Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H(2)O(2)) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H(2)O(2), as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

Role of the Spore Coat Layers in Bacillus subtilis Spore Resistance to Hydrogen Peroxide, Artificial UV-C, UV-B, and Solar UV Radiation

Spores of Bacillus subtilis possess a thick protein coat that consists of an electron-dense outer coat layer and a lamellalike inner coat layer. The spore coat has been shown to confer resistance to lysozyme and other sporicidal substances. In this study, spore coat-defective mutants of B. subtilis (containing the gerE36 and/or cotE::cat mutation) were used to study the relative contributions of spore coat layers to spore resistance to hydrogen peroxide (H₂O₂) and various artificial and solar UV treatments. Spores of strains carrying mutations in gerE and/or cotE were very sensitive to lysozyme and to 5% H₂O₂, as were chemically decoated spores of the wild-type parental strain. Spores of all coat-defective strains were as resistant to 254-nm UV-C radiation as wild-type spores were. Spores possessing the gerE36 mutation were significantly more sensitive to artificial UV-B and solar UV radiation than wild-type spores were. In contrast, spores of strains possessing the cotE::cat mutation were significantly more resistant to all of the UV treatments used than wild-type spores were. Spores of strains carrying both the gerE36 and cotE::cat mutations behaved like gerE36 mutant spores. Our results indicate that the spore coat, particularly the inner coat layer, plays a role in spore resistance to environmentally relevant UV wavelengths.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Failure to decontaminate Glomus clarum NT4 spores is due to spore wall-associated bacteria

 Exposure of spores of Glomus clarum NT4 to solutions of chloramine-T (2.5–10% w/v) for 10–120 min failed to fully decontaminate all spores. Scanning electron microscopy did not show the presence of contaminants on treated spores, but transmission electron microscopy revealed bacterial cells embedded within the outer spore wall layer. Bacteria that remained protected within the spore walls were detected only when the spores were placed on appropriate media. Nutrient agar and tryptic soy agar supported relatively high levels of contaminant growth and were regarded as good media for assessing contamination, whereas the detection of contaminant growth on water agar required prolonged incubation. Contamination and germination of G. clarum NT4 spores following decontamination treatments were dependent on spore age. Generally, lower concentrations of chloramine-T and shorter incubation periods were required to reduce contamination of freshly harvested spores than of mature spores. Exposure to 10% chloramine-T for 120 min was required to reduce the levels of contamination of mature spores to ≤10%. Unfortunately, spore germination was compromised by rigorous decontamination treatments, thus the success of any decontamination procedure should be evaluated prior to its routine use. Moreover, if the interpretation of experimental results rests on the assumption of true surface sterility of VAMF spores, we suggest that the axenic condition of spores be confirmed prior to experimentation on a medium that encourages contaminant growth. Accepted: 12 July 1995     

Intradiurnal periodicity of fungal spore concentrations (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) in Cracow, Poland

Aerobiological monitoring enables the definition of seasonal fungal spore concentrations and also intradiurnal time when the highest concentrations of spores could cause or increase allergy symptoms. These data are useful to estimate symptoms of disease, duration of infection and how advanced the illness is in people suffering from fungal allergens. The aim of the study was to compare the concentrations of fungal spores (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) during dry and rainy periods and to analyse their intradiurnal changes. Average daily spore concentrations in dry and rainy periods were compared, using z test, separately for each taxon, season and for a combined 3-year period. Intradiurnal periodicity of fungal spore concentrations was analysed on the basis of three complementary diagrams. These spore concentrations were presented using three curves for all, dry and rainy days in 1997–1999 (April–November). The spore percentage in particular hours was normalized in relation to the daily spore sum accepted as 100%. Two further diagrams enabled the more precise analysis of the highest concentrations in dry days. Daily Botrytis and Cladosporium spore concentrations did not show significant differences between dry and rainy periods. In the case of Didymella and Ganoderma spore concentrations, there were no significant differences between both weather types in the single years, although there was a significant difference when a 3-year period was considered. The differences between daily concentrations of Alternaria spores in dry and rainy periods occurred in 1997 and in a 3-year period. Intradiurnal periodicity of spore concentrations was different for ‘dry’ and ‘wet’ fungal spores. Dry spores are released from the spore-producing parts of the fungus under conditions of decreasing humidity and increasing airflow. Examples of dry spores are those from Alternaria, Cladosporium and Botrytis. Wet spores, such as those from many Ascomycetes (Didymella) and Basidiomycetes (Ganoderma), are released into the atmosphere by processes related to humidity conditions or rain. The highest concentrations of ‘dry’ spores were observed early in the afternoon, while highest values of ‘wet’ spore concentrations occurred in the predawn hours. Statistically non-significant differences between daily spore concentrations in dry and rainy periods of single seasons were found except for Alternaria. Statistically significant differences could occur when the studied period was longer than one season (Alternaria, Didymella, Ganoderma). The highest concentrations of Alternaria, Botrytis and Cladosporium spores were recorded at noon and early in the afternoon. Concentrations of Didymella and Ganoderma spores were highest in the predawn hours.     

