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.     

The fine structure of the frozen-etched spore of Nosema apis Zander

The mature spore of Nosema apis possesses a thick spore coat and a particle-bearing spore membrane. Within the spore membrane, in the anterior portion of the spore, is the highly laminated polaroplast. The fractured face of the lamella is granular. The convex face of the polar filament membrane carries few particles, while the concave face bears many densely packed particles. The nucleus of the mature spore is centrally located, and pores were observed on the nuclear envelope.     

Preliminary Observations on the Fine Structure of the Pansporoblast of Thelohania bracteata (Strickland, 1913) (Microsporida, Nosematidae) as Revealed by Freeze-Etching Electron Microscopy

SYNOPSIS. The ultrastructure of a microsporidan pansporoblast was observed with freeze-etching electron microscopy. The cross-fractured face of ovoid mature spores, with the upper part of the spore coat fractured off, revealed the spore membrane; the convex face had many small depressions and the concave face bore fine particles. In cross-section the spore-coat was highly laminated and about 0.5 μ in diameter.
In the cytoplasm of the pansporoblast, fluid-filled and finger-print-life profiles of vesicles were observed. The vesicles were approximately 180 nm in diameter and laminated, each lamella being about 15–18 nm thick. In addition to these vesicles, a population of elevations, each with an average diameter of 40 nm, was evenly distributed in the pansporoblast among the spores. No other cytoplasmic organelles were observed within the pansporoblast. The pansporoblast wall was about 15–19 nm thick with particles 15–18 nm in diameter on its outer surface.     

Effect of polymyxin on the outer membrane of Salmonella typhimurium: freeze-fracture studies.

Polymyxin-caused projections on the cell surface of Salmonella typhimurium were seen as depressions in the outer concave fracture face and as protrusions in the outer convex fracture face, indicating participation of both leaflets of the outer membrane in these projections.     

Ultrastructural changes in the spermatid nuclear envelope of Periplaneta americana during spermiogenesis

The nuclear envelope in the earliest stage of spermatid development possesses double membranes with pores aggregated in certain areas and arranged in a hexagonal pattern. The convex face of the outer nuclear membrane is relatively smooth and the convex face of the inner nuclear membrane carries many particles which are arranged in net-like pattern. In the later developmental stages, nuclear pores were not observed, the convex face of the inner nuclear membrane being covered with densely packed particles and the convex face of the outer nuclear membrane having a rough appearance. During the final stage of spermatid development, the cross-fractured face of the nucleus reveals the profiles of the nuclear fibres, the granular appearance of the convex face of the outer nuclear membrane, and the convex face of the inner nuclear membrane carrying more densely packed particles.     

SPORE WALL ULTRASTRUCTURE IN THE LIVERWORT ATHALAMIA HYALINA

Mature spores of Athalamia hyalina (Marchantiales, Cleveaceae) were examined with both scanning and transmission electron microscopes. Single, hollow, dome-like projections, sometimes having small pores and a coarsely granular surface texture, stud the spore surface, usually in a pattern of concentric circles. In section, the spore wall has an intine and two-layered exine. Intine-like material separates some lamellae of the inner exine, which is joined to the outer exine around the dome bases. Inner exine lamellae are composed of thin (5–6 nm), closely parallel membrane-like subunits. The outer exine is formed from a single large highly modified and doubly-coated lamella, the undulations of which form the surface domes. Dome cavities often are filled with a loose network of granular material.     

Appendage Development in Clostridium bifermentans

The appendages of Clostridium bifermentans UK-A 1003 spores were shown to originate from a substance located just exterior to the outer forespore membrane. The dense spore coat develops along the periphery of this material, and, as the appendages develop in the cytoplasm, the coalescing spore coat intervenes between the appendages and their origin. Freeze etching revealed that the appendages are in the form of distinct fibers in proximity to the mature spore body. These fibers form a network around the spore, seemingly encasing it and insuring that the appendages remain attached to the mature, free spore. The inner wall of each appendage tubule is lined with fibers whereas the outer surface is smooth. The developing exosporium contained several layers consisting of small (3 nm) globular subunits; the outer exosporial surface is composed of relatively unstructured material.     

