Effect of proteases,phospholipases and polysaccharide-splitting enzymes on plasma membrane particles and on the synthesis of the fibrillar cell wall component in yeast protoplasts

Plasma membrane particles demonstrable by the freeze-etching technique, play, according to some authors, a role in the cell wall synthesis. On a model of yeast protoplast capable of regenerating the cell wall we studied the morphology of plasma membrane particles and the synthesis of the fibrillar cell wall component following a treatment with various enzymes and with lysolecithin. The enzymes used included proteases (trypsin, papain, pronase), polysaccharide-splitting enzymes (snail enzyme complex, mannosidase), phospholipases (A, C, D) and lipase. Upon treating living protoplasts with these substances in no case did we observe any morphologically demonstrable change in the particle structure or in their distribution in the plasma membrane. The fibrillar cell wall component was synthetized even in the presence of proteases and phospholipases. If the plasma membrane particles are assumed to represent enzyme systems synthesizing the cell wall component then in living protoplasts they are not located on the outer plasma membrane face or else are protected by some mechanism against the action of the corresponding enzymes.     

Nutrition and metabolism of marine bacteria. XVI. Formation of protoplasts, spheroplasts, and related forms from a gram-negative marine bacterium

When cells of a marine pseudomonad were washed and suspended in 0.5 m sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 m NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 m NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up alpha-aminoisobutyric acid by a Na(+)-dependent process.     

Subcellular fractionation of the Gram negative rumen bacterium, Butyrivibrio S2, by protoplast formation, and localisation of lipolytic enzymes in the plasma membrane

Treatment of the anaerobic, Gram negative general fatty acid auxotroph Butyrivibrio S2 with lysozyme in low molarity buffers resulted in the formation of protoplasts, some of which retained the original rod-shaped morphology of the organism. The protoplasts were stabilised by the presence of Mg²⁺ ions. Most of the phospholipase A and C and galactolipase activity of the cells was retained by the protoplasts. Electron microscopy and chemical markers were used to monitor the separation of plasma membrane and cell wall fragments by density gradient centrifugation after osmotic lysis of protoplasts. Phospholipase and galactolipase activities were demonstrated in a subcellular fraction which contained only fragments of plasma membrane.     

Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.     

Cytochalasin D attenuates the desensitisation of pressure-stimulated vesicle fusion in guard cell protoplasts

Fusion of vesicular membranes with the plasma membrane during pressure-driven swelling of guard cell protoplasts was studied using patch clamp capacitance measurements. Hydrostatic pressure pulses were applied via the patch pipette and resulted in an immediate and linear increase in membrane capacitance, a parameter proportional to the surface area. In any given protoplast, pressure-stimulated increases in membrane capacitance could be provoked repetitively. However, the rate of rise in capacitance upon the same strength of stimulation decreased exponentially with time (tau = 4 min) for subsequent pressure stimuli. This process was the result of a desensitisation of the plasma membrane to mechanical forces. Incubation of guard cell protoplasts in cytochalasin D, which depolymerises actin filaments, nearly abolished this desensitisation process. These results suggest that membrane stretch initiates a reactive process that may fortify or stabilise the plasma membrane of guard cell protoplasts.     

Isolation and characterization of forespores from Bacillus megaterium

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.     

Cryobehavior of the plasma membrane in protoplasts isolated from cold-acclimated