Structure of Micrococcus denitrificans and M. halodenitrificans Revealed by Freeze-etching

S ummary . After freeze-etching, cells of Micrococcus denitrificans and M. halodenitrificans revealed structures similar to those observed in ultrathin sections. Both organisms had a similar cell wall structure. The cell wall was double layered, the smooth surface of which had a delicate granular structure. The cytoplasmic membrane was in 2 parts, both covered with spherical particles 8–12 nm diam. The cytoplasmic membrane possessed rod-shaped invaginations (100–300 × 30–50 nm). The cytoplasm of both species contained inclusions of poly-β-hydroxybutyric acid.     

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.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium.

The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmemberane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar.     

Cell wall synthesis in Allomyces macrogynus

Scanning electron microscopy and freeze-etching/cleaving have been employed to examine events in the synchronized development of gametophytic germlings of the aquatic Phycomycete Allomyces macrogynus. Motile spores were induced to start synchronized development and the sequence of surface changes associated with the encystment process was studied. Time course studies show that small vesicles (apparently blebbed off from the gamma-particles) start to accumulate on the surface of the plasma membrane after 6 min of synchronized growth at the same time as the first cell wall material can be detected. The vesicles increase in number during encystment. After 15 min of synchronized growth the number of vesicles decrease and after 20 min of growth no vesicle can be observed on the cell surface. During this period the cell surface appears increasingly smooth, probably due to cell wall formation. In freeze-etching/cleaving electron micrographs from this period, both intact and what appear to be ruptured vesicles outside the cell surface, can be observed. The intact vesicle has a characteristic surface pattern presumably of membrane particles. This surface view of the encystment processes supports the hypothesis that the gamma-particles through gamma vesicle formation participate in the cell wall synthesis during encystment in Allomyces.     

Die Feinstruktur der Zellwand und Cytoplasmamembran von Clostridium nigrificans,dargestellt mit Hilfe der Gefrierätz- und Ultradünnschnittechnik

Zusammenfassung Der mit Hilfe der Gefrierätztechnik dargestellte Feinbau der Zellwand und Cytoplasmamembran von Clostridium nigrificans wurde mit den Ergebnissen der chemischen Fixation verglichen. Die Zellwand von chemisch fixierten Zellen zeigt einen Aufbau aus drei stark und zwei schwach elektronenstreuenden Schichten, wobei die innerste, der Cytoplasmamembran angrenzende Schicht nicht immer darstellbar ist. Die Cytoplasmamembran hat eine asymmetrische Elementarmembran-Struktur. Mit Hilfe der Gefrierätztechnik konnte die Zellwand in drei Schichten aufgespalten werden: Einer äußeren aus globulären, rechtwinkelig geordneten, etwa 9 nm messenden Partikeln aufgebauten Schicht und zwei darunter liegenden, 15 und 5 nm dicken Schichten. Die Cytoplasmamembran wird in jungen Kulturen vollkommen, in alten teilweise mit 5–15 nm großen Teilchen bedeckt. An Stellen wo die Teilchen abgespalten wurden oder entsprechend locker angeordnet waren, konnte die Oberfläche der Cytoplasmamembran sichtbar gemacht werden. Sie ist sowohl auf der der Zellwand als auch auf der dem Cytoplasma zugekehrten Seite mit systemlos verteilten, scheinbar verschieden tief eingebetteten Teilchen bedeckt.

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The fine structure of the cell wall and cytoplasmic membrane of Clostridium nigrificans demonstrated by means of freeze-etching and chemical fixation techniques

Summary A comparison was made between the results obtained using the freeze-etching technique in demonstrating the fine structure of the cell wall and the cytoplasmic membrane of Clostridium nigrificans with those using the chemical fixation technique. In chemically fixed cells, the cell wall appears to consist of three dense layers separated by two layers of low electron scattering power, whereby it is not always possible to observe that layer immediately bordering on the cytoplasmic membrane. The cytoplasmic membrane has an asymmetrical unit membrane structure. It was possible, to separate the cell wall in three distinct layers using the freeze-etching technique. The outermost is composed of globular, rectangularly arranged particles, approximataly 9 nm in size, the two inner layers are 15 and 5 nm wide respectively. The cytoplasmic membrane is covered with particles 5 to 15 nm in size, in younger cultures completely, in older cultures partially. On those places where the particles have been split off or where they are loosly arranged, it was possible to observe the surface of the cytoplasmic membrane. It appears to be covered with unsystematically scattered particles imbedded at different depths, not only on that side turned to the cell wall but also on that facing the cytoplasm.