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.     

Fine Structure of Bacillus megaterium during Microcycle Sporogenesis

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-β-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.     

Ultrastructural changes occurring during germination and outgrowth of spores of the thermophile Bacillus acidocaldarius.

Spores of the thermophilic, acidophilic, Bacillus acidocaldarius were covered by a thick outer coat and a laminated inner coat (5.5 nm periodicity). Small membranous vesicles were present in the spore core and they disappeared as germination proceeded. After depolymerization of the cortex, and a 30% increase in spore diameter a localized gap appeared in the laminated inner coat only. This inner coat gap was narrow and could be the whole length of the spore. The germ cell appeared to grow, or to be pushed towards the inner coat gap, at which stage the outer coat disappeared in the same localized area. As the vegetative cell grew out the spore coat fell away, with loose cortical material still attached to it. The young germ cell developed a large spherical electron dense inclusion body in the cytoplasm, at the same time as the ribosomal and nuclear areas became distinct.     

Dynamics of the assembly of a complex macromolecular structure--the coat of spores of the bacterium Bacillus subtilis

The coat is the outermost layer of spores of many Bacillus species, and plays a key role in these spores' resistance. The Bacillus subtilis spore coat contains > 70 proteins in four distinct layers: the basement layer, inner coat, outer coat and crust. In this issue of Molecular Microbiology, McKenney and Eichenberger study the dynamics of spore coat assembly using GFP-fusions to 41 B. subtilis coat proteins. A key finding in the work is that formation of the spore coat is initiated by the apparently simultaneous assembly of foci of proteins from all four coat layers on the developing spore just as forespore engulfment by the mother cell begins. The expansion of these foci before completion of forespore engulfment then sets up the scaffold to which coat proteins added later in sporulation are added. This study provides new understanding of the mechanism of the assembly of a multi-protein, multi-lamellar structure.     

Metal-binding sites in germinating fern spores (Onoclea sensibilis)

Summary During germination of the spore of the sensitive fernOnoclea sensibilis L. the nucleus migrates from a central position to the proximal face and then to one end of the ellipsoidal spore. An asymmetric cell division follows giving rise to a small cell which differentiates immediately into a rhizoid, and a large cell which divides further to give rise to the prothallus. The proximal face of the spore coat is differentiated from the remainder of the spore by its ability to bind nickel ions under certain conditions and by its staining with a sulfide-silver procedure which localizes heavy metals. The inner portion of the exine at the proximal face is differentiated from the outer part by its ability to stain with sulfide-silver at specific periods during germination. The exine at the proximal face also contains pore-like structures 50 nm in diameter which extend from the inner layer of the exine to the outer surface. Sulfide-silver staining material appears to be extruded through the pores at specific periods during germination. The percentage of spores showing nickel-binding and sulfide-silver stainability increases sharply during the first two to four hours of imbibition, then decreases sharply during the following two hours. This is followed by a second rise in staining at 8 to 12 hours of imbibition.The role of the ion-binding sites in the exine is discussed in relation to the stable polarity of the spore.Publishing prior to 1984 asAlix R. Bassel     

Ultrastructural changes occurring during germination and outgrowth of spores of the thermophile Bacillus acidocaldarius

Spores of the thermophilic, acidophilic, Bacillus acidocaldarius were covered by a thick outer coat and a laminated inner coat (5.5 nm periodicity). Small membranous vesicles were present in the spore core and they disappeared as germination proceeded. After depolymerization of the cortex, and a 30% increase in spore diameter, a localized gap appeared in the laminated inner coat only. This inner coat gap was narrow and could be the whole length of the spore. The germ cell appeared to grow, or to be pushed towards the inner coat gap, at which stage the outer coat disappeared in the same localized area. As the vegetative cell grew out the spore coat fell away, with loose cortical material still attached to it. The young germ cell developed a large spherical electron dense inclusion body in the cytoplasm, at the same time as the ribosomal and nuclear areas became distinct.     

