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The first ∼10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca(2+) but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores of Bacillus cereus and Bacillus megaterium, as well as of several isogenic Bacillus subtilis strains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca²+ and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time, T(lag), at which spores began release of the great majority of their CaDPA during the germination of B. subtilis spores with different nutrient germinants and also increased the variability of T(lag) values. (iii) The time period, ΔT(release), between T(lag) and the time, T(release), at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values of I(release), the intensity of a spore's DIC image at T(release), than did untreated spores and had much longer time periods, ΔT(lys), for the reduction in I(release) intensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases in T(lag) and ΔT(release) were also observed when wet-heat-treated B. subtilis spores were germinated with the nonnutrient dodecylamine, while the change in I(release) was less significant. (vi) The effects of wet-heat treatment on nutrient germination of B. cereus and B. megaterium spores were generally similar to those on B. subtilis spores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

Clostridium perfringens type A isolates carrying a chromosomal copy of the enterotoxin (cpe) gene are involved in the majority of food poisoning (FP) outbreaks, while type A isolates carrying a plasmid-borne cpe gene are involved in C. perfringens-associated non-food-borne (NFB) gastrointestinal diseases. To cause diseases, C. perfringens spores must germinate and return to active growth. Previously, we showed that only spores of FP isolates were able to germinate with K(+) ions. We now found that the spores of the majority of FP isolates, but none of the NFB isolates, germinated with the cogerminants Na(+) and inorganic phosphate (NaP(i)) at a pH of approximately 6.0. Spores of gerKA-KC and gerAA mutants germinated to a lesser extent and released less dipicolinic acid (DPA) than did wild-type spores with NaP(i). Although gerKB spores germinated to a similar extent as wild-type spores with NaP(i), their rate of germination was lower. Similarly, gerO and gerO gerQ mutant spores germinated slower and released less DPA than did wild-type spores with NaP(i). In contrast, gerQ spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaP(i) germination. In sum, this study identified NaP(i) as a novel nutrient germinant for spores of most FP isolates and provided evidence that proteins encoded by the gerKA-KC operon, gerAA, and gerO are required for NaP(i)-induced spore germination.

The endospores formed by strains of type A Clostridium perfringens that produce the C. perfringens enterotoxin (CPE) are known to be more resistant to heat and cold than strains that do not produce this toxin. The high heat resistance of these spores allows them to survive the cooking process, leading to a large number of food-poisoning cases each year. The relative importance of factors contributing to the establishment of heat resistance in this species is currently unknown. The present study examines the spores formed by both CPE(+) and CPE(-) strains for factors known to affect heat resistance in other species. We have found that the concentrations of DPA and metal ions, the size of the spore core, and the protoplast-to-sporoplast ratio are determining factors affecting heat resistance in these strains. While the overall thickness of the spore peptidoglycan was found to be consistent in all strains, the relative amounts of cortex and germ cell wall peptidoglycan also appear to play a role in the heat resistance of these strains.

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in a 1:1 chelate with calcium ion (Ca-DPA) comprises 5 to 15% of the dry weight of spores of Bacillus species. Ca-DPA is important in spore resistance to many environmental stresses and in spore stability, and Ca-DPA levels in spore populations can vary with spore species/strains, as well as with sporulation conditions. We have measured levels of Ca-DPA in large numbers of individual spores in populations of a variety of Bacillus species and strains by using microfluidic Raman tweezers, in which a single spore is trapped in a focused laser beam and its Ca-DPA is quantitated from the intensity of the Ca-DPA-specific band at 1,017 cm(-1) in Raman spectroscopy. Conclusions from these measurements include the following: (i) Ca-DPA concentrations in the spore core are >800 mM, well above Ca-DPA solubility; (ii) SpoVA proteins may be involved in Ca-DPA uptake in sporulation; and (iii) Ca-DPA levels differ significantly among individual spores in a population, but much of this variation could be due to variations in the sizes of individual spores.

