Morphological and Chemical Studies of the Spores and Parasporal Bodies of Bacillus laterosporus

Spores of Bacillus laterosporus were studied to determine the chemical and morphological nature of their basophilic canoe-shaped parasporal bodies. An unusually high phosphorus content of these spores compared to other Bacillus species appeared to be associated with the parasporal body. Preparations of these "canoes" still attached to the spore coats were indeed high in phosphorus, but also in nitrogen. They were free of lipide-soluble and nucleic acid phosphorus and stained for protein. Some 50 per cent of the total nitrogen, but only 6 to 10 per cent of the total P were liberated by extraction with alkali-thioglycollate (pH 11.5) or alkali alone (pH 12.2–12.5). Proteinaceous material was recovered from these alkaline extracts and electron microscopy indicated that there had been a marked loss of "canoe" substance. Extraction with acid, removed some 80 per cent of the phosphorus associated with the "canoes" as orthophosphate. Chromatographic analyses for amino acids indicated some 14 ninhydrin-positive spots in the canoe-coat preparations whereas the whole spores contained at least 16.     

FOWLER'S BACILLUS AND ITS PARASPORAL BODY

Fowler's bacillus is one of several organisms which form a non-viable inclusion or parasporal body during the process of sporulation. This body is globular and may be as large as or larger than the spore. Its position in the cell is not random; the spore is terminal and the body paracentral, lying between the spore and the remaining vegetative cell chromatin bodies. On completion of sporulation both spore and body are contained within an exosporium. The sequence in the development of the cell structures was followed in ultrathin sections of material fixed in permanganate. When sporulation is well advanced the body begins to grow from a single crystal, then presumably as a result of some disorientation in the growth process it develops as a multicrystalline body with the lattices orientated at different angles. When the body approximates the spore in size, a lamella coat is formed and an exosporium develops which eventually encircles the body and the spore. Other lamella systems microscopically similar to those surrounding the parasporal body develop free in the cytoplasm outside the exosporium. In both of these systems the number of lamellae is variable. The spore coat of Fowler's bacillus, consisting of an outer lamella layer and an inner unresolved amorphous layer has been found microscopically identical to the spore coat of B. cereus. In both organisms the lamella layer of the spore coat consists, in contrast to the other lamella systems, of a regular number of lamellae. Physiological tests would indicate that Fowler's bacillus is a variety of B. cereus.     

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.     

Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation

Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.     

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.     

Morphological and chemical studies of the spores and parasporal bodies of Bacillus laterosporus

Spores of Bacillus laterosporus were studied to determine the chemical and morphological nature of their basophilic canoe-shaped parasporal bodies. An unusually high phosphorus content of these spores compared to other Bacillus species appeared to be associated with the parasporal body. Preparations of these "canoes" still attached to the spore coats were indeed high in phosphorus, but also in nitrogen. They were free of lipide-soluble and nucleic acid phosphorus and stained for protein. Some 50 per cent of the total nitrogen, but only 6 to 10 per cent of the total P were liberated by extraction with alkali-thioglycollate (pH 11.5) or alkali alone (pH 12.2-12.5). Proteinaceous material was recovered from these alkaline extracts and electron microscopy indicated that there had been a marked loss of "canoe" substance. Extraction with acid, removed some 80 per cent of the phosphorus associated with the "canoes" as orthophosphate. Chromatographic analyses for amino acids indicated some 14 ninhydrin-positive spots in the canoe-coat preparations whereas the whole spores contained at least 16.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Survival of Bacillus thuringiensis Spores in Soil

Bacillus thuringiensis spores and parasporal crystals were incubated in natural soil, both in the laboratory and in nature. During the first 2 weeks, the spore count decreased by approximately 1 log. Thereafter, the number of spore CFU remained constant for at least 8 months. B. thuringiensis did not lose its ability to make the parasporal crystals during its residence in soil. Spore survival was similar for a commercial spore-crystal preparation (the insecticide) and for laboratory-grown spores. In contrast to these results, spores that were produced in situ in soil through multiplication of added vegetative cells survived for only a short time. For spore additions to soil, variations in soil pH had little effect on survival for those spores that survived the first 2 weeks of incubation. Also without effect were various pretreatments of the spores before incubation in soil or nutritional amendment or desiccation of the soil. Remoistening of a desiccated soil, however, caused a decrease in spore numbers. Spores incubated in soil in the field did not show this, but the degree of soil desiccation in nature probably never reached that for the laboratory samples. The good survival of B. thuringiensis spores after the first 2 weeks in soil seemed to be a result of their inability to germinate in soil. We found no evidence for the hypothesis that rapid germination ability for spores in soil conferred a survival advantage.     

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus cereus, Bacillus thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. The diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.     