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.     

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 Oocyst Wall of Eimeria nieschulzi

SYNOPSIS. Oocysts of Eimeria nieschulzi from the laboratory rat, Rattus, norvegicus , were studied by scanning and transmission electron microscopy. Oocysts had a rough outer wall with apparent random depressions. The oocyst wall is composed of 2 layers: an osmiophilic outer layer consisting of a rough external and smooth internal surface, and a relatively thick, electron-lucent inner layer. The outer layer is composed of a dense, coarsely granular matrix. The inner layer consists of homogeneous fine granular material interspersed with coarse osmiophilic granules and contains one closely applied membrane on the outermost surface. Several raised lenticular areas are seen on the coarse outer surface of the inner layer. These layers are 102 (75–128) and 176 (135–204) nm thick, respectively.
The sporocyst wall is thin, consisting of 3 to 4 unit membranes, and measures 27 (18–34) nm thick.     

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.     

The fine structure of the endogenous stages of Eimeria labbeana: 2. Mature macrogamonts and young oocysts.

The fine structure of the mature macrogamonts and intracellular oocysts of Eimeria labbeana from the ileal mucosa of experimentally infected Pigeons (Columbia livia) was investigated and described. The macrogamont reached a maximum size of 12.0 x 9.5 mum (average equals 10.8 x 8.8 mum), and was located within a narrow parasitophorus vacuole. Most of the macrogamonts were limited by two membranes. Intravacuolar tubules, 1.2 mum long and 58 nm in diameter, established direct connections between the parasite and the host cell. Each tabule was composed of 9 subunits arranged around the central lumen. Cytoplasmic canaliculi were composed of bundles of microtubule-like structures (8-10 nm wide). Type 1 wall-forming bodies reached a maximum size of 1.8 x 1.5 mum, and many had centric or eccentric electron transparent portions within them. They were frequently seen lodged within peripherally-located mitochondria. Type 2 wall-forming bodies averaged 1.5 mum in diameter. The role of the two types of wall-forming bodies in forming the outer and inner layers of the wall of the oocyst was similar to that in other species of Eimeria. The oocyst wall was 0.2 mum thick and composed of a limiting membrane (20 nm thick), an outer layer (75 nm thick), and an inner layer (100 nm thick).     

Hexagonal periodicity in the outer membrane of Bacteroides buccae

In Bacteroides buccae, a hexagonally arranged periodic structure was found in the outer membrane (OM), in addition to hexagonal lattices present in its external surface layer (S-layer). This crystalline OM protein (COMP) was present as patches on the concave fracture face (the outer leaflet) of the OM in freeze-fractured cells. Occasionally, hexagonally arranged structures could also be seen on the convex fracture face of the OM as 'fingerprints' of the COMP. The OM proteins were isolated and analysed by gel electrophoresis. The major band protein had an apparent molecular mass of 17 kDa. Whether the minor band proteins are also components in the structure of the COMP remains to be elucidated. Other oral Gram-negative anaerobic rods studied did not show any periodicity in their OM.     

ULTRASTRUCTURAL STUDY ON SPORE WALL MORPHOGENESIS IN LYCOPODIUM CLAVATUM (LYCOPODIACEAE)

Spore wall morphogenesis of Lycopodium clavatum was observed by transmission electron microscopy. The spore plasma membrane indicates the reticulate spore sculpture shortly after meiosis. The mature spore wall of this species consists of two layers, inner endospore and outer exospore. There is no perispore in the sporoderm of this species. The exospore formation begins during the tetrad stage; and this layer is divided into two distinct sublayers, an outer lamellar layer and an inner granular layer. The lamellar layer is formed on the sculptured spore plasma membrane. Additional lamellae attach to this layer in a centripetal direction. For that reason, this layer may be derived from spore cytoplasm. The granular layer is formed only in the proximal region following lamellar layer formation, and it also may be derived from spore cytoplasm. The endospore is formed lastly and seems to be derived from spore cytoplasm as well. Accordingly, the spore sculpture of this species may be under the genetic control of the spore nucleus.