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.
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). 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 or simulated planetary conditions. Among fungal and bacterial spores collected at high altitudes up to 77 km, pigmented forms dominated. It has already 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.
Generally, Bacillus endospores are 5–50 times more resistant to UV radiation than their corresponding vegetative cells. 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 (5-thyminyl-5,6-dihydrothymine) as the main photoproduct induced by UV-C (reviewed in; (ii) a DNA repair pathway specific for the spore photoproduct; (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; and (iv) the presence of a thick spore protein coating consisting of an electron dense outer coat layer and a lamellar inner coat layer.
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. A melanin-like pigment was found to be produced during sporulation in minimal medium. 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 ΔcotA mutant spores. 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. During sporulation of B. megaterium spores, a red pigment, associated with the membrane, was identified to be consistent with a carotenoid structure. 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 λ > 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. 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. Partial characterization of the pigments was reached by spectrophotometry of the pigment extract.
2Materials and methods
2.1Microorganism and growth conditions
The following Bacillus strains were obtained from the German Collection of microorganisms and cells, DSMZ, Brunswick, Germany: B. atrophaeus DSM 675 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 with spores of light-gray coloration. Spores were harvested from a culture in a sporulation medium 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 μg/ml), lysozyme (200 μg/ml) and DNAse (2 μg/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 1010spores/ml) were stored in aqueous suspension at 4 °C.
2.2UV 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 μl 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 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 Student's t test. Differences with P values 0.05 were considered statistically significant.
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. 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).
3.1UV-inactivation of spores differing in endogenous pigmentation
The aim of this study was to determine whether bacterial endospores are protected by their pigments against 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.
Table 1. Curve characteristics after UV-irradiation (data from Fig. 2)
IC = inactivation constant; n= extrapolation number; p= statistical significance of difference of data of strain × compared to strain 675; p < 0.05= significant difference
(3.38 ± 0.17) × 10−6
1.19 ± 0.13
(3.72 ± 0.18) × 10−5
1.14 ± 0.13
(6.81 ± 0.34) × 10−5
1.17 ± 0.12
(1.02 ± 0.06) × 10−3
3.23 ± 0.34
(1.01 ± 0.08) × 10−3
5.28 ± 0.56
(1.46 ± 0.07) × 10−3
5.47 ± 0.71
(1.09 ± 0.06) × 10−2
1.62 ± 0.18
(1.25 ± 0.10) × 10−2
3.29 ± 0.26
(1.22 ± 0.07) × 10−2
2.08 ± 0.27
3.2Characteristics of endogenous pigments
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 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 β-carotene in hexane, for example, has an absorption maximum at 452.46 and 480.47 nm. One reason for the shift in the absorption maxima of the spore extract to shorter wavelengths compared to pure β-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).
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 planets, for example Mars. 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 λ < 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) 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.
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. 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 β-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) supports 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. 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. 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.