FEMS Microbiology Ecology 51 (2005) 231–236
www.fems-microbiology.org
Role of pigmentation in protecting Bacillus sp. endospores
against environmental UV radiation
Ralf Moeller
a
a,b,*
, Gerda Horneck a, Rainer Facius a, Erko Stackebrandt
b
Department of Radiation Biology, German Aerospace Center, Institute of Aerospace Medicine, Linder Hoehe D-51147 Cologne, Germany
b
German Collection of Microorganism and Cell Cultures, Brunswick, Germany
Received 22 June 2004; received 23 August 2004; accepted 26 August 2004
First published online 21 September 2004
Abstract
Bacillus endospores show different kinds of pigmentation. Red-pigmented spores of Bacillus atrophaeus DSM 675, dark-gray
spores of B. atrophaeusT DSM 7264 and light-gray spores of B. subtilis DSM 5611 were used to study the protective role of the pigments in their resistance to defined ranges of environmental UV radiation. Spores of B. atrophaeus DSM 675 possessing a dark-red
pigment were 10 times more resistant to UV-A radiation than those of the other two investigated strains, whereas the responses to
the more energetic UV-B and UV-C radiation were identical in all three strains. The methanol fraction of the extracted pigment
from the spores absorbs in the associated wavelength area. These results indicate that the carotene-like pigment of spores of B.
atrophaeus DSM 675 affects the resistance of spores to environmental UV-A radiation.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Bacillus endospores; Resistance; UV-radiation; Pigments; Photoprotection
1. Introduction
Bacillus endospores are highly resistant to a variety of
environmental stresses, such as toxic chemical agents,
desiccation, high and low pressure, temperature extremes and high doses of ionizing or UV radiation (reviewed in [1]). They are ubiquitous, inhabit soils and
rocks and are easily disseminated by wind and water.
Their high resistance to environmental extremes makes
these spores also ideal model systems for testing their responses to extraterrestrial conditions, such as outer
space [2] or simulated planetary conditions [3]. Among
fungal and bacterial spores collected at high altitudes
up to 77 km [4], pigmented forms dominated. It has al*
Corresponding author. Tel.: +49 2203 601 3145; fax: +49 2203
61970.
E-mail address: [email protected] (R. Moeller).
URLs: http://www.dlr.de/Strahlenbiologie, http://www.dsmz.de.
ready been suggested in this early work that endogenous
pigments, such as carotenoids and melanins, might provide a selective advantage to these microorganisms by
shielding from environmental UV radiation, which at
these high altitudes comprises the full extraterrestrial
spectrum, including the UV-C and full UV-B range. In
fact, endogenous pigment production such as melanin
has been shown to protect various microorganisms
against oxidative damage caused by UV or ionizing
radiation by scavenging free radicals [5].
Generally, Bacillus endospores are 5–50 times more
resistant to UV radiation than their corresponding vegetative cells [6]. This high resistance of Bacillus endospores to UV radiation has been ascribed to several
spore-specific attributes: (i) an altered conformation
of their DNA (A-form) caused by the presence of a
group of small, acid-soluble proteins (SASP) binding
to the DNA, thereby leading to an altered photochemistry of the DNA with the so-called spore photoproduct
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.08.008
232
R. Moeller et al. / FEMS Microbiology Ecology 51 (2005) 231–236
(5-thyminyl-5,6-dihydrothymine) as the main photoproduct induced by UV-C (reviewed in [6]; (ii) a
DNA repair pathway specific for the spore photoproduct [7]; (iii) the accumulation of dipicolinic acid (DPA)
as the Ca2+ chelate in the dormant spore core accounting for approximately 10% of the total spore dry weight
[8]; and (iv) the presence of a thick spore protein coating consisting of an electron dense outer coat layer and
a lamellar inner coat layer [9].
Endospores of Bacillus sp. show a broad spectrum
of pigmentation [10–13]. In many cases, the nomenclature refers to the coloration of an organism, e.g. B.
subtilis var. niger acquired its name from its black pigment [14]. A melanin-like pigment was found to be
produced during sporulation in minimal medium [15].
In B. subtilis 168 spores, a CotA-dependent brownish
pigmentation, especially produced in the presence of
copper, resulted in an increase in resistance to UV-B
and UV-A radiation by one to two orders of magnitude as compared with the non-pigmented spores of
the DcotA mutant spores [16]. The brownish photoprotective pigment was suggested to be a melanin-like
compound. A mutant of B. thuringiensis, producing
melanin, was significantly more resistant to UV radiation at 254 and 366 nm than its non-pigmented parental strain, thereby increasing its insecticidal activity
under field conditions [17]. During sporulation of B.
megaterium spores, a red pigment, associated with
the membrane, was identified to be consistent with a
carotenoid structure [13]. It was assumed that this pigment plays a role in membrane stabilization as well as
in photoprotection against UV radiation.
