Structure and Chemical Composition of Prospheroplast Envelopes of Saccharomyces cerevisiae and Hansenula anomala

Cells of Saccharomyces cerevisiae and Hansenula anomala were digested with snail enzyme under conditions yielding prospheroplasts. Surrounding envelopes were isolated after lysis of prospheroplasts in distilled water. The envelope material was embedded and sectioned for electron microscopy, and thin, hollow structures still retaining the elongated form of the original cells were seen. The envelopes were of low electron density in sections stained with uranyl magnesium acetate and lead citrate, but were more electron-dense when stained with phosphotungstic acid. Shadowed preparations of prospheroplast envelopes revealed structures resembling ghosts. These "ghosts" were similar to the original cells in form and size but seemed to be very thin. Varying numbers of anular structures (bud scars) were found on them. Chemical analyses of the envelope indicated that an alkali-soluble glucan was a major constituent. The results show that the prospheroplast envelope is part of the original cell wall of the yeast and is located in close apposition to the cytoplasmic membrane.     

Subunit Cell Wall of Sulfolobus acidocaldarius

The cell wall of Sulfolobus acidocaldarius has been isolated. Cells were mechanically disrupted with a French press, and the cytoplasmic membrane was removed by extracting cell-envelope fragments with Triton X-100. The Triton-insoluble cell wall material retained the characteristic subunit structure when examined in the electron microscope. Isolated cell wall fragments formed in open sheets that were easily separated from cytoplasmic contamination. Chemical studies showed that the Triton-insoluble cell wall fragments consisted of lipoprotein with small amounts of carbohydrate and hexosamine. The amino acid composition indicated a highly charged hydrophobic cell surface. The presence of diaminopimelic acid with only traces of muramic acid indicates that the cell envelope does not have a rigid peptidoglycan layer. The results of chemical analyses and electron microscopy suggest a wall-membrane interaction stabilizing the cell envelope. The chemical and physical properties of this type of cell envelope would appear to form the basis for a new major division of bacteria with the definitive characteristics of a morphologically distinct subunit cell wall devoid of peptidoglycan.     

Basal structure and attachment of flagella in cells of Proteus vulgaris

Abram, Dinah (Purdue University, Lafayette, Ind.), Henry Koffler, and A. E. Vatter. Basal structure and attachment of flagella in cells of Proteus vulgaris. J. Bacteriol. 90:1337-1354. 1965.-The attachment of flagella to cells of Proteus vulgaris was studied electron microscopically with negatively stained and shadow-cast preparations of ghosts from standard cultures and from special cultures that produced "long forms." The flagellum, the basal portion of which is hooked, arises within the cell from a nearly spherical structure, 110 to 140 A in diameter. This structure appears to be associated with the cytoplasmic membrane; it may be a part of the membrane or a separate entity that lies just beneath the membrane. Flagella associated with cell walls free from cytoplasmic membrane frequently have larger bodies, 200 to 700 A in diameter, associated with their base. These structures probably consist at least partly of fragments of the cytoplasmic membrane, a portion of which folds around a smaller structure. Flagella in various stages of development were observed in long forms of P. vulgaris cells grown at low temperature. The basal structure of these flagella was similar to that of the long or "mature" flagella. Strands connecting the basal structures were observed in ghosts of long forms; these strands appear to be derived from the cytoplasmic membrane. Flagella were found to be attached to fragments of cell wall and to cytoplasmic membrane in a similar manner as they are attached to ghosts. In isolates of flagella that have been separated from the cells mechanically, the organelles often terminate in hooks which almost always appear naked, but have a different fine structure than the flagellum proper.     

Light- and electron-microscopic study of the endolymphatic sac of the tree frog,Hyla arborea japonica

Summary The endolymphatic sac of the tree frog and its crystals were observed by light- and electron microscopy. Scanning electron microscopy revealed that the crystals have a faceted body and two pointed ends. Light- and transmission electron microscopy revealed that the endolymphatic sac is composed of many small chambers. In their lumina, numerous ghosts of crystals that resulted from decalcification were observed. The ghosts were demarcated by a linear dense material or embedded in a flocculent substance. The epithelium of the endolymphatic sac is simple squamous or cuboidal and peculiar cytoplasmic granules are found in most cells. The granules are surrounded by a limiting membrane and have varying electron density. Some granules contain a core and/or tubular structures. Vacuoles containing large ghosts are also found in the epithelial cells. These ghosts were quite similar to those in the lumen and sometimes coexist with cell debris. The fine structure of the endolymphatic sac and its crystals is discussed.     

