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 metabolically 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 peptidoglycan spore cortex, and abundant intracellular constituents such as the calcium chelate of dipicolinic acid and small, acidsoluble 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 photochemistry 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 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 recombination pathway, which requires at least two homologous chromosomes, 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 considered here. This pathway as it occurs in eukaryotic cells requires a DNA end-binding component called Ku (Ku70 and Ku80) (58). The fir...

The scenario of lithopanspermia describes the viable transport of microorganisms via meteorites. To test the first step of lithopanspermia, i.e., the impact ejection from a planet, systematic shock recovery experiments within a pressure range observed in martian meteorites (5-50 GPa) were performed with dry layers of microorganisms (spores of Bacillus subtilis, cells of the endolithic cyanobacterium Chroococcidiopsis, and thalli and ascocarps of the lichen Xanthoria elegans) sandwiched between gabbro discs (martian analogue rock). Actual shock pressures were determined by refractive index measurements and Raman spectroscopy, and shock temperature profiles were calculated. Pressure-effect curves were constructed for survival of B. subtilis spores and Chroococcidiopsis cells from the number of colony-forming units, and for vitality of the photobiont and mycobiont of Xanthoria elegans from confocal laser scanning microscopy after live/dead staining (FUN-I). A vital launch window for the transport of rock-colonizing microorganisms from a Mars-like planet was inferred, which encompasses shock pressures in the range of 5 to about 40 GPa for the bacterial endospores and the lichens, and a more limited shock pressure range for the cyanobacterium (from 5-10 GPa). The results support concepts of viable impact ejections from Mars-like planets and the possibility of reseeding early Earth after asteroid cataclysms.

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple ␣/␤-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-␣ and/or SASP-␤) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-␥) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that ␣/␤-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-␥ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and ␣/␤-type small, acid-soluble spore proteins (␣/␤-type SASP), the last two of which protect spore DNA (6,42,46,48,52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33,47,48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of re...

The role of DNA repair by nonhomologous end joining (NHEJ), homologous recombination, spore photoproduct lyase, and DNA polymerase I and genome protection via ␣/␤-type small, acid-soluble spore proteins (SASP) in Bacillus subtilis spore resistance to accelerated heavy ions (high-energy charged [HZE] particles) and X rays has been studied. Spores deficient in NHEJ and ␣/␤-type SASP were significantly more sensitive to HZE particle bombardment and X-ray irradiation than were the recA, polA, and splB mutant and wild-type spores, indicating that NHEJ provides an efficient DNA double-strand break repair pathway during spore germination and that the loss of the ␣/␤-type SASP leads to a significant radiosensitivity to ionizing radiation, suggesting the essential function of these spore proteins as protectants of spore DNA against ionizing radiation.Endospores of the gram-positive bacterium Bacillus subtilis are highly resistant to inactivation by environmental stresses, such as biocidal agents and toxic chemicals, desiccation, pressure and temperature extremes, and high fluences of UV radiation (reviewed in references 44, 45, and 61) and are a powerful biodosimetric system for terrestrial environmental monitoring and astrobiological studies (44). On Earth, understanding extreme spore resistance to ionizing radiation is important in the areas of food preservation, medical sterilization, and decontamination from bioterror attack (4, 17, 47; reviewed in references 42 and 43). Off Earth, spore radiation resistance is important both in space flight and in ground-based simulations, in order to obtain information on the biological damage produced by exposure to space conditions (23,25,26,44). Onboard several spacecraft (Apollo 16, Spacelab 1, LDEF, D2, and FOTON), spores of Bacillus subtilis were exposed to selected parameters of space, such as space vacuum and different spectral ranges of solar UV radiation and cosmic rays, applied separately or in combination (5, 9, 19-21, 23, 24, 26). Especially, the radiation environment on Earth, on Mars, in low-Earth orbit, and in deep space is typified by a wide variety of primary particles covering an extended range of energies. Galactic cosmic rays (GCR) are charged particles that originate from sources beyond our solar system. The distribution of GCR is believed to be isotropic throughout interstellar space. The spectrum of the GCR consists of 98% protons and heavier ions (baryon component) and 2% electrons and positrons (lepton component). The baryon component is composed of 87% protons, 12% helium ions (alpha particles), and the remaining 1% heavy ions of charge 3 from lithium to 92 from uranium. Due to their high abundance, iron ions are highly penetrating, giving them a large potential for radiobiological damage (3,21).While the UV photochemistry of spore DNA and repair of UV damage to DNA during germination are well characterized (34,44,(59)(60)(61), there has been relatively little work on the nature of DNA damage in spores caused by ionizing radiation, the protective role of...

The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica. All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm2 to 108 J/cm2). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications.IMPORTANCE Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further investigation.

Bacterial spores (Bacillus subtilis), cyanobacteria (Chroococcidiopsis sp.), and lichen (Xanthoria elegans) embedded in martian analogue rock (gabbro) were exposed to shock pressures between 5 and 50 GPa which is the range of pressures observed in martian meteorites. The survival of Bacillus subtilis and Xanthoria elegans up to 45 GPa and of Chroococcidiopsis sp. up to 10 GPa supports the possibility of transfer of life inside meteoroids between Mars and Earth and it implies the potential for the transfer of life from any Mars-like planet to other habitable planets in the same stellar system

Bacillus endospores show different kinds of pigmentation. Red-pigmented spores of Bacillus atrophaeus DSM 675, dark-gray spores of B. atrophaeus(T) 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.

Bacteria will accompany humans in our exploration of space, making it of importance to study their adaptation to the microgravity environment. To investigate potential phenotypic changes for bacteria grown in space, Escherichia coli was cultured onboard the International Space Station with matched controls on Earth. Samples were challenged with different concentrations of gentamicin sulfate to study the role of drug concentration on the dependent variables in the space environment. Analyses included assessments of final cell count, cell size, cell envelope thickness, cell ultrastructure, and culture morphology. A 13-fold increase in final cell count was observed in space with respect to the ground controls and the space flight cells were able to grow in the presence of normally inhibitory levels of gentamicin sulfate. Contrast light microscopy and focused ion beam/scanning electron microscopy showed that, on average, cells in space were 37% of the volume of their matched controls, which may alter the rate of molecule–cell interactions in a diffusion-limited mass transport regime as is expected to occur in microgravity. TEM imagery showed an increase in cell envelope thickness of between 25 and 43% in space with respect to the Earth control group. Outer membrane vesicles were observed on the spaceflight samples, but not on the Earth cultures. While E. coli suspension cultures on Earth were homogenously distributed throughout the liquid medium, in space they tended to form a cluster, leaving the surrounding medium visibly clear of cells. This cell aggregation behavior may be associated with enhanced biofilm formation observed in other spaceflight experiments.