So far, in most studies on the UV resistance of B. subtilis spores monochromatic UV radiation at 254 nm
from a germicidal UV source was used. However, environmental UV radiation is polychromatic and comprises
the full spectrum of UV-A and UV-B radiation at wavelengths of k > 290 nm. To understand the spore responses to the terrestrial UV radiation climate, recent
studies have used solar simulators or natural insolation
as the UV source. In this case, the photochemistry of the
spore DNA and the DNA repair processes during germination appeared quite different from those after exposure to UV-C radiation [18]. The role of the spore coat
layers and DPA in the resistance of B. subtilis spores
to environmental UV radiation has been recently investigated using polychromatic UV sources [8,9]. We have
been interested in understanding the role of endogenous
pigments in the resistance of Bacillus spores to environmental UV radiation. We thus selected from the DSMZ
culture collection Bacillus strains that form spores comprising different kinds of pigmentation, i.e. white, gray
or red spores. We studied their inactivation by polychromatic UV(A + B) and UV-A radiation from a solar simulator and also by monochromatic UV-C radiation at
254 nm for comparison with earlier literature data. Par-
tial characterization of the pigments was reached by
spectrophotometry of the pigment extract.
2. Materials and methods
2.1. Microorganism and growth conditions
The following Bacillus strains were obtained from the
German Collection of microorganisms and cells,
DSMZ, Brunswick, Germany: B. atrophaeus DSM 675
[19] producing red pigmented spores, B. atrophaeusT
DSM 7264, formerly known as Bacillus subtilis var. niger producing spores of dark-gray color, and B. subtilis
DSM 5611 [14] with spores of light-gray coloration.
Spores were harvested from a culture in a sporulation
medium [20] after 4 days of incubation at 37 C, when
a sporulation rate of over 90% was reached. Free spores
were purified by centrifugation (10,000 rpm, 200 min at
4 C) and treatment with MgSO4 (2,5 lg/ml), lysozyme
(200 lg/ml) and DNAse (2 lg/ml) for 30 min at 37 C
in order to destroy the residual vegetative cells. The enzymes were inactivated by heat (80 C) for 10 min. After
repeated centrifugation and washing in distilled water,
the purified spores (about 1010 spores/ml) were stored
in aqueous suspension at 4 C.
2.2. UV irradiation experiments
Spores in aqueous suspension (107 spores/ml) were
exposed to UV-C radiation from a mercury low-pressure lamp (NN 8/15, Heraeus, Berlin, Germany) with
a major emission line at 253.65 nm (Fig. 1), and to defined spectral ranges of UV-(A + B) or UV-A radiation
obtained by use of a metal halogenide-high-pressure
lamp (solar simulator SOL 2, Dr. Hönle AG, München,
Germany) and optical filter combinations (Fig. 1). During irradiation the spore suspension was stirred continuously to ensure homogeneous exposure. The spectral
irradiance was measured by use of a double monochromator (Bentham DM 300). After UV radiation at defined fluences, 100 ll from the aqueous suspension was
taken for further analysis. Survival was determined from
appropriate dilutions in distilled water as colony forming ability (CFA) after growth overnight on nutrient
broth agar (Difco Detroit, USA) at 37 C. The surviving
fraction was determined from the quotient N/N0, with
N = the number of colony formers of the irradiated sample and N0 that of the non-irradiated controls. Plotting
the logarithm of N/N0 as a function of fluence, survival
curves were obtained. To determine the curve parameters,
the following relationship was used: ln N/N0 = IC ·
F + n within N = colony formers after UV-irradiation;
N0 = colony formers without UV-irradiation; IC = inactivation constant (m2/J); n = extrapolation number, i.e.
the intercept with the ordinate of the extrapolated
R. Moeller et al. / FEMS Microbiology Ecology 51 (2005) 231–236
233
Fig. 1. Spectral irradiances in the UV-(A + B) and UV-A range at the sample site, obtained from SOL 2 with a combination of optical filters. (I) UV(A + B): WG 305 3 mm and UG 11 1m (Schott); (II) UV-A (WG 335 3 mm and UG 11 1 mm (Schott). For comparison the (III) terrestrial UV
spectrum is also shown. The inset shows the emission lines of the UV-C source.
semi-log straight-line. The inactivation constant was
determined from the slope of the fluence–effect-curves.