Substructure of the Cytoplasmic Membrane of Bacillus megaterium I. Method for the Fractionation of “Ghosts”

A method of fractionation of "ghosts" was devised to identify the chemical components of the cytoplasmic membrane. The method consists of dialyzing the "ghosts" against distilled water, and then dissolving the ghosts in dilute alkali. The ghosts were fractionated into four fractions by use of differential centrifugation. The components of each fraction were analyzed in detail. The ratio of lipid to protein and content of carbohydrate were found to be different for the four fractions. The main two fractions (fractions 2 and 3) contained several types of materials. Fraction 2, which is soluble in alkali and sedimentable at 105,000 x g, contained protein and lipid in the ratio of 2:1; ribonucleic acid was not detectable. Under the electron microscope, the ghosts appeared to have released the cell's cytoplasmic contents, but many small dense particles (about 100 A in diameter) remained adherent to the membrane surface. On the other hand, fraction 2 appeared to be made up only of a membrane structure. No 100 A particles were visible in this fraction. From these results, fraction 2 seemed to be pure membrane material.     

Cross wall synthesis and the arrangement of the wall polymers in the cell wall of Staphylococcus spp

The growing process and the fine structure of the cross wall of Staphylococcus were investigated by electron microscopy. Examination of the tangentially sectioned cross wall revealed that it was initially synthesized as a thin cell wall layer by an invaginated cytoplasmic membrane. The wall thickness soon increased by additional synthesis of the wall from the cytoplasmic membrane located at the side region of the cross wall. Scanning electron microscopic observation of sodium dodecyl sulfate-treated and mechanically separated cross walls revealed that the outer surface of the cross wall exhibits regular circular structures and the inner surface showed has an irregular surface. This indicates that cell wall materials were arranged in a regular circular manner in the initially synthesized thin layer. It is conceivable that in Staphylococcus spp. two cell wall synthesizing systems are present: wall-elongation synthesis in which wall materials are arranged in a regular circular manner and wall-thickening synthesis in which wall materials are arranged in an irregular manner.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Effect of the lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells

Abstract The effects of iturin A, at fungicidal concentrations, on yeast cells were studied by scanning electron microscopy and by transmission electron microscopy. A depression, observed in each iturin A-treated cell, was the consequence of the release of electrolytes and other cytoplasmic components. Iturin A passes through the cell wall and disrupts the plasma membrane with the formation of small vesicles and the aggregation of intramembranous particles. Moreover, iturin A passes through the plasma membrane and interacts with the nuclear membrane and probably with membranes of other cytoplasmic organelles.     

Ultrastructural effects of silicic acid on primary lung fibroblasts in tissue culture

Transmission and scanning electron microscopic examination of primary lung fibroblasts exposed in tissue culture to polymeric silicic acid (PSA) revealed profound cellular changes in the cell surface membranes, resulting in rapid endocytosis of affected membranes and formation of multivesicular bodies. Exposure to monomeric silicic acid did not appear to exhibit any immediate adverse effects. Appearance of numerous cytoplasmic vacuoles within 1 h of PSA exposure was easily visible by light microscopy. Electron microscopy revealed that PSA exposure caused formation of an 'osmiophilic' cell surface membrane. Numerous osmiophilic cytoplasmic blebs on the surface and subsequent endocytotic vesicles appeared to collapse and aggregate into multivesicular bodies. This study provides ultrastructural evidence of the direct interaction between lung fibroblasts and polymeric silicic acid, which has a dramatic effect the surface membrane, its subsequent internalization and cytoplasmic processing. This interaction could be one of the key steps in the damaging effects of silica containing dust.     

Ultrastructure of Bacterial Cells Infected with Bacteriophage PM2, a Lipid-containing Bacterial Virus

The cytological pattern of infection of a host pseudomonad with PM2, a lipid-containing bacterial virus, was investigated by electron microscopy. Normal and infected cells frequently contain a myelin figure, which is found in the nucleoid region or at the periphery of the cell. The most striking finding in this investigation was that completed virions are found in the cell adjacent to or in association with the cytoplasmic membrane. This localization is precise; virions are not found elsewhere in infected cells. The completed virions occasionally appear to be attached to the cytoplasmic membrane. The virus contains a darkly staining core surrounded by a tripartite envelope of a thickness of approximately 70 A, which is identical to the thickness of the cytoplasmic membrane. Lysing cells appear to undergo extensive damage of the cytoplasmic membrane prior to rupture of the L layer of the cell wall.     

Electron Microscopy of Young Candida albicans Chlamydospores

One- to three-day-old cultures of Candida albicans bearing chlamydospores were grown and harvested by a special technique, free of agar, and prepared for ultramicrotomy and electron microscopy. These young chlamydospores exhibited a subcellular structure similar to that of the yeast phase, e.g., cytoplasmic membrane, ribosomes, and mitochondria. Other structural characteristics unique to chlamydospores were a very thick, layered cell wall, the outer layer of which was continuous with the outer layer of the suspensor cell wall and was covered by hair-like projections; membrane bound organelles; and large lipoid inclusions. Only young chlamydospores less than 3 to 4 days old exhibited these ultrastructural characteristics.     