     

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.     

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.     

Ultrastructural, physiological, and cytochemical characterization of cores in group D streptococci.

Cores are large, rod-shaped structures that have been found almost exclusively in group D streptococci, measure 0.1 to 0.16 mum in diameter, and extend the width or length of cells. This study has shown that cores are produced in the cells at a reproducible point in early stationary growth after extensive mesosomal formation and after the pH has dropped below 6.5. When cells containing cores were introduced into a fresh medium with a pH above 6.5, the structures disappeared within 5 min. The structures were not found in young, logarithmically growing cells but formed in these cells upon autolysis or treatment with penicillin. Cores that were forming or disintegrating appeared to have a lamellar substructure. When chloramphenicol was added to the medium before the culture reached stationary phase, no cores were found in the cells. Cytochemical studies indicated that cores contain protein and are not composed of cell wall material or other polysaccharides that contain 1,2-glycol groups.     

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.     

The fine structure of the pellicle ofEuglena gracilis as revealed by freeze-etching

Summary The ultrastructure of the pellicle ofEuglena gracilis (Klebs) Z strain was studied using the freeze-etching technique and the results were correlated with data obtained from thin sections of fixed material.Examination of freeze-etched pellicles reveals an outer particulate layer and an inner striated layer. The particles of the outer layer measure approximately 150 Å in diameter. The striations of the inner layer are about 50 Å wide and are separated from each other by about 35 Å. A broad repeating pattern is also visible with a periodicity of about 450 Å. When deep etching is employed, a smooth outer layer is seen covering the particulate layer. This is probably the outer surface of the plasma membrane. Mucilage is present on the outer surface of the cell and is seen as a substructure of threads superposed on the smooth layer of plasma membrane.Thin sectioning also shows a striated layer interior to the plasma membrane. This appears to be identical to the striated layer seen after freeze-etching.     

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.     

Tolerance to antimicrobial agents and persistence of Escherichia coli and cyanobacteria

Bacterial persistence is the tolerance of a small part of a cell population to bactericidal agents, which is attained by a suppression of important cell functions and subsequent deceleration or cessation of cell division. The growth rate is the decisive factor in the transition of the cells to the persister state. A comparative study of quickly growing Escherichia coli K-12 strain MC 4100 and cyanobacteria Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413 growing slowly was performed. The cyanobacterial cells, like E. coli cells, differed in sensitivity to antimicrobial substances depending on the growth phase. Carbenicillin inhibiting the synthesis of peptidoglycan, a component of the bacterial cell wall, and lincomycin inhibiting the protein synthesis gave rise to nucleoid decay in cells from exponential cultures of Synechocystis 6803 and did not influence the nucleoids in cells from stationary cultures. Carbenicillin suppressed the growth of exponential cultures and had no effect on cyanobacterial stationary cultures. A suppression of Synechocystis 6803 growth in the exponential phase by lincomycin was stronger than in the stationary phase. Similar data were obtained with cyanobacterial cells under the action of H2O2 or menadione, an inducer of reactive oxygen species production. Slowly growing cyanobacteria were similar to quickly growing E. coli in their characteristics. Persistence is a characteristic feature of cyanobacteria.     

Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins.

Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed. One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion. It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures. The E. coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly. The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein. In contrast, the hemolysin which accumulated intracellularly in the E. coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase. Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface. It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells.     

Temperature-dependent structural rearrangements in the cytoplasmic membranes of Escherichia coli cells]

By the freeze-etching method, in has been shown that E. coli plasma membranes undergo a structural transition in the range of temperatures within 0 and 20 degrees C which could be observed as redistribution of intramembrane particles with free-zone formation. The onset of temperature interval of this transition (20 degrees) well correlate with the break in the Arrhenius curves characterizing the cell membrane permeability for free nucleotides and for respiration intensity.     