Bacteria of various Bacillus species are able to grow in media with very high osmotic strength in part due to the accumulation of low-molecular-weight osmolytes such as glycine betaine (GB). Cells of Bacillus species grown in rich and minimal media contained low levels of GB, but GB levels were 4- to 60-fold higher in cells grown in media with high salt. GB levels in Bacillus subtilis cells grown in minimal medium were increased approximately 7-fold by GB in the medium and 60-fold by GB plus high salt. GB was present in spores of Bacillus species prepared in media with or without high salt but at lower levels than in comparable growing cells. With spores prepared in media with high salt, GB levels were highest in B. subtilis spores and > or =20-fold lower in B. cereus and B. megaterium spores. Although GB levels in B. subtilis spores were elevated 15- to 30-fold by GB plus high salt in sporulation media, GB levels did not affect spore resistance. GB levels were similar in wild-type B. subtilis spores and spores that lacked major small, acid-soluble spore proteins but were much lower in spores that lacked dipicolinic acid.

Prior studies have described a morphologically diverse group of intestinal microorganisms associated with surgeonfish. Despite their diversity of form, 16S rRNA gene surveys and fluorescent in situ hybridizations indicate that these bacteria are low-G+C gram-positive bacteria related to Epulopiscium spp. Many of these bacteria exhibit an unusual mode of reproduction, developing multiple offspring intracellularly. Previous reports have suggested that some Epulopiscium-like symbionts produce dormant or phase-bright intracellular offspring. Close relatives of Epulopiscium, such as Metabacterium polyspora and Clostridium lentocellum, are endospore-forming bacteria, which raises the possibility that the phase-bright offspring are endospores. Structural evidence and the presence of dipicolinic acid demonstrate that phase-bright offspring of Epulopiscium-like bacteria are true endospores. In addition, endospores are formed as part of the normal daily life cycle of these bacteria. In the populations studied, mature endospores were seen only at night and the majority of cells in a given population produced one or two endospores per mother cell. Phylogenetic analyses confirmed the close relationship between the endospore-forming surgeonfish symbionts characterized here and previously described Epulopiscium spp. The broad distribution of endospore formation among the Epulopiscium phylogenetic group raises the possibility that sporulation is a characteristic of the group. We speculate that spore formation in Epulopiscium-like symbionts may be important for dispersal and may also enhance survival in the changing conditions of the fish intestinal tract.

Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.

Thermal resistance in heat-sensitive, dipicolinic acid (DPA)-less spores of Bacillus cereus Ht-8 heated in sucrose solutions increased at and above a concentration of 2 M sucrose. The decimal reduction times at 75 degrees C for spores heated in 0.0, 1.8, 2.2, and 2.6 M sucrose were 2.0, 2.8, 4.5, and 12 min, respectively. Maltose, fructose, and glucose increased heat resistance above that observed in water but did not elevate resistance to the level observed with sucrose at the same osmolality. Cation-induced loss of thermal resistance in chemically sensitized spores was reversed in the presence of sucrose. Spores germinated in brain heart infusion were resistant when heated in sucrose. In the presence of sucrose, spores exhibited an increase in optical density at 700 nm. Electron micrographs of the DPA-less spores suspended in 2.2 M sucrose revealed a shrinkage of outer coats and exosporium membranes. The results suggested that the osmotic property of sugars increased thermal resistance in DPA-less spores. The osmotic pressure exerted by sugars may be similar to the pressure that usually exists within the cortex of normal spores containing DPA and may cause the dehydration of the protoplast and the consequent thermal resistance. The role of dehydration and the nonessential nature of DPA for thermal resistance in spores were confirmed.

A gas-liquid chromatographic procedure has been developed to quantitate dipicolinic acid in bacterial spores. The culture, washed from a plate, was hydrolyzed with acid containing the internal standard, pyridine-2,4-dicarboxylate, and then extracted into methyl isobutyl ketone. The internal standard and dipicolinic acid were then extracted into a small volume of trimethylphenylammonium hydroxide. Injection of the resultant quaternary ammonium salts into a gas chromatograph yielded, via thermal decomposition, the methyl ester derivatives of the dipicolinic acid and the internal standard. The amount of dipicolinic acid in the sample was determined from a standard curve. The method was sensitive to 100 ng of dipicolinic acid per sample and was 1,000 to 5,000 times more sensitive than the commonly used methods. Preparation of the sample required less than 1.5 h and less than 15 min of the analyst's time.

