Studies on the bacterial spore coat 6 effects of alkali extraction on the spore of Bacillus thiaminolyticus.

Thin sections of the spore of Bacillus thiaminolyticus Matsukawa and Misawa show a characteristic surface structure with five ridges, and a series of three district layers. The outer layer of the spore coat was peeled off by SDS sonic treatment, and than the middle layer was solubilized by alkali extraction of the SDS sonic-treated spore. The spores subjected to these treatments were still refractile, heat resistant, and contained dipicolinic acid, but lost their resistance to mechanical shock.     

The spore wall and polar tube proteins of the microsporidian Nosema grylli: the major spore wall protein is released before spore extrusion

Incubation of Nosema grylli spores in alkaline--saline solution (10 mM KOH, 170 mM KCl) leads to solubilization of the major spore wall protein of 40 kDa (p40). Both the compounds of this solution are crucial for p40 solubilization. After spore incubation in 170 mM KCl no proteins were released in the medium. In contrast, 10 mM KOH causes a release of many spore proteins but only a small amount of p40. A long storage of spores (over a year) in water or 0.02% sodium azide results in a sharp decrease of p40 content. Specific polyclonal antibodies were obtained by immunization of rabbits with isolated p40. The specificity of serum was confirmed by immunoblotting. IFA showed reliable reaction on the envelopes of sporonts and sporoblasts, whereas only part of spores reacted with antibodies. This distinction may be due to changing surface antigens during spore maturation. Solubilization of p40 under alkaline conditions could be associated with spore extrusion, since a subsequent transfer of spores to neutral solution leads to their discharge. Subsequent wash of discharged spores with 1-3% SDS, 9 M urea and treatment by 100% 2-ME result in solubilization of protein of 56 kDa (p56). The maximum concentration of 2-ME is important for isolation of pure p56. Evidence has been provided that p56 is a protein of N. grylli polar tubes. Treatment of discharged spores by 2-ME in the presence of SDS results in solubilization of four additional proteins with molecular weights about 46, 34, 21 and 15 kDa.     

The Bacillus anthracis spore

In response to starvation, Bacillus anthracis can form a specialized cell type called the spore, which is the infectious particle for the disease anthrax. The spore is largely metabolically inactive and can resist a wide range of stresses found in nature. In spite of its dormancy, the spore can sense the presence of nutrient and rapidly return to vegetative growth. These properties help the spore to persist for long periods of time in the environment, survive host defenses after entering the body, and cause disease when the correct location in the host is reached. The anatomy of the spore is unique among bacteria, being comprised of a series of specialized concentric shells, each of which provides specific critical functions. Surrounding the spore core (which houses the chromosome) is a peptidoglycan layer important for spore dormancy, a protein shell that resists a variety of toxic molecules, and finally an exterior protein and glycoprotein layer that, among other functions, mediates interactions with surfaces, including those encountered by the spore within the host. Detailed molecular analysis of these shells has shed considerable light on how each layer determines specific spore properties. Future work, especially on the outermost spore layer, is likely to advance therapeutics, methods for spore decontamination and other critical biodefense technologies.     

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 prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Myxococcus xanthus protein C is a major spore surface protein.

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.     

Formation of the Dictyostelium spore coat

The spore coat forms as a rigid extracellular wall around each spore cell during culmination. Coats purified from germinated spores contain multiple protein species and an approximately equal mass of polysaccharide, consisting mostly of cellulose and a galactose/N-acetylgalactosamine polysaccharide (GPS). All but the cellulose are prepackaged during prespore cell differentiation in a regulated secretory compartment, the prespore vesicle. The morphology of this compartment resembles an anastomosing, tubular network rather than a spherical vesicle. The molecules of the prespore vesicles are not uniformly mixed but are segregated into partially overlapping domains. Although lysosomal enzymes have been found in the prespore vesicle, this compartment does not function as a lysosome because it is not acidic, and a common antigen associated with acid hydrolases is found in another, acidic vesicle population. All the prespore vesicle profiles disappear at the time of appearance of their contents outside of the cell; this constitutes an early stage in spore coat formation, which can be detected both by microscopy and flow cytometry. As an electron-dense layer, the future outer layer of the coat, condenses, cellulose can be found and is located immediately beneath this outer layer. Certain proteins and the GPS become associated with either the outer or inner layers surrounding this middle cellulose layer. Assembly of the inner and outer layers occurs in part from a pool of glycoproteins that is shared between spores, and unincorporated molecules loosely reside in the interspore matrix, a location from which they can be easily washed away. When the glycosylation of several major protein species is disrupted by mutation, the coat is assembled, but differences are found in its porosity and the extractibility of certain proteins. In addition, the retention or loss of proteolytic fragments in the mutants indicates regions of spore coat proteins that are required for association with the coat. Comparative examination of the macrocyst demonstrates that patterns of molecular distributions are not conserved between the macrocyst and spore coats. Thus spore coat assembly is characterized by highly specific intermolecular interactions, leading to saturable associations of individual glycoproteins with specific layers and the exclusion of excess copies to the interspore space.     

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.     

