The existence of a dispensable fibrillar layer on the wall surface of mycelial but not yeast cells of Aureobasidium pullulans

Abstract Wall surface ultrastructure of Aureobasidium pullulans was studied by freeze-etching. Yeast cells had a smooth wall surface as in typical yeast species. Mycelial cells and chlamydospores had an extra layer on the wall surface made mostly of fibrils. The fibrils were 20 nm in diameter, and thicker than typical major fungal wall skeletal fibrils of β-glucan and chitin. This layer was apparently easily detached from the wall proper, presumably as a result of enzymic activity or by physical means, suggesting that it is a physiologically dispensable wall component.     

Ultrastructure of the Cell Envelope of Escherichia coli B After Freeze-Etching

The cell envelope of Escherichia coli B was investigated with the freeze-etching technique. A considerable gain in visible structural detail over more conventional electron microscopic techniques was obtained. The inner surface of the plasma membrane revealed a smooth surface sparsely studded with particles measuring from 5 to 10 nm in diameter, whereas the outer surface of the plasma membrane showed many more particles of corresponding diameter. The freeze-etched cell wall appeared to be a multilayered structure. The innermost layer could be observed as a profile studded with closely packed elements of about 10 nm in diameter. External to this layer was a smooth surface bordering the outermost cell wall layer. When frozen in the absence of glycerol the outermost surface observed in the cell wall was smooth, but when grown in the presence of glycerol it had a "wavy" appearance with small particles attached to it. The observations support current concepts on the ultrastructure of the enterobacterial cell envelope.     

Surface Structure of Intact Cells and Spheroplasts of Pseudomonas aeruginosa

This report describes the ultrastructural features of Pseudomonas aeruginosa after freeze-etching of intact cells and enzymatically prepared spheroplasts. Freeze-etching of intact cells revealed two convex layers of the cell wall and particles within the hydrophobic interior of the cell membrane. Areas of the membrane free of particles were sometimes elevated in the form of rather large dome-shaped structures. Spheroplasts were formed from intact cells by the addition of trypsin to a reaction mixture of lysozyme and ethylenediaminetetraacetic acid. Spheroplasts contained the outer lipoid layer of the cell wall. It was possible to observe this cell wall layer in freeze-etch preparations of spheroplasts. The spheroplast membrane like that of intact cells was cleaved along a central plane to expose particles and particle-free areas.     

Structure of Escherichia coli After Freeze-Etching

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.     

Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques

Remsen, C. C. (Swiss Federation Institute of Technology, Zurich, Switzerland), and D. G. Lundgren. Electron microscopy of the cell envelope of Ferrobacillus ferrooxidans prepared by freeze-etching and chemical fixation techniques. J. Bacteriol. 92:1765-1771. 1966.-A comparison was made of the fine structure of the cell envelope of the gram-negative bacterium Ferrobacillus ferrooxidans when cells were prepared for microscopy by freeze-etching and chemical fixation techniques. Cell envelopes of chemically fixed cells appeared as five separate layers distinguishable by their location and electron density. Frozen-etched cells showed a three-layered complex with each layer measuring approximately 100 A in thickness. The latter technique is considered to be "artifact-free" and, as a technique, yields purely morphological information on the natural state. The three layers revealed by freeze-etching are: the outer layer, a lipoprotein-lipopolysaccharide layer; the middle layer, a layer composed of globular protein attached to fibrillar mucopeptide; and the innermost layer, the cytoplasmic membrane. The latter was covered with 100 to 120 A particles. The relationship of the aforementioned layers to those seen in chemically fixed cells is discussed.     

On the morphology and ultrastructure of the cell wall of Spirulina platensis

The cell wall of the blue-green alga Spirulina platensis was studied with the electron microscope using ultra-thin sectioning, shadowing, carbon-replication or freeze-etching techniques for specimen preparation. The cell wall could be resolved into four layers, L-I through L-IV. The L-I and L-III layers contain fibrillar material. The septum is a three-layered wall: an L-II layer sandwiched between L-I layers. The shape in vitro of isolated septa might be an artifact due to the preparation technique used. Certain structural properties of the septum seem to allow tangential stretching; they might be reflected in the flexible gliding mobility of Spirulina species. The outer, L-IV layer contains material longitudinally arranged along the trichome axis.     

Freeze-Etching of Azotobacter vinelandii: Examination of Wall, Exine, and Vesicles

The structure of the cell wall, the arrangement of the cyst exine, and the origin and distribution of intine vesicles in Azotobacter vinelandii ATCC 12837 were examined by freeze-etching and conventional electron microscopic techniques. In the vegetative organism the cell wall appears to have a woven texture which disappears during cyst formation. The exine is composed of two different types of material: the outer layer is a fibrous, amorphous layer, and the numerous inner layers form the basic hexagonal structures which unite to form the cyst coat. The presence of intine vesicles in the encysting organism was confirmed in frozen-etched cells. The appearance of frozen-etched cells and cysts and the distribution of capsular material indicate that extracellular polysaccharide is an important factor in cyst formation.     

Comparative ultrastructure of selected oral streptococci: thin-sectioning and freeze-etching studies.

The ultrastructure of Streptococcus mutans, serotypes a-e, S. sanguis, S. mitis, and S. salivarius HHT, were examined by the techniques of thin-sectioning and freeze-etching. The cell walls varied in width between 15 and 46 nm and were covered with an electron-dense fibrillar or fuzz layer. The cytoplasmic membrane was in close association with numerous mesosomes which were, in turn, either closely associated or in contact with the bacterial chromosome. In freeze-etch replicas, the outermost layer of the cell wall was fibrous, and the cytoplasmic membrane was covered with particles composed of several subunits. Both particle-clusters and particle-free areas occurred on the surfaces of the cytoplasmic membrane, as well as a crystalline array in the ground plasm of the cell.     

Demonstration by freeze-etching of a single cleavage plane in the cell wall of a gram-negative bacterium

In the examination of protoplasts of a gram-negative bacterium classified as a Pseudomonas sp. by freeze-etching, we found a smooth external surface which is not seen if the preparations are not "etched." This external structure is seen as a sleeve surrounding and connecting the cells in unetched preparations, and we present evidence that it is a eutectic formed during the freezing of the specimen. In the system used in this study, the four layers of the cell wall of a gram-negative bacterium can be removed from the cell. The single cell wall cleavage plane is not affected by the removal of the loosely bound outer layer or of the peptidoglycan layer, but it is lost when the outer double track layer and the underlying soluble layer are simultaneously removed. Thus, we conclude that it is one of these two layers which is responsible for the cleavage plane which exposes variable areas of a smooth surface in the cell wall. This cell wall cleavage plane is more likely to deflect the actual cleavage of the frozen cell when cells are relatively old or when they are suspended in sucrose.     

A structural comparison of cyst and germ tube wall inPhytophthora palmivora

Summary An electron microscopic analysis of germinating cysts ofPhytophthora palmivora involving freeze-etching, thin sectioning, and replica techniques reveals that both cyst and hyphal wall comprise a two-phase system with a fibrillar and an amorphous component. The cyst wall is fibrillar throughout with the fibrils tightly interwoven and embedded in an amorphous matrix on the internal side of the wall. The hyphal wall consists of a fibrillar inner layer with the fibrils lightly covered by some amorphous material and an amorphous outer layer devoid of any fibrillar material. Both cyst and germ tube walls are wholly or partially covered by a fluffy coat of variable thickness. In the zone of germ tube emergence cyst wall and germ tube wall overlap and are tightly apposed. Thus, the germ tube wall is not a simple extension of the cyst wall but a new structural entity separated from the cyst wall by a thin line of demarcation.     

THE ULTRASTRUCTURE OF SCENEDESMUS (CHLOROPHYCEAE). I. SPECIES WITH THE “RETICULATE” OR “WARTY” TYPE OF ORNAMENTAL LAYER1

The ultrastructure of the wall layers and ornamentative features of Scenedesmus pannonicus and S. longus are described using carefully correlated freeze-etched replicas, thin sections and scanning electron micrographs. The two species arc enclosed by different types of ornamented layers, S. pannonicus by the tightly filling, “warty” layer and S. longus by the loosely fitting, “reticulate” layer, held off the coenobium by 2 types of tubular propping spikelets and rosettes. The reticulate layer has an intricate substructure, especially when studied with freeze-etching. Its inner and outer surfaces appear different, as is its attachment to the 2 types of spikelets. Whole cells of S. longus subjected to acetolysis lack the cellulose wall and cytoplasm, but all other surface features survive, including the Trilaminar Sheath (TLS); this ornamentation cannot be “pectic.” The cellulose wall and ornamentation is unaffected by boiling water alone. Boiling in 6n NaOH removes the surface ornamentation, but the TLS and wall remain; the possibility that these features contain silica is discussed. The terminal spines of both species consist of closely packed spikelets enclosed within a skin of hexagonally-packed subunits. Similar subunits are seen in the propping spikelets of S. longus, and in the rows or “combs” of laterally fused spikelets of S. pannonicus. The warty layer of S. pannonicus is tightly appressed to the TLS except close to where the cells are joined, where it is suspended free. It is composed of a layer of globular subunits, and small indentations form the warts. Single, evenly distributed warts characterize the freely suspended sections of the warty layer, and the layer that encloses young coenobia soon after they have been formed: in contrast, the warts are clumped over the surface of older and larger colonies. Some of the single warts form characteristic double rows, but these latter remain single even on older cells. The surface structure of the warty layer, TLS, and plasmalemma are revealed by the freeze-etching process.     

Superficial cell-wall layers on Spirillum "Ordal" and their in vitro reassembly.

The cell envelope of Sporillum sp. strain "Ordal" (possibly a variety of S. anulus) demonstrated multiple superficial wall layers which were diverse in their macromolecular arrays. Negative staining and freeze-etching techniques revealed an outer hexagonally packed layer and an inner tetragonally packed layer. However, both thin sections and freeze-etched cleavages of the wall showed that each of these regular structures rested upon a backing layer, and that there was a delicate amorphous layer overlying the outer hexagonal array. Rotary integration, optical deffraction, and reconstruction of image were used to clarify measurements of each array and to verify the validity of a diagrammatic model of the outer hexagonal system. The integrity of these layers required suitable cations (Ca2+ appeared essential) and pH (pH less than or equal to 4.6 dissociated most superficial layers). These observations aided in the development of a low-pH cationic-substitution technique, in which Na+ replaced essential Ca2+, for extraction of the layers from the cell surface. Dialysis to remove Na+ and restoration of Ca2+ initiated in vitro reassembly of the superficial layer components until regularly structured assembly products were formed.     

Ultrastructure and adhesion properties of Ruminococcus albus.

Morphological studies have shown that cells of the anaerobic rumen bacterium Ruminococcus albus have electron-translucent granules of reserve carbohydrate in their cytoplasm, and that they have a polysaccharide "coat" layer external to their gram-negative cell wall. This coat layer, which stains specifically with ruthenium red, forms a compact mat of fibers adjacent to the cell, and fibrous elements also project as much as 0.6 mum from the cells. These radial fibers are clearly visualized by freeze-etching, and can be seen to extend throughout the extensive intercullular space in centrifuged pellets of these bacteria. Cells of R. albus adhere to cellulose fibers added to the culture medium, and the coat material is seen to mediate this adhesion in addition to its function in the general protection of these cells.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

Ultrastructural specialization at the host-pathogen interface in rust-infected flax

Summary Ultrastructure of the association between the rust fungus, Melampsora lini, and a compatible variety of flax, Linum usitatissimum, was studied to clarify the structural relationships and interactions at the site of host penetration and at the host-parasite interface. Results of freeze-etching as well as a special section-staining procedure consisting of periodate-chromate-phosphotungstate (PACP) are shown with a host-parasite combination for the first time. The host plasma membrane is invaginated by the fungus and forms a continuous boundary around the fungal haustoria which penetrate the host cells. No morphological continuities are observed linking the protoplasts of host and fungus. With both freeze-etching and the PACP stain, the invaginated portion of the host plasma membrane at the host-parasite interface shows distinctive features that are not characteristic of the non-invaginated portion of the membrane. This localized specialization of host plasma membrane in response to the fungus appears as a significant and consistent feature of the host-parasite interaction. The host plasma membrane is separated from the haustorial wall by an amorphous layer of sheath material which covers the body but not the neck of the haustorium. This sheath provides the environment in which the haustorium exists and functions during the course of the host-parasite association. Occasionally, a collar of wall-like material derived from the host cell forms around the haustorial neck. The collar is continuous with the host wall and is distinct and discontinuous from the haustorial sheath. In fewer than 5% of the infected cells this wall material encases entire haustoria. The fungal wall is structurally specialized around the site of host penetration, and it becomes intimately associated with the host wall where the fungus penetrates into the lumen of the host cell. During penetration, the host and fungal walls appear to be fused so that the interface between them is not clearly delineated. The haustorial wall is continuous, via the haustorial neck, with the wall of the haustorial mother cell which lies outside the host cell. Different staining properties reveal this wall continuum to consist of several well-defined regions having different structure or composition. A ring of fungal wall material midway along the haustorial neck stains densely with lead citrate, but is preferentially etched away by periodic acid. The neck ring denotes a transition in the staining reaction of the fungal wall, from that present in the region of host penetration to that of the wall surrounding the haustorium. The findings demonstrate specialization of the fungal wall in the area of host penetration as well as specialization of the host plasma membrane at the host-parasite interface to a degree not previously realized from ultrastructural information.     

Ultrastructure of the sporangiospore of Piptocephalis unispora (Mucorales).

Sporangiospore structure in Piptocephalis unispora Benjamin was studied using light microscopy, freeze-etching, scanning and transmission electron microscopy, and compared with that of other members of the Mucorales. A merosporangial wall, plasmalemmal invaginations, and wall protuberances were demonstrated in sections and their possible significance discussed.     

Wall ultrastructure in regenerating protoplasts ofAspergillus nidulans

Summary The ultrastructure of the wall formed by regenerating protoplasts ofAspergillus nidulans was investigated by electron microscopy using shadowing and freeze-etching techniques. The first evidence of wall formation by the protoplasts was seen in the development of a microfibrillar network composed of chitin. As the protoplasts develop further amorphous material was deposited on the outer surface of the skeletal net.     

Cell wall anchoring to cytoplasmic membrane of Candida albicans

Abstract Cell wall ultrastructure of the opportunistic pathogenic yeast Candida albicans was investigated by stereoscopic freeze-etching technique. Three wall layers were distinguishable by this technique. No clear periplasmic space was evident. Bilayer membrane invaginations were extensive. The outermost regions of the membrane invaginations were lined with thin, spine-like fibrils, which extended into the cell wall. We suggest that the fibrils along the invaginations are involved in anchoring the cell wall to the membrane.     

Ultrastructure of the sporangiospore of Piptocephalis unispora (Mucorales)

Sporangiospore structure in Piptocephalis unispora Benjamin was studied using light microscopy, freeze-etching, scanning and transmission electron microscopy, and compared with that of other members of the Mucorales. A merosporangial wall, plasmalemmal invaginations, and wall protuberances were demonstrated in sections and their possible significance discussed.     

Fine structure of swarmers of Cladophora and Chaetomorpha

Summary Naked swarmers of Cladophora have been collected and wall synthesis and development have been followed using the techniques of freeze-etching and sectioning. Swarmers frozen after 9 hours liberation have lost their flagella, developed the characteristic fibrous layer and show the initial stages of wall production. Both the first formed (randomly oriented) and the later (more ordered) microfibrils appear to have a distinct granular texture. Occasionally linear arrays of granules up to 4 m long may be seen. After 5 days settling a thick wall composed of almost transversely oriented microfibrils is present and a rhizoid is pushed out. Also characteristic of this stage is the central localisation of cell components and peripheral vacuolar distribution. Longitudinally oriented microtubules also reappear at this stage having been absent during carlier wall formation.A possible relationship between the cortical microtubules of the motile swarmer and the development of the fibrous layer is suggested.