores of recA, ykoU,
ykoV, and ykoU ykoV strains were remarkably more sensitive
than wild-type spores, up to a factor of 7.9 in the case of ykoU
ykoV spores (Tables 2 and 3). The observed differences in
X-ray resistance of the mutant versus wild-type spores, deter-
mined as the repair capacity, were much more pronounced for
X rays than for UV radiation (Table 3). These differences in
sensitivity of the spores to X rays showed a tendency similar to
that observed for vegetative cells of the B. subtilis mutants (57).
However, Weller et al. (57) reported that in stationary-phase
B. subtilis cells, ykoU mutants were more X ray sensitive than
ykoV mutants; interestingly, in spores we observed the opposite
(Fig. 2; Tables 2 and 3).
Spores are clearly much more resistant than their vegetative
counterparts to extended desiccation both at atmospheric pres-
sure and in vacuo (9–11, 29, 32, 39). When typical laboratory
vacuum systems are used for simulated extreme desiccation,
wild-type spores often exhibit no detectable killing (9, 10). A
major reason for spore resistance to these processes is protec-
tion of spore DNA by SASP. SASP-deficient spores are much
more sensitive to extended desiccation, and killing by this
treatment is accompanied by mutagenesis and DNA damage
NOTES 3309
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FIG. 3. Survival curves of B. subtilis strains in response to ultrahigh
vacuum (10⫺7 Pa). Strains: 168 (wild type) (F), recA mutant (E), ykoU
mutant (Œ), ykoV mutant (‚), ykoU ykoV mutant (■), and splB mutant
(ƒ). Data are averages and standard deviations (n ⫽ 3).
e.g., single-strand breaks and DNA-protein cross-links (11, 49).
Previous studies of spores exposed to extreme desiccation via
ultrahigh vacuum indicated that wild-type spores were quite
resistant while DNA repair-deficient spores showed significant
sensitivity to extended dryness via vacuum (reviewed in refer-
ence 32). Although significant killing of wild-type spores was
not observed, the survivors exhibited significant mutagenesis
(29), indicating that DNA was targeted. In the present study,
spores were exposed to ultrahigh vacuum (10⫺7 Pa) for up to
30 days and assayed for survival (Fig. 3). Strict exponential
inactivation kinetics were observed for all strains (Fig. 3). After
30 days of vacuum-induced extreme desiccation, wild-type and
splB spores both showed high levels of survival (33.4% and
19.8%, respectively), which were not statistically different (Ta-
bles 2 and 3). In sharp contrast, spores carrying the recA, ykoU,
ykoV, or ykoU ykoV mutations were dramatically more sensi-
tive to high vacuum (Fig. 3), ranging from 77-fold (recA spores)
to ⬎16,000-fold (ykoU ykoV spores) as measured after 30 days
(Tables 2 and 3). The extreme sensitivity of ykoU ykoV-defi-
cient spores to high vacuum-induced desiccation is entirely
consistent with the observation that exposure to high vacuum
induces SSB and DSB formation in DNA (9, 10, 20, 32). Thus,
NHEJ is an extremely important determinant of spore survival
of the ultrahigh vacuum prevailing in space, a critical prereq-
uisite for interplanetary transport of spores by natural pro-
cesses or human spaceflight activities (15, 18, 20, 32).
Recent data indicated that ykoU and ykoV are functional in
the ionizing radiation stress response of B. subtilis, Mycobac-
terium tuberculosis, and Mycobacterium smegmatis (1, 7, 38, 57,
58). Current models of bacterial NHEJ propose that multiple
subunits of YkoV bind to DSB ends and recruit YkoU ligase to
join the ends in an ATP-dependent manner (3, 17, 38, 58). It is
worthwhile to note that dormant spores do not contain detect-
able amounts of endogenous ATP; rather, high-energy phos-
phate is stored in the spore as a large depot of 3-phosphogly-
ceric acid (3-PGA). In the first minutes of spore germination,
3-PGA is rapidly converted to ATP by the lower branch of
3310 NOTES
glycolysis (45, 51). Thus, the activity of the NHEJ pathway is
dependent upon reactivation of ATP generation during spore
germination.
The above results raise a question about the potential role of
ykoW in the ykoWVU operon in NHEJ. According to the Subti-
List database (http://genolist.pasteur.fr/SubtiList) (26), the pu-
tative ykoW product is similar to sensory box proteins and/or
diguanylate cyclase/phosphodiesterases potentially involved in
DNA repair. In this regard, it is interesting that Mun et al. (27)
reported that nonhomologous ends were removed by a DNA
phosphodiesterase to eliminate radiolytic products (e.g., thy-
mine glycol adducts) at broken DNA ends in Deinococcus
radiodurans. In addition, YkoV ligases themselves from a num-
ber of bacteria are reported to encode both DNA polymerase
and DNA end-processing activities (reviewed in reference 3).
These observations point to the intriguing possibility that the
ykoW product may also function to prepare DSB ends for
resection, a possibility that is currently being addressed.
In conclusion, when faced with prolonged exposure to harsh
environments, dormant bacterial spores must repair accumu-
lated DSB to ensure their survival and genome integrity. Our
results lend support to the hypothesis that NHEJ is a key
strategy used during spore germination to repair DSB caused
by terrestrial UV-B and UV-A radiation, as well as UV, ion-
izing radiation, and ultrahigh vacuum-induced extreme desic-
cation encountered during exposure to space.
(These results will be included in the Ph.D. thesis of Ralf
Moeller and in the research reports of the HIMAC project
[17B463].)
We are grateful to A. Green and R. E. Yasbin for the generous
donation of strains and to J. Drescher for his excellent technical as-
sistance.
This study was supported in part by grant NNA06CB58G from the
NASA Planetary Protection Office to W.L.N.
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