The distribution of a number of key elements in Bacillus cereus T spores was determined by high-resolution scanning electron probe X-ray microanalysis. To circumvent the redistribution of soluble or weakly bound elements, freeze-dried cryosections of spores, which had been rapidly frozen in 50% aqueous polyvinyl pyrrolidone, were employed. The sections were examined by using a modified Philips EM400 electron microscope fitted with a field emission gun, scanning transmission electron microscopy attachment, and a computer-linked energy-dispersive X-ray microanalysis system. X-ray maps for selected elements and the corresponding electron image were produced simultaneously by scanning the cryosections with a fine electron beam in a raster pattern, using the scanning transmission electron microscopy attachment. The results indicated that almost all of the calcium, magnesium, and manganese, together with most of the phosphorus, was located in the core region. An unexpectedly high concentration of silicon was found in the cortex/coat layer. Granules containing high concentrations of calcium, manganese, and phosphorus were demonstrated in spores containing reduced levels of dipicolinic acid. Spot mode analyses, in which a stationary beam was located over the region of interest in the spore cryosection, confirmed the results obtained with the scanning mode and also provided a more accurate quantitation of the elemental concentrations on a dry weight bases.

Isolated stage III forespores of Bacillus megaterium ATCC 19213 in aqueous suspensions were nearly as dehydrated as mature spores, as indicated by low dextran-impermeable volumes of ca. 3.0 ml per g (dry weight) of cells compared with values of ca. 2.6 for mature spores and 7.3 for vegetative cells. The forespores lacked dipicolinate, had only minimal levels of calcium, magnesium, manganese, potassium, and sodium, and were more heat sensitive than vegetative cells. The effective homogeneous conductivities and dielectric constants measured over a frequency range of 1 to 200 MHz indicated that the inherent conductivities of the forespores were unusually low, in keeping with their low mineral contents, but that the forespores could be invaded by environmental ions which could penetrate dielectrically effective membranes. Overall, our findings support the view that the dehydration of a forespore during stage III of sporogenesis may be the result of ion movements out of the forespore into the sporangium.

The lysyl-transfer ribonucleic acid synthetase (LRS) and tryptophanyl-transfer ribonucleic acid synthetases (TRS) (l-lysine:tRNA ligase [AMP], EC 6.1.1.6; and l-tryptophan:tRNA ligase [AMP], EC 6.1.1.2) have been purified 60- and 100-fold, respectively, from vegetative cells and spores of Bacillus subtilis. There are no significant differences between the corresponding spore and vegetative enzymes with respect to their elution characteristics from columns of phosphocellulose or hydroxylapatite, their molecular weight (~130,000 for LRS and ~87,000 for TRS as determined by gel filtration), their kinetic constants for substrates (in the amino acid-dependent adenosine triphosphate-pyrophosphate exchange reaction), and the kinetics of inactivation by heat and by antibody. The Mg(2+) requirement for optimal enzyme activity of the corresponding spore and vegetative enzyme differ slightly. Mutants having defective (temperature sensitive) vegetative LRS or TRS activities produce spores in which these enzymes are also defective. The mutant spores are more heat sensitive than the parental type, but contain normal levels of dipicolinic acid. They germinate normally at the restrictive temperature (43 C), but are blocked at specific developmental stages in outgrowth. No modification in temperature sensitivity phenotype occurs during outgrowth, nor is there a change in molecular weight of the two enzymes. The implication is that the LRS and TRS activities of the vegetative and spore stages are each coded (at least in part) by the same structural gene. The temperature sensitivity of mutant spores is discussed with respect to those factors which are involved in the formation of the heat-resistant state.

The localization of dipicolinic acid in dormant spores of Bacillus subtilis was examined by an immunoelectron microscopy method with colloidal gold-immunoglobulin G complex. The colloidal gold particles were distributed mainly in the core regions of dormant spores and were not observed in those of germinated or autoclaved spores. This result clearly demonstrates that dipicolinic acid is localized in the cores of dormant spores.