Formation and organization of the spore coat ofDictyostelium discoideum

Macromolecular components of the spore coat ofDictyostelium discoideum have been localized by gold-labeled affinity cytochemistry. The outer electron-dense layer is the residence of three prominent glycoproteins that express a fucose-dependent epitope, whereas the inner electron-dense layer includes SP85 and the galactose/N-acetylgalactosamine-containing polysaccharide (GPS). The cellulosic layers are interposed between them. The outer-layer glycoproteins and the GPS also can be found in the interspore fluid, which is usually lost during collection of the spores. Assembly of the spore coat, examined over time, showed that all components, except for the cellulose, are found in an internal secretory vesicle population. All components are found in each vesicle but are not uniformly intermixed within them. Cellulose does not appear until after the outer electron-dense layer of the spore coat has been organized following secretion. The GPS is excluded from the outer dense layer and largely from the cellulosic layer, being more concentrated in the inner layer. SP85 remains localized in the inner dense layer near the cell surface with a circumferentially focal distribution. The distinct distributions of these macromolecular species in the mature spore coat are foreshadowed by their mosaic distribution in the prespore vesicles from which they originate.     

Fine Structure of the Bacillus thuringiensis Spore

The thin-sectioned spore of Bacillus thuringiensis resembles that of Bacillus cereus in fine structure. Planar inclusions occur between the exosporium and spore coat and are structured differently from the parasporal crystal outside the exosporium.     

Temporal Formation and Immunolocalization of an Endospore Surface Epitope During Pasteuria penetrans Sporogenesis

The synthesis and localization of an endospore surface epitope associated with the development of Pasteuria penetrans was determined using a monoclonal antibody (MAb) as a probe. Nematodes, uninfected or infected with P. penetrans, were harvested at 12, 16, 24, and 38 days after inoculation (DAI) and then examined to determine the developmental stage of the bacterium. Vegetative growth of P. penetrans was observed only in infected nematodes harvested at 12 and 16 DAI, whereas cells at different stages of sporulation and mature endospores were observed at 24 and 38 DAI. ELISA and immunoblot analysis revealed that the adhesin-associated epitope was first detected at 24 DAI, and increased in the later stages of sporogenesis. These results indicate that the synthesis of adhesin-related proteins occurred at a certain developmental stage relative to the sporulation process, and was associated with endospore maturation. Immunofluorescence microscopy indicated that the distribution of the epitope is nearly uniform on the periphery of each spore, as defined by parasporal fibers. Immunocytochemistry at the ultrastructural level indicated a distribution of the epitope over the parasporal fibers. The epitope also was detected over other structures such as sporangium and exosporium during the sporogenesis process, but it was not observed over the cortex, inner-spore coat, outer-spore coat, or protoplasm. The appearance of the adhesin epitope first at stage III of sporogenesis and its presence on the parasporal fibers are consistent with an adhesin-related role in the attachment of the mature endospore to the cuticle of the nematode host.     

Peptide Ligands That Bind Selectively to Spores of Bacillus subtilis and Closely Related Species

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.     

Spore coat architecture of Clostridium novyi NT spores

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these studies document the presence of proteinaceous growth spirals in a biological organism.     

Characterization of a Novel <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Phenotype Possessing Multiple Appendages Attached to a Parasporal Body

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore–crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 μm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

Changes of Ultrastructure in Spore Coat of Bacillus thiaminolyticus during Germination and Outgrowth

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L -alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at a prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Composition and Ultrastructure of Streptomyces venezuelae

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.     

Ultrastructural Analysis of Spores and Parasporal Crystals Formed by Bacillus sphaericus 2297

Bacillus sphaericus 2297, growing from a boiled, relatively nontoxic spore inoculum, increased about 30-fold in toxicity for mosquito larvae during early exponential growth but showed an approximately 1,000-fold toxicity increase during the late-exponential phase, as spores began to appear in the culture. The development of spores in the bacterial cells was accompanied by the formation of parasporal crystals. These parasporal crystals appeared during stage III as the forespore septum engulfed the incipient forespore. The paraspores were separated from the forespores by a branch of the exosporium across the cell. Measurements of the parasporal substructure revealed a 6.3-nm distance between the striations. When spores and paraspores were fed to mosquito larvae and the larvae were fixed 15 min after feeding, it was found that the spores remained relatively unchanged but that the matrix of the paraspores was dissolved. After dissolution of the paraspore matrix, a meshlike envelope remained which retained the paraspore shape and which was often in contact with the cross-cell portion of the exosporium. The parasporal crystals may be a source of the mosquito larval toxin in this strain of B. sphaericus, but proof will require their isolation from other cellular components.     

FORMATION AND STRUCTURE OF THE SPORE OF BACILLUS COAGULANS

Spore formation in Bacillus coagulans has been studied by electron microscopy using an epoxy resin (Araldite) embedding technique. The developmental stages from the origin of the initial spore septum to the mature spore were investigated. The two forespore membranes developed from the double layer of cytoplasmic membrane. The cortex was progressively deposited between these two membranes. The inner membrane finally became the spore protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer spore coat itself. In the mature spore the completed integuments around the spore protoplasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium was observed. The method of formation of the cortex and the spore coats is discussed.