Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.     

Dielectric properties of native and decoated spores of Bacillus megaterium.

A general model for use in interpreting dielectric data obtained with bacterial endospores is developed and applied to past results for Bacillus cereus spores and new results for Bacillus megaterium spores. The latter were also subjected to a decoating treatment to yield dormant cells with damaged outer membranes that could be germinated with lysozyme. For both spore types, core ions appeared to be completely immobilized, and decoating of B. megaterium spores did not affect this extreme state of electrostasis in the core. The cortex of B. megaterium appeared to contain a high level of mobile ions, in the cortex of B. cereus. The outer membrane-coat complex of B. megaterium acted dielectrically as an insulating layer around the cortex, so that native dormant spores showed a Maxwell-Wagner dispersion over the frequency range from about 1 to 20 MHz. The decoating treatment resulted in a shift in the dispersion to frequencies below the range of observation. Increases in cell conductivity in response to increases in environmental ionic strength indicated that the coats. of B. megaterium could be penetrated by environmental ions and that they had an inherent fixed charge concentration of about 10 to 20 milliequivalents per liter. In contrast, the dispersion for B. cereus spores was very sensitive to changes in environmental ion concentration, and it appeared that some 40% of the spore volume could be penetrated by environmental ions and that these ions traversed a dielectrically effective layer, either the exosporium or the outer membrane. It appears that dormancy is associated with extreme electrostasis of core ions but not necessarily of ions in enveloping structures and that the coat-outer membrane complex is dielectrically effective but not required for maintenance of extreme electrostasis in the core.     

Sensitivity of Three Selected Bacterial Species to Ozone

THE MINIMAL LETHAL CONCENTRATION OF OZONE IN WATER WAS DETERMINED FOR THREE BACTERIAL SPECIES: Escherichia coli, Bacillus cereus, and Bacillus megaterium. A contact period of 5 min was selected. The lethal threshold concentration for the cells of B. cereus was 0.12 mg/liter while that for E. coli and B. megaterium was 0.19 mg/liter. Low concentrations of ozone were ineffective when organic matter was present to interfere with the action of ozone on the bacterial cells. Also determined during the study was the sensitivity of spores of B. cereus and B. megaterium to ozone in water. The threshold concentration required to kill the spores of both species was 2.29 mg/liter. The cells and spores of these organisms exhibited the "all-or-none" die-away phenomenon normally associated with ozone treatment.     

Effects of heat-, CaCl2- and ethanol-treatments on activation of Bacillus spores

The effects of heat, CaCl2, and ethanol on activation of Bacillus spores were determined by monitoring the absorbance decrease during germination in inosine. Bacillus cereus T, B. subtilis A and B. megaterium QM B1551 spores were activated by heat- and CaCl2-treatments. Ethanol activated B. megaterium and B. subtilis spores yet did not activate B. cereus spores. CaCl2- and ethanol-activations were less effective than heat-activation as judged by optimal germination rates and germination extents. The presence of CaCl2 during heat-treatment inhibited heat-activation of all three Bacillus spores without affecting viability or dipicolinic acid content of the spores. The electrophoretic patterns of coat plus outer membrane proteins extracted from Bacillus spores treated with CaCl2 and heat in the presence of CaCl2 were similar to each other and were distinctively different from the patterns of proteins from unactivated spores or the spores treated with heat and/or ethanol.     

Effects of added germination agents on loss of optical density in electron-irradiated spores.

Spores of Bacillus megaterium ATCC 14581, subjected to partial-cell iradiation, were exposed to either lysozyme, H2O2, or glucose in an attempt to reduce or eliminate the nonmonotonic behavior in curves of percentage of germination versus energy, obtained when such spores were resuspended in phosphate buffer alone. Except at the lower doses. H2O2 effectively eliminated this anomalous dip in these curves, whereas lysozyme amplified it greatly. Glucose was generally ineffective. Coinciding with the increases in optical density when lysozyme was present was the formation of an occluding product.     

Effects of added germination agents on loss of optical density in electron-irradiated spores.

Spores of Bacillus megaterium ATCC 14581, subjected to partial-cell iradiation, were exposed to either lysozyme, H2O2, or glucose in an attempt to reduce or eliminate the nonmonotonic behavior in curves of percentage of germination versus energy, obtained when such spores were resuspended in phosphate buffer alone. Except at the lower doses. H2O2 effectively eliminated this anomalous dip in these curves, whereas lysozyme amplified it greatly. Glucose was generally ineffective. Coinciding with the increases in optical density when lysozyme was present was the formation of an occluding product.     

Spore heat resistance and specific mineralization.