The significance of the difference of the fluence–
effect-curves was statistically analyzed using StudentÕs t
test. Differences with P values 60.05 were considered
statistically significant.
2.3. Pigment characterization
The pigment properties of the spores were analyzed
spectrophotometrically from a spore extract. For the
isolation of the pigments, 1010 spores of each Bacillus
sp. strain were centrifuged and resuspended in 5 ml of
50 mM Tris–HCl (pH 8,0), containing 8 M urea, 1% sodium dodecyl sulfate, 10 mM EDTA, and 50 mM
dithiothreitol, incubated for 90 min at 37 C and treated
with ultrasonication for 30 min at 37 C in order to remove the spore coats [9]. After washing twice in distilled
water, the pellet of the decoated spores was resuspended
in 5 ml methanol to extract the pigment fraction. After
60 min shaking at 200 rpm, the supernatant containing
the pigments was analyzed spectrophotometrically
(Hitachi, Tokyo, Japan).
environmental UV-radiation. Spores of three strains,
which show different kinds and extents of pigmentation,
namely B. atrophaeus DSM 675 (red spores), B. atrophaeusT DSM 7264 (dark-gray spores) and B. subtilis DSM
5611 (light-gray spores) were thus exposed to polychromatic UV-A or UV-(A + B) radiation, and also to UV-C
radiation for comparison with literature data. From the
fluence–effect curves of inactivation (Fig. 2), the inactivation constants (ICs) were derived (Table 1). From
the ratio of the ICs it can be seen that the red pigmented
endospores of B. atrophaeus DSM 675 showed about
10–20 times higher resistance to UV-A radiation than
spores of the type strain of B. atrophaeusT DSM 7264
or the less pigmented endospores of B. subtilis DSM
5611 (Table 1). Only for this spectral range, namely
polychromatic UV-A radiation, a statistical difference
was observed between the inactivation kinetics of spores
of strain 675 and spores of the other two strains used.
There was no significant difference in the resistance of
the spores of the three strains against UV-C or UV(A + B) radiation. The data suggest a protective effect
against UV-A radiation by the red pigments of the
spores of B. atrophaeus DSM 675, but not against the
more energetic UV-B and UV-C ranges.
3. Results
3.2. Characteristics of endogenous pigments
3.1. UV-inactivation of spores differing in endogenous
pigmentation
The aim of this study was to determine whether bacterial endospores are protected by their pigments against
For the initial characterization of these endogenous
pigments, the absorption spectrum of the pigments extracted from the endospores was measured (Fig. 3).
Spores of all three strains were subjected to identical
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R. Moeller et al. / FEMS Microbiology Ecology 51 (2005) 231–236
Fig. 2. Fluence effects curves of inactivation of spores of B. atrophaeus DSM 675, B. atrophaeusT DSM 7264, and B. subtilis DSM 5611 after
exposure to UV-A (a), UV-(A + B) (b) and UV-C (c).
Table 1
Curve characteristics after UV-irradiation (data from Fig. 2)
Strain
IC (m2/J)
UV-A
675
7264
5611
(3.38 ± 0.17) · 10
(3.72 ± 0.18) · 10
(6.81 ± 0.34) · 10
UV-(A + B)
675
7264
5611
(1.02 ± 0.06) · 10 3
(1.01 ± 0.08) · 10 3
(1.46 ± 0.07) · 10 3
UV-C
675
7264
5611
(1.09 ± 0.06) · 10
(1.25 ± 0.10) · 10
(1.22 ± 0.07) · 10
6
5
5
2
2
2
n
Ratio ICX/IC675
p
1.19 ± 0.13
1.14 ± 0.13
1.17 ± 0.12
1.00
11.01
20.45
1.000
0.002
0.001
3.23 ± 0.34
5.28 ± 0.56
5.47 ± 0.71
1.00
0.99
1.43
1.000
0.446
0.834
1.62 ± 0.18
3.29 ± 0.26
2.08 ± 0.27
1.00
1.15
1.12
1.000
0.179
0.410
IC = inactivation constant; n = extrapolation number; p = statistical significance of difference of data of strain · compared to strain 675;
p < 0.05 = significant difference
extraction procedures, namely a methanol extraction of
the decoated spores. Only the fraction isolated from B.
atrophaeus DSM 675 spores showed a pronounced
absorption in the UV range with two strong peaks at
377 and 398 nm and two weaker peaks at 338 and 355
nm. These absorption maxima of the pigment extract
found in methanol are in the UV-A range (Fig. 3) and
could well be the reason for the increased UV-A resistance of the spores of B. atrophaeus DSM 675 compared
to those of the other strains tested. The spectrum of the
pigment extract shows many similarities in the absorption behavior to carotenoid terpenes (inset in Fig. 3).