The biochemical and physiological basis of density heterogeneity in Renografin of Bacillus subtilis W23 spores was determined by analysis of metals, macromolecules, and dipicolinic acid in the two density classes of the population. Germination rate and heat resistance were measured in both density classes. Atomic absorption spectrophotometry revealed that heavy spores (density = 1.335 g/ml) have 30% more calcium than light spores (density = 1.290 g/ml). Other metals found in greater amounts in heavy spores were manganese and potassium. However, light spores had more sodium than heavy spores. The amounts of carbohydrates, nucleic acids, and proteins were the same in both types of spores, but light spores contained more lipids, whereas heavy spores had 30% more dipicolinic acid than light spores. Calcium and lipid were excluded as causes of the heterogeneity in density in that alteration of their contents in spores did not detectably affect the density of these spores. Spores of two densities were genetically similar. Furthermore, light density spores arose earlier during sporulation than heavy spores as determined by releasing refractile forespores at various times during sporulation. We concluded that light spores represent an incomplete stage in development because they became heavy when reinoculated into spent sporulation medium. This must involve the additional accretion or synthesis of dipicolinic acid.

The release of calcium and dipicolinic acid from spores of Bacillus megaterium KM during L-alanine-induced triggering of germination has been studied using a new, simple, and rapid assay for dipicolinic acid capable of detecting a concentration of 0.5 micron. The release of both calcium and dipicolinate started within seconds of exposure of the spores to L-alanine, thus preceding other measurable changes associated with germination. From the earliest times, the two substances were released in equimolar quantities, although later in germination calcium predominated.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.

A chemically defined medium which allowed germination, outgrowth, and subsequent resporulation of Bacillus cereus T spores, without intervening cell division (microcycle sporogenesis), is described. No medium replacement was required. The second-stage spores were heat-stable and had similar germination characteristics and dipicolinic acid content to primary spores. Deoxyribonucleic acid (DNA) replication began soon after germination and there was a doubling in the DNA content of the cells within 2 hr.

The spore appendages of Clostridium taeniosporum NI were removed from the spores by sonic treatment and were isolated by using discontinuous sucrose gradients. The amino acid composition of the appendages, which are elaborations of the spore coat, was similar to but not identical with the amino acid composition of the coats. Approximately 80% of the appendage dry weight was composed of 17 common amino acids, whereas 68% of the spore coat dry weight was amino acids. Mole ratios of the amino acids differed between the appendages and spore coats. The appendages contained neither diaminopimelic acid nor hydroxyproline. Glucosamine was an abundant constituent but muramic acid was absent. Approximately 10% of appendage dry weight consisted of three sugars, one of which was glucose. Phosphorus content was high and dipicolinic acid was absent. Appendage fine structure was not affected by common buffers, dilute acids and bases, hydrogen bond-breaking agents, certain proteolytic enzymes, or lysozyme.

Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.

Bacillus fastidiosus, which requires uric acid or allantoin, grows and sporulates on a simple medium containing 59.5 mM uric acid, 5.7 mM K(2)HPO(4), and 2% agar in distilled water. Seventy to ninety percent sporulation was achieved in 96 h. Spores obtained on this medium do not need a heat shock prior to germination. The necessary germination conditions for this organism are 30 C, phosphate or this(hydroxymethyl)aminomethane buffer at pH 7.0, and 5.95 mM uric acid. Sporulation occurred earlier (48 h) and with higher frequency (greater than 99%) when Mn(2+) was added to the growth medium. However, these spores germinated only after heat activation (70 C, 30 min). The effectiveness of heat activation was directly dependent upon the concentration of Mn(2+) in the growth medium; 10(-5) M Mn(2+) was the minimal concentration for the effect. This phenomenon was not found upon addition of Ca(2+), Mg(2+), Fe(2+), Zn(2+), or Cu(2+) to the medium. The Mn(2+) content of the spores depended upon the concentration of Mn(2+) in the sporulation medium. There was a significant difference in heat resistance between spores harvested from unsupplemented medium and those harvested from medium supplemented with 5 x 10(-5) M Mn(2+). A D(85 C) value of 6.5 min was determined with the former, whereas the latter had a value of 17.0 min. Very little change in either Ca(2+) or dipicolinic acid content was detected in spores harvested from various Mn(2+)-supplemented media. Thus Mn(2+) may play a role in the inducement of the heat-shock requirement and the formation of spores with increased heat resistance.