Spores of Bacillus megaterium ATCC 19213, Bacillus subtilis niger and Bacillus stearothermophilus ATCC 7953 were converted to fully demineralized, but viable, H forms by controlled acid titration. H forms were more heat sensitive than were native forms, but z values were greater for killing of H spores than those for native spores. Therefore, the differences in heat sensitivity between native and H forms decreased with increasing killing temperature. The increase in heat sensitivity associated with demineralization did not appear to be due to damage to cortex lytic enzymes of the germination system because it could not be moderated by decoating heated H spores and plating them on medium with added lysozyme. H spores could be remineralized by means of back titration with appropriate base solutions. The remineralized spores, except for the Na form, were then more heat resistant than were H spores. Ca and Mn were more effective in restoring resistance than were Mg and K. Generally, the remineralized forms (except for the Na form) had z values greater than those of the native forms but still less than those of the H forms. At lower killing temperatures, the reinstatement of resistance could be related to the extent of remineralization. However, at higher killing temperatures, only a fraction of the mineral was effective in restoring resistance, and higher levels of remineralization did not result in greater resistance. Mineralization is clearly an important factor in spore heat resistance, but the relationship between resistance and mineralization is complex and dependent on killing temperature.     

Formation of Protoplasts from Resting Spores

Coat-stripped spores suspended in hypertonic solutions and supplied with two essential cations can be converted into viable protoplasts by lysozyme digestion of both cortex and germ cell wall. Calcium ions are necessary to prevent membrane rupture, and magnesium ions are necessary for changes indicative of hydration of the core, particularily the nuclear mass. Since remnant spore coat covered such protoplasts of Bacillus subtilis and the germ cell wall of B. cereus spores is not lysozyme digestible, coatless spores of B. megaterium KM were more useful for these studies. Lysozyme digestion in cation-free environment produced a peculiar semi-refractile spore core free of a cortex but prone to rapid hydration and lytic changes on the addition of cations. Strontium could replace Ca(2+) but Mn(2+) could not replace Mg(2+) in these digestions. When added to the spores, dipicolinic acid and other chelates appeared to compete with the membrane for the calcium needed for stabilization during lysozyme conversion to protoplasts. It is argued that calcium could function to stabilize the inner membrane anionic groups over the anhydrous dipicolinic acid-containing core of resting spores.     

Spore heat resistance and specific mineralization

Spores of Bacillus megaterium ATCC 19213, Bacillus subtilis niger and Bacillus stearothermophilus ATCC 7953 were converted to fully demineralized, but viable, H forms by controlled acid titration. H forms were more heat sensitive than were native forms, but z values were greater for killing of H spores than those for native spores. Therefore, the differences in heat sensitivity between native and H forms decreased with increasing killing temperature. The increase in heat sensitivity associated with demineralization did not appear to be due to damage to cortex lytic enzymes of the germination system because it could not be moderated by decoating heated H spores and plating them on medium with added lysozyme. H spores could be remineralized by means of back titration with appropriate base solutions. The remineralized spores, except for the Na form, were then more heat resistant than were H spores. Ca and Mn were more effective in restoring resistance than were Mg and K. Generally, the remineralized forms (except for the Na form) had z values greater than those of the native forms but still less than those of the H forms. At lower killing temperatures, the reinstatement of resistance could be related to the extent of remineralization. However, at higher killing temperatures, only a fraction of the mineral was effective in restoring resistance, and higher levels of remineralization did not result in greater resistance. Mineralization is clearly an important factor in spore heat resistance, but the relationship between resistance and mineralization is complex and dependent on killing temperature.     

Mineralization and heat resistance of bacterial spores.

The heat resistances of the fully demineralized H-form spores of Bacillus megaterium ATCC 19213, B. subtilis var. niger, and B. stearothermophilus ATCC 7953 were compared with those of vegetative cells and native spores to assess the components of resistance due to the mineral-free spore state, presumably mainly from dehydration of the spore core, and to mineralization. Mineralization greatly increased heat resistance at lower killing temperatures but appeared to have much less effect at higher ones.     

Surface hydrophobicity of spores of Bacillus spp

The surface hydrophobicity of 12 strains of Bacillus spp. was examined in a hexadecane-aqueous partition system. Mature and germinated spores of Bacillus megaterium QM B1551 transferred to the hexadecane layer, while vegetative and sporulating cells did not. Wild-type spores were more hydrophobic than spores of an exosporium-deficient mutant of B. megaterium QM B1551, although the mutant spores were shown to be hydrophobic to some extent by using increased volumes of hexadecane. This result suggests that the exosporium is more hydrophobic than the spore coat and that the surface hydrophobicity of spores depends mainly on components of the exosporium. The surface hydrophobicity of spores of nine other species of Bacillus was also examined, and spores having an exosporium were more hydrophobic than those lacking an exosporium. Thus measurement of the hydrophobicity of spores by the hexadecane partition method may provide a simple and rapid preliminary means of determining the presence or absence of an exosporium.     