A pure b-carotene in hexane, for example, has an
absorption maximum at 452.46 and 480.47 nm [21].
One reason for the shift in the absorption maxima of
the spore extract to shorter wavelengths compared to
pure b-carotene in hexane could be the number of conjugated C@C bounds, which will be discussed below.
However, so far, it is not known whether the extract is
composed of a single pigment or of a mixture of different
pigments. Nevertheless, it is important to note that none
of the extracts from spores of the other strains, B.
atrophaeusT DSM 7264 or B. subtilis DSM 5611, which
were obtained by the same method, showed any absorption in the UV-A region (Fig. 3).
4. Discussion
B. subtilis is the best-characterized spore-forming
microorganism and is often used as a model system
for studying the resistance of bacterial endospores to
environmental extremes. Solar UV radiation is a major
source of lethal damage to spore DNA not only in the
terrestrial environment but even more so in extraterrestrial environments such as outer space [1,2] or on other
R. Moeller et al. / FEMS Microbiology Ecology 51 (2005) 231–236
235
Fig. 3. Absorption spectrum of the pigment fraction extracted from the endospores and absorption spectrum of pure b-carotene in hexane from [21]
as inset.
planets, for example Mars [3]. Whereas the extraterrestrial UV-spectrum comprises the full range from the vacuum-UV to UV-A, the solar UV reaching the surface of
the Earth is cut off for wavelengths k < 290 nm due to
the shielding by the stratospheric ozone layer. Hence,
UV-A radiation (315–400 nm) makes up the major portion of the terrestrial UV radiation climate. Although
the effectiveness of UV-A in inactivating B. subtilis
spores is orders of magnitude lower than UV-B (1–2 orders of magnitude) or UV-C (4 orders of magnitude) [22]
its role in affecting biological integrity should not be neglected. For example, UV-A is believed to play an
important role in mutation induction and DNA damage
[23].
The absorption of the extracted pigment in the UV-A
range and its spectral pattern are similar to the group of
carotenoid terpenes. The absorption maximum of UV
absorbing organic components such as polyenic pigments are depending on the number of the C@C bonds
in conjugation. With decreasing number of C@C bonds
in conjugation, the absorption maxima shift towards
shorter wavelengths [24]. This may be the reason why
the absorption maxima of the pigment, isolated from
spores of strain 675 are found at shorter wavelengths
than those of pure b-carotene. In that case the UV-A
protection by a carotene-like pigment of B. atrophaeus
spores (DSM 675) may be attributable to the following
mechanisms: (i) the pigment provides a UV-A screen
thereby shielding the sensitive spore components such
as the spore DNA, against radiation in this UV region.
The absorption spectrum of the pigment (Fig. 3) sup-
ports this assumption. (ii) The pigment, as an antioxidant scavenges reactive oxygen species generated by
UV-A radiation in the spores. Reactive oxygen intermediates such as hydrogen peroxide or superoxide anions,
target several cellular components, including the DNA.
As a result of this interaction with the DNA the phosphodiester backbone of the DNA may break leading
to single or double strand fission [8]. However, in order
to protect the DNA from such short-living radicals, the
pigment needs to be located close to the DNA, i.e. within the spore core, which is very unlikely. UV-A may also
induce indirectly other DNA photoproducts, such as
7,8-dihydro-8-oxoguanine [25]. In addition to DNA,
reactive oxygen species may also attack other cellular
components leading to lipid peroxidation or protein
inactivation. Little is known about the role of such
non-DNA damage induced by UV-A in spore inactivation. (iii) Carotenoids as products of the isoprenoid biosynthesis may stabilize the spore membranes; in this
case, the pigment may play a role in preventing lipid peroxidation through its antioxidative potential. To answer
this question, more information are required on the
location of the pigment within the spore and characterization by mass spectroscopic analysis such as LC-(APcI)MS analyses of the extracted UV screening
compounds.
The results shown above suggest that the development of endogenous pigments has provided an evolutionary advantage during sporulation, protecting the
spores against the harmful environmental UV-A
radiation.
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R. Moeller et al. / FEMS Microbiology Ecology 51 (2005) 231–236
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