Resistance and recovery studies on ultraviolet-irradiated spores of Bacillus pumilus.

A spore suspension model and a procedure for recovering ultraviolet (UV)-irradiated spores of Bacillus pumilus were investigated. A most-probable-number tube dilution method using double-strength Trypticase soy broth was found to be superior to the agar plate method for recovering optimal numbers of spores irradiated with sublethal doses of UV energy. Aqueous suspensions of B. pumilus survived UV doses up to 108,000 ergs/mm2 as determined by a most-probable-number recovery and estimation procedure. Resistance and stability data were consistent and reproducible, indicating the dependability of this method for recovering UV-damaged spores. The procedures used to collect information concerning resistance characteristics for two strains of B. pumilus are discussed.     

Proteins involved in formation of the outermost layer of Bacillus subtilis spores

To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.     

Microbial survival in the stratosphere and implications for global dispersal

Spores of Bacillus subtilis were exposed to a series of stratosphere simulations. In total, five distinct treatments measured the effect of reduced pressure, low temperature, high desiccation, and intense ultraviolet (UV) irradiation on stratosphere-isolated and ground-isolated B. subtilis strains. Environmental conditions were based on springtime data from a mid-latitude region of the lower stratosphere (20 km). Experimentally, each treatment consisted of the following independent or combined conditions: −70°C, 56 mb, 10–12% relative humidity and 0.00421, 5.11, and 54.64 W/m² of UVC (200–280 nm), UVB (280–315 nm), UVA (315–400 nm), respectively. Bacteria were deposited on metal coupon surfaces in monolayers of ~1 × 10⁶ spores and prepared with palagonite (particle size < 20 μm). After 6 h of exposure to the stratosphere environment, 99.9% of B. subtilis spores were killed due to UV irradiation. In contrast, temperature, desiccation, and pressure simulations without UV had no effect on spore viability up through 96 h. There were no differences in survival between the stratosphere-isolated versus ground-isolated B. subtilis strains. Inactivation of most bacteria in our simulation indicates that the stratosphere can be a critical barrier to long-distance microbial dispersal and that survival in the upper atmosphere may be constrained by UV irradiation.     

Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial and Solar UV Radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Role of dipicolinic acid in survival of Bacillus subtilis spores exposed to artificial and solar UV radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Consequences ofSporidiobolus pararoseus killer toxin action on sensitive cells

Sensitive cells ofRhodotorula araucariœ treated with killer toxin produced by two strains of the red basidiomycetous yeastSporidiobolus pararoseus were stained with methylene blue and released UV absorbing material into the growth medium. They also behaved as permeabilized cells in a high-frequency field. All these phenomena indicate permeabilization of the sensitive cells.     

Single-spore elemental analyses indicate that dipicolinic acid-deficient Bacillus subtilis spores fail to accumulate calcium

Dipicolinic acid (pyridine-2,6-carboxylic acid; DPA) is a major component of bacterial spores and has been shown to be an important determinant of spore resistance. In the core of dormant Bacillus subtilis spores, DPA is associated with divalent calcium in a 1:1 chelate (Ca–DPA). Spores excrete Ca–DPA during germination, but it is unknown whether Ca and DPA are imported separately or together into the developing spore. Elemental analysis by scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM–EDS) of wild-type spores and mutant spores lacking the ability to synthesize DPA showed that DPA-less spores also lacked calcium, suggesting that the two compounds may be co-imported.     

Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase

An extracellular tannase (tannin acyl hydrolase) was isolated from Paecilomyces variotii and purified from cell-free culture filtrate using ammonium sulfate precipitation followed by ion exchange and gel filtration chromatography. Fractional precipitation of the culture filtrate with ammonium sulfate yielded 78.7% with 13.6-folds purification, and diethylaminoethyl–cellulose column chromatography and gel filtration showed 19.4-folds and 30.5-folds purifications, respectively. Molecular mass of tannase was found 149.8 kDa through native polyacrylamide gel electrophoresis (PAGE) analysis. Sodium dodecyl sulphate–PAGE revealed that the purified tannase was a monomeric enzyme with a molecular mass of 45 kDa. Temperature of 30 to 50°C and pH of 5.0 to 7.0 were optimum for tannase activity and stability. Tannase immobilized on alginate beads could hydrolyze tannic acid even after extensive reuse and retained about 85% of the initial activity. Thin layer chromatography, high performance liquid chromatography, and ¹H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic acid.     

Effect of a small, acid-soluble spore protein from Clostridium perfringens on the resistance properties of Bacillus subtilis spores

Alpha/beta-type small, acid-soluble spore proteins (SASP) are essential for the resistance of DNA in spores of Bacillus species to damage. An alpha/beta-type SASP, Ssp2, from Clostridium perfringens was expressed at significant levels in B. subtilis spores lacking one or both major alpha/beta-type SASP (alpha- and alpha- beta- strains, respectively). Ssp2 restored some of the resistance of alpha- beta- spores to UV and nitrous acid and of alpha- spores to dry heat. Ssp2 also restored much of the resistance of alpha- spores to nitrous acid and restored full resistance of alpha- spores to UV and moist heat. These results further indicate the interchangeability of alpha/beta-type SASP in DNA protection in spores.     

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.     

Effect of initiated spores on the resistance of nongerminated resting forms of Bacillus cereus remaining in the suspension to the action of damaging agents

The elevated resistance of a Bacillus cereus spore suspension against the action of UV was found to depend on the quantity of resting forms initiated in the suspension prior to an irradiation. The resistance against UV increased 80-50 times if 60-90% of spores were initiated in the suspension as compared to that of the original resting forms. When suspensions containing 40% of non-germinated B. cereus spores were kept at 4 degrees C for 14 days, the latter became 10 and 14 times more resistant to elevated temperature (90 degrees C) and chloramine (2.5%), respectively, as compared to control intact spores. The higher resistance of non-germinated spores against the action of physical and chemical damaging agents was registered within the entire period of experiments (over three months). This phenomenon was not observed if ca. 100% of spores were initiated in a suspension. The resistance of initiated spores against the action of UV was 40 times lower than that of B. cereus resting forms.     

Ultrastructure and properties of Paecilomyces lilacinus spores

Strains of the filamentous soil fungus Paecilomyces lilacinus are currently being developed for use as biological control agents against root-knot, cyst, and other plant-parasitic nematodes. The inoculum applied in the field consists mainly of spores. This study was undertaken to examine the size, ultrastructure, and rodlet layers of P. lilacinus spores and the effect of the culture method on structural and functional spore properties. A rodlet layer was identified on aerial spores only. Other differences noted between aerial spores and those produced in submerged culture included the size and appearance of spores and thickness of spore coat layers when examined with transmission electron microscopy. The two spore types differed in UV tolerance, with aerial spores being less sensitive to environmentally relevant UV radiation. Also, viability after drying and storage was better with the aerial spores. Both spore types exhibited similar nematophagous ability.     

Nuclear Staining of Colletotrichum Gloeosporioides F. SP. Malvae Conidia with Fluorescent and Nonfluorescent Stains

Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores.     

Transitory germinative excision repair in Bacillus subtilis.

Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells. These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1). When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA. This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them. The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair.     

Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).