Immunological detection of the GerA spore germination proteins in the spore integuments of Bacillus subtilis using scanning electron microscopy

Abstract To clarify the molecular mechanisms that trigger spore germination of Bacillus subtilis , the location of GerA proteins (GerAA, GerAB and GerAC), which were reported to be putative gene products of a receptor for one of the germinants, l-alanine, was investigated by immunological techniques using anti-GerA peptide antibodies. Four antibodies were raised against the corresponding epitopes, two in GerAA, one in GerAB and the other in GerAC molecules. The binding of all four antibodies to the inner surface of the cortex-less spore coat fragments could be seen by scanning immunoelectron microscopy with colloidal gold particles. The result agreed with the fact, previously reported, that the colloidal gold particles were visualized just inside the spore coat layer by transmission immunoelectron microscopy using another anti-GerAB peptide antibody.     

Ultrastructural study of spore wall morphogenesis inOphioglossum thermale kom. var.nipponicum (Miyabe et Kudo) nishida

Spore wall morphogenesis ofOphioglossum thermale var.nipponicum was examined by transmission electron microscopy. The spore wall of this species consists of three layers: endospore, exospore, and perispore. The spore wall development begins at the tetrad stage. At first, the outer undulating lamellar layer of the exospore (Lo) is formed on the spore plasma membrane in advance of the inner accumulating lamellar layer (Li) of the exospore. Next, the homogeneous layer of the exospore (H) is deposited on the outer lamellar layer. Both lamellar layers may be derived from spore cytoplasm; and the homogeneous layer, from the tapetum. Then the endospore (EN) is formed. It may be derived from spore cytoplasm. The membranous perispore (PE), derived from the tapetum, covers the exospore surface as the final layer. Though the ornamentation of this species differs distinctly from that ofO. vulgatum, the results mentioned above are fundamentally in accordance with the data obtained fromO. vulgatum (Lugardon, 1971). Therefore, the pattern of spore wall morphogenesis appears to be very stable in the genusOphioglossum.     

Effect of depletion of FtsY on spore morphology and the protein composition of the spore coat layer in Bacillus subtilis

Bacillus subtilis FtsY is a homolog of the alpha-subunit of mammalian signal recognition particle (SRP) receptor, and is essential for protein translocation and vegetative cell growth. An FtsY conditional null mutant (strain ISR39) can express ftsY during the vegetative stage but not during spore formation. Spores of ISR39 have the same resistance to heat and chloroform as the wild-type, while their resistance to lysozyme is reduced. Electron microscopy showed that the outer coat of spores was incompletely assembled. The coat protein profile of the ftsY mutant spores was different from that of wild-type spores. The amounts of CotA, and CotE were reduced in spore coat proteins of ftsY mutant spores and the molecular mass of CotB was reduced. In addition, CotA, CotB, and CotE are present in normal form at T(8) of sporulation in ftsY mutant cells. These results suggest that FtsY has a pivotal role in assembling coat proteins onto the coat layer during spore morphogenesis.     

Fine structure of resting spore formation and germination in Entomophthora virulenta

The fine structure during the formation and germination of resting spores of Entomophthora virulenta is described. There were many microbodies in contact with oil droplets, and the microbodies appeared to participate in spore germination. The mature resting spore had an epispore layer with two regions and an endospore layer with five regions. Dictyosomes, numerous vesicles, and lomasomes were produced during the formation of the endospore layer. Prior to spore germination, the single large oil droplet separated into numerous small oil droplets, and the new cell wall was formed beneath the endospore layer which gradually disintegrated possibly by enzymatic actions. The germ tube perforated the epispore layer mainly by mechanical pressure.     

Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface

The exosporium is the outermost layer of the Bacillus anthracis spore. The predominant protein on the exosporium surface is BclA, a collagen-like glycoprotein. BclA is incorporated on the spore surface late in the B. anthracis sporulation pathway. A second collagen-like protein, BclB, has been shown to be surface-exposed on B. anthracis spores. We have identified sequences near the N-terminus of the BclA and BclB glycoproteins responsible for the incorporation of these proteins into the exosporium layer of the spore and used these targeting domains to incorporate reporter fluorescent proteins onto the spore surface. The BclA and BclB proteins are expressed in the mother cell cytoplasm and become spore-associated in a two-step process involving first association of the protein with the spore surface followed by attachment of the protein in a process that involves a proteolytic cleavage event. Protein domains associated with each of these events have been identified. This novel targeting system can be exploited to incorporate foreign proteins into the exosporium of inactivated, spores resulting in the surface display of recombinant immunogens for use as a potential vaccine delivery system.     

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.     

红盖鳞毛蕨孢子发育的超微结构和细胞化学研究

采用透射电镜和细胞化学技术对红盖鳞毛蕨(Dryopteris erythrosora(Eaton)O.Ktze.)的孢子发育过程进行了研究,根据超微结构和细胞化学特征可将其孢子发育过程分为3个阶段:(1)孢子母细胞及其减数分裂阶段:孢子母细胞壳在孢原细胞末期开始形成,位于孢子母细胞及其减数分裂形成的四分体外侧,PAS反应显示其为多糖性质,与胼胝质壁为同功结构;在减数分裂形成的四分孢子之间产生孢子外壳,从功能、形成位置和时间上看与胼胝质壁相似,但苏丹黑B反应显示其可能含有脂类物质,与孢子母细胞壳和胼胝质壁不同。(2)孢子外壁形成阶段:外壁为乌毛蕨型(Blechnoidal-type),由薄的多糖性质的外壁内层和表面平滑的孢粉素外壁外层构成;小球参与外壁外层的形成,组织化学分析显示小球的中央区域和外壁外层内侧部分由红色(多糖)变为黄色,小球的表面区域和外壁外层部分始终被染成黑色(脂类),可知小球与外壁同步发育。(3)孢子周壁形成阶段:周壁为凹陷型(Cavate-type),包括2层,内层薄,紧贴外壁,外层隆起形成孢子脊状褶皱纹饰的轮廓,以少见的向心方向发育;苏丹黑B和PAS反应观察周壁被染成橙色,推测其可能由多糖等成分构成;孢子囊壁细胞参与周壁的形成。本研究为揭示蕨类植物孢子发生的细胞学机制提供了新资料。     

Electron microscopic examination of the dormant spore and the sporulation of Paenibacillus motobuensis strain MC10

We previously reported a new species Paenibacillus motobuensis. The type strain MC10 was stained gram-negative, but had a gram-positive cell wall structure and its spore had a characteristic star shape. The spore and sporulation process of P. motobuensis strain MC10 were examined by electron microscopy using the technique of freeze-substitution in thin sectioning. The structure of the dormant spore was basically the same as that of the other Bacillus spp. The core of the spore was enveloped with two main spore components, the cortex and the spore coat. In thin section, the spore showed a star-shaped image, which was derived from the structure of the spore coat, which is composed of three layers, namely the inner, middle and outer spore coat. The middle coat was an electron-dense thick layer and had a characteristic ridge. By scanning electron microscopic observation, the ridges were seen running parallel to the long axis of the oval-shaped spore. The process of sporulation was essentially the same as that of the other Bacillus spp. The forespore was engulfed by the mother cell membrane, then the spore coat and the cortex were accumulated in the space between the mother cell membrane and forespore membrane. The mother cell membrane seemed to participate in the synthesis of the spore coat. MC10 strain showed almost identical heat resistance to that of B. subtilis.     

Characterization of spore appendages from Bacillus cereus strains

AIMS: Further characterization and comparison of spore appendages from Bacillus cereus strains. METHODS AND RESULTS: Appendages were isolated from 10 B. cereus strains from the food industry and food-borne outbreaks. The appendage proteins were dissolved in sample buffer containing 2% SDS and 5% mercaptoethanol at 100 degrees C, and subjected to SDS-PAGE. None of the appendages showed identical protein patterns. Western blots, using antibodies raised against a 3.5 kDa appendage protein, showed that the majority of the appendage proteins reacted with the antibody. Removal of the appendages by sonic treatment of the spores did not alter their heat resistance. The appendages were digested by proteinase K, pepsin, and the enzymes in the detergent Paradigm 10, but not by trypsin or chymotrypsin. Spore adhesion to stainless steel was scarcely affected by removal of the appendages. Digestion of adhered intact spores (with appendages) with Paradigm 10 showed a high degree of variation. CONCLUSIONS: Spore appendages from B. cereus are complex proteinaceous structures that differ among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Information about spore appendages and their involvement in spore adhesion is crucial for improving cleaning methods used for control of bacterial spores in the food industry.     

Ultrastructural analysis of the freeze-etched spore envelope of the microsporidian,Nosema apis zander

The outer limiting layer of the spore coat ofNosema apis is relatively smooth. The inner limiting layer shows two fractured faces, the concave face carrying many stud-like projections, 120 nm long and 50 nm high, while the convex face carries numerous depressions which are complementary to the projections. In addition, the convex face bears 7 nm particles. In between the outer and inner limiting layers lies the thick homogeneous portion of spore coat which is comprised of numerous microfibres, each 9 nm in diameter. These microfibres resemble those in the freeze-etched host endocuticle. Next to the inner limiting layer of the spore coat are double spore membranes. The convex faces of these spore membranes have a dense population of particles, each 7 nm in diameter.     

Aspects of spore production in the red algaCeramium

Summary Tetraspore development from the post-meiotic to the mature stage has been studied using light and electron microscopy and histochemistry. The structure of the mature carpospore is identical to that of the tetraspore suggesting a similar developmental sequence.The tetrasporangial wall consists of 3 main fibrillar layers, the origin of the inner of which appears to be the wall-plasmalemma interface. The development of furrows cleaving the protoplast into 4 results in the formation of new plasmalemma and subsequently new wall fibrils. The Golgi apparatus is important in the formation of two well-defined substances. The first is fibrillar and is secretedvia vacuole-like structures into the sporangial wall. After spore release, this functions as a protective mucilaginous layer. The second has a distinctive fine structural morphology and probably functions as an adhesive.Observations on spore releasein vivo reveals a similar process for both types of spore. Each spore is surrounded by mucilage which may assist in initial attachment prior to the secretion of the adhesive.     

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.