Dipicolinic acid-less spores of a Bacillus cereus T mutant in 33 mm sodium bicarbonate (pH 8.5) underwent pseudogermination (germination-like changes) accompanied by ribonucleic acid leakage and loss of viability.

Heat activation of bacterial spores at low pH was investigated in detail. Unlike activation of spores in distilled water at a neutral pH, activation at low pH involves two superimposed processes: enhanced activation and death. Low-pH-activated spores failed to germinate in d-alanine, in contrast to spores activated at neutral pH, owing to the abolition of alanine-racemase activity. Morphological and permeability changes such as release and partial disruption of spores were dipicolinic acid-observed during low-pH activation. The kinetics pattern of low-pH activation, as well as the change in properties of the spores thereafter, suggest that the mechanism of low-pH activation differs from that of other kinds of heat-activation.

A study was made of certain cations present in hemolymph and alimentary tract tissues of healthy and diseased European chafer larvae and the spores of Bacillus popilliae collected from diseased hemolymph. The major ions found in the hemolymph, in order of decreasing abundance, were potassium, magnesium, sodium, and calcium. Hemolymph of diseased larvae contained relatively higher concentrations of sodium, potassium, magnesium, iron, and zinc than hemolymph of healthy larvae. Concomitantly, the concentrations of ions were lower in the mid-gut and anterior intestinal tissues of diseased larvae. Only sodium decreased slightly in the diseased tissues of the rectum and rectal sac; other ions remained unchanged or increased. Little or no manganese or copper was detected in the hemolymph or tissues. The major cations of spores of B. popilliae were sodium, calcium, and magnesium. Small amounts of potassium, manganese, copper, iron, and zinc were detected in the spores. Based on calcium and dipicolinic acid determinations of the spores, sufficient calcium was found to allow for the formation of calcium dipicolinate in the expected concentrations.

Sporulation of Bacillus megaterium Km (ATCC 13632) was synchronized by a technique employing three 10% transfers. The culture was harvested when 60% of the cells contained spore forms. Dihydrodipicolinic acid synthetase was purified 150-fold by ammonium sulfate fractionation at pH 6.5, heating for 15 min at 45 C at pH 6.0, ammonium sulfate fractionation at pH 6.0, and subsequent chromatography on diethylaminoethyl cellulose. During the final stage of the purification procedure, the enzyme exhibited sensitivity to refrigeration temperatures. The enzyme had a pH optimum of 7.65 in imidazole buffer. The apparent K(m) values were 4.6 x 10(-4) and 5.0 x 10(-4)m for beta-aspartyl semialdehyde and pyruvate, respectively. All attempts to demonstrate cofactor requirements were unsuccessful. Sulfhydryl inhibiting reagents and lysine did not inhibit the enzymatic reaction. The enzyme exhibited maximal thermal resistance at pH 10.5. The thermal stability of the enzyme at 75 C was increased more than 1,800-fold by the addition of 0.3 m pyruvate. The E(a) was 67,300 cal/mole for the thermal denaturation of the enzyme. At 60 C, the DeltaF, DeltaH, and DeltaS values for the thermal denaturation of the enzyme were 22,250, 66,700, and 133 cal per mole per degree, respectively.

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.

Treatment with urea-mercaptoethanol of purified spores of Bacillus thuringiensis, other Bacillus species, and Clostridium roseum solubilizes a protein fraction between 5 and 12% of the dry weight of the spores. This fraction behaves identically to the crystal protein of B. thuringiensis on acrylamide-gel electrophoresis. The protein from all of the Bacillus species shows partial homology with crystal protein, using the Ouchterlony immunodiffusion technique. A further fraction, similar in amount, can be removed from spores of B. thuringiensis by the addition of sodium lauryl sulfate to the urea-mercaptoethanol. Spores of B. thuringiensis extracted in these ways show no difference when compared to untreated spores with respect to viability or resistance to heat and ultraviolet-irradiation. The extracted spores do show differences in their germination requirements and their susceptibility to phase-darkening by lysozyme. It is concluded that an urea-mercaptoethanol-soluble protein or class of protein is a widespread component of bacterial spores, possibly located in the spore coat, and that this protein may be related to the crystal protein of B. thuringiensis.