Identification of germination gene of Bacillus megaterium

Glucose, KNO3, proline and leucine initiate the spore germination of B. megaterium ATCC 12872, but not of B. megaterium ATCC 19213. In order to isolate the gene concerning germination of B. megaterium ATCC 12872, we constructed its gene library in plasmid vector, and introduced into B. megaterium ATCC 19213. We obtained a transformant whose spores differed from those of the wild type strain with respect to germinability. Spores of this transformant could be germinated by glucose, proline or leucine. The recombinant plasmid prepared from this transformant was found to carry 2 kilobase pairs fragment of B. megaterium ATCC 12872 DNA. This fragment may contain the gene encoding the protein which plays an important role in germination.     

Method to sensitize bacterial spores to subsequent killing by dry heat or ultraviolet irradiation

Hydrogen peroxide and ultraviolet irradiation are known to interact synergistically for killing of bacterial spores. Synergy could be demonstrated with spores of Bacillus megaterium ATCC19213 adsorbed to filter paper strips or glass coverslips treated first with the peroxide and then dried for as long as 48 h prior to UV irradiation. This delayed action was considered to be due to absorption of the peroxide by the spores in an active but not readily vaporized form, which could become sporicidal also if the spores were heated to 50 degrees C. B. megaterium spores mixed with 0.1% (32.6 mM) H(2)O(2) solution appeared to absorb as much as 15 micromol/mg dry weight or about 0.5 mg/mg, but only a third to half of the peroxide could be recovered by water washing. A part of the unrecovered peroxide was degraded in reactions resulting in measurable production of oxygen. Degradation was not reduced by heating the spores to 65 degrees C or by azide and so appeared to be non-enzymatic. Spores of the anaerobe Clostridium sporogenes were also sensitized to ultraviolet killing by H(2)O(2) treatment followed by drying. They appear to absorb less peroxide, only about 2 micromol/mg, but had lower capacities to degrade H(2)O(2) so that nearly all of the peroxide could be recovered by washing with water. The findings presented should be helpful in the design of new methods for synergistic killing of spores by H(2)O(2) and UV irradiation or dry heat, especially involving, for example, packaging materials.     

Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Protoplast dehydration correlated with heat resistance of bacterial spores.

Water content of the protoplast in situ within the fully hydrated dormant bacterial spore was quantified by use of a spore in which the complex of coat and outer (pericortex) membrane was genetically defective or chemically removed, as evidenced by susceptibility of the cortex to lysozyme and by permeability of the periprotoplast integument to glucose. Water content was determined by equilibrium permeability measurement with 3H-labeled water (confirmed by gravimetric measurement) for the entire spore, with 14C-labeled glucose for the integument outside the inner (pericytoplasm) membrane, and by the difference for the protoplast. The method was applied to lysozyme-sensitive spores of Bacillus stearothermophilus, B. subtilis, B. cereus, B. thuringiensis, and B. megaterium (four types). Comparable lysozyme-resistant spores, in which the outer membrane functioned as the primary permeability barrier to glucose, were employed as controls. Heat resistances were expressed as D100 values. Protoplast water content of the lysozyme-sensitive spore types correlated with heat resistance exponentially in two distinct clusters, with the four B. megaterium types in one alignment, and with the four other species types in another. Protoplast water contents of the B. megaterium spore types were sufficiently low (26 to 29%, based on wet protoplast weight) to account almost entirely for their lesser heat resistance. Corresponding values of the other species types were similar or higher (30 to 55%), indicating that these spores depended on factors additional to protoplast dehydration for their much greater heat resistance.     

Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus"

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP. Genes coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.     

L-alanine and inosine enhancement of glucose triggering in Bacillus megaterium spores

Both rate and extent of germination of Bacillus megaterium 14581 (ATCC) spores are considerably augmented when L-alanine and inosine are added to the glucose commonly used as triggering agent for this strain. This enhancement does not arise from heterogeneity in germination requirements of the dormant spore, but is rather a consequence of the combined action of glucose and either or both of the added reagents on a sizeable fraction of spores unable to germinate in glucose alone. Nearly half of the spores that eventually germinate in the mixture of germinants used are either triggered by glucose or are sensitized by it to subsequent triggering by L-alanine and inosine in the first 10 s of imbibition. For a good number of these spores, then, triggering consists of a sequence of separable events.     

Resonance Raman scattering by bacterial spores

Abstract The laser Raman spectra of lyophilized spores of 5 species (6 strains) of Bacillus were examined. Under the experimental conditions employed, only Bacillus megaterium species showed 3 intense Raman bands at 1515, 1157 and 1007 cm⁻¹. Spores of other species, despite their high content of dipicolinic acid, did not show distinct Raman signals.
The strong, scattering bands at 1515 and 1157 cm⁻¹ in the spectra of spores of B. megaterium may be attributable to conjugated double-bond systems, probably of membrane-associated carotenoids. Their high intensities are due to resonance enhancement.