The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,grown in different media

In order to study the ultrastructure of the cell surface and plasma membrane of Schizosaccharomyces pombe as a function of growth conditions we investigated exponential and stationary phase cells grown in rich and minimal medium.Electron microscopic preparation techniques based on rapid cryofixation (without cryoprotectants) were used. The intramembraneous aspects of the plasma membrane were described by freeze fracturing. For the first time the dynamic surface structures could be directly analyzed by freeze drying in the scanning electron microscope and in thin section of freeze substituted samples. This preparation techniques reveal hair-like structures on the surface of yeast cells. The hairs of cells grown in the rich medium are longer than those grown in the minimal medium. A mutant defective in the structure of a cell surface galactomannoprotein (acid phosphatase) reveals (under conditions of maximal acid phosphatase expression) a cell surface structure that differs from the wild type. It is likely that the hairs represent the peripheral galactomannan layer or part of it.On the membrane fracture faces the number, shape, distribution and state of aggregation of the intramembraneous particles are different between membranes of growing and non-growing cells and between cells grown under different physiological conditions. In the minimal medium corresponding periodical structures on the plasmic and exoplasmic fracture faces were observed, which clearly differ between exponential and stationary phase cells. The number, length and depth of plasma membrane invaginations increase as the cells go from the exponential phase to the stationary phase. Short and flattened invaginations are filled with thin periodic structures.     

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.     

Distribution of newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus of an Escherichia coli lac-lpp strain.

The insertion of newly synthesized lipoprotein molecules into the cell wall of Escherichia coli was studied topographically by immunoelectron microscopy. Lipoprotein was briefly induced with isopropyl-beta-D-thiogalactopyranoside in cells carrying lac-lpp on a low-copy-number plasmid in an E. coli lpp host. Specific antibodies bound to the newly inserted lipoprotein molecules, which were exposed at the cell surface after treatment of the cells with Tris-EDTA, were detected with a protein A-gold probe. The average distribution of the gold particles over the cell surface of noninduced cells was determined for cells induced for 5 and 10 min. Analysis of 250 to 350 cells showed that the distribution of newly synthesized lipoprotein over the cell surface was homogeneous in both cases. The binding of lipoprotein to the peptidoglycan layer was studied by the same technique, and visual inspection again revealed a homogeneous distribution of bound lipoprotein over the entire sacculus surface. It is therefore concluded that free lipoprotein is inserted equally over the entire cell wall of E. coli, while binding to peptidoglycan also occurs over the entire cell surface. The rate of lipoprotein synthesis increased with cell length in nondividing cells, whereas it was constant in cells which had initiated constriction. Analysis of cells having different amounts of lipoprotein in their cell wall revealed that the cell shape depended on the total lipoprotein content of the cell. Cells having no or only a small amount of lipoprotein grew as spheres, whereas cells with increasing numbers of lipoprotein molecules gradually changed their shape to short rods.     

Localization of the cholesterol oxidase in Rhodococcus erythropolis IMET 7185 studied by immunoelectron microscopy.

Rhodococcus erythropolis IMET 7185 produces an inducible cholesterol oxidase (COD) which can easily be extracted by treatment of cells with 0.1% Triton X-100. The yield of the enzyme was 3.3 U/g wet wt from induced cells, which is about 5 times more than from non-induced cells. A study of the location of COD on intact cells and on ultrathin sections by means of immunogold electron microscopy revealed the following distribution, which corresponds with the biochemical results: (1) COD, which is extractable by the detergent, was localized in a distance up to 80 nm above the cell surface. It belongs to a surface layer, which only became visible after lysine/glutaraldehyde treatment and staining with ruthenium red, indicating a high carbohydrate content. (2) Non-extractable COD was found on the cell surface in a shorter distance to the cell wall as well as within the cell wall, in the cytoplasmic membrane and in the peripheral cytoplasm. In the latter clusters of gold particles on some places suggest the presence of larger amounts of insoluble enzyme.