Cytoplasmic fibrils in living cultured cells

Summary By a combined light and electron microscopic study, the structure and behavior of the stress fibers of cultured rat embryo cells are described. From an analysis of movie records of living cells it is seen that the stress fibers are in a state of flux, continually altering their dimensions and dispositions within the cell. However, compared to most other cellular movements, these rates of change are slow. By electron microscopy it is shown that the stress fibers consist of bundles of close packed elongate 75 Å filaments, arranged just beneath the plasma membrane adjacent to the cell's plane of attachment and that similar filaments, forming a loose framework, permeate the cytoplasmic matrix. On the basis of careful light and electron microscopic comparisons, it is concluded that the filamentous structure shown within the cytoplasm of glutaraldehyde/osmium-fixed cells is a generally accurate representation of the structure of the living cell cytoplasm. It seems likely that the stress fibers are concerned in stabilizing areas of cellular attachment as well as with resisting forces that stretch the cell. The suggestion is made that, by controlling cytoplasmic viscosity and responding to cytoplasmic microtubules, the diffuse framework of filaments helps to determine the form of the cell and that, by a coordinate dynamic activity of its filaments, it provides the motive power for the various forms of cellular and intracellular movements.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

ELECTRON MICROSCOPY OF CELLULAR DIVISION IN ESCHERICHIA COLI.

Conti, S. F. (Brookhaven National Laboratory, Upton, N. Y.) and M. E. Gettner. Electron microscopy of cellular division in Escherichia coli. J. Bacteriol. 83:544-550. 1962.-Exponentially growing cells of Escherichia coli were fixed in formalin, exposed to uranyl nitrate, dehydrated at low temperatures with ethanol, and embedded in methacrylate. Polymerization was carried out at -70 C, by exposure of specimens to radiation from a cobalt(60) source. Electron micrographs revealed that cellular division occurs by the centripetal growth of the cell wall. The fine structure of the cytoplasm, nuclear apparatus, cell wall, and cytoplasmic membrane was also studied.     

Microorganism in the hyphae of mycorrhiza-forming fungus]

A microorganism with the cell wall and cytoplasmic membrane was found when the endotrophic mycorrhiza of pea was studied by electron microscopy. The wall of the microorganism was in a close contact with the cytoplasm of the fungus. No changes were found in the ultrastructure of the fungus and microorganism caused by their interaction.     

Fine Structure of Strictly Anaerobic,Non-Spore-Forming,Gram-Negative Bacilli

The cell division of a strain of Bacteroides convexus was examined by the ultrathin sectioning and the electron microscopy. The cell division was initiated by the invagination of the cytoplasmic membrane from the opposite sites at the middle of the cell. The constriction of the cell wall also occurred simultaneously or soon after the initiation of the invagination of the cytoplasmic membrane. A short septum structure similar to those of gram positive bacteria originated within the base of mesosome. The two mesosomes arising from the opposite sites fused at the center of the cell. After the tips of invaginating outer membrane reached to the middle between cell center and original outer membrane, the mesosomes were reduced gradually and finally disappeared. In this stage of the cell division, a transverse septum was usually completed. The invagination of the outer membrane proceeded progressively and finally fused at the center of the division plane.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

Fine Structure of Selected Marine Pseudomonads and Achromobacters

The fine structure of more than 20 marine pseudomonads and more than 15 achromobacters was examined. Under the conditions extant, clear differences between members of these two groups were seen. The pseudomonads displayed the characteristic gram-negative morphology: the cell wall was irregularly undulant and the cytoplasmic membrane more nearly planar, ribonucleoprotein (RNP) particles were loosely packed throughout the periphery of the cytoplasm, and the deoxyribonucleic acid (DNA) was axially disposed. Cell division appeared to be by constriction. Some strains characteristically produced evaginations or blebs of the cell wall. Occasionally, thick, densely stained ring structures were seen which are possibly analogous to mesosomes. In contrast, the achromobacters demonstrated a regularly undulant outer cell wall element and a planar inner wall. The cytoplasmic membrane was thin and not readily observed. RNP particles were densely stained and tightly packed in the cytoplasm; the DNA was most often lobate in disposition. Cellular division was mediated by the formation of a septum which consisted of the cytoplasmic membrane and the inner element of the cell wall. Mesosomes were observed in all of the strains examined. Dense inclusion bodies were also seen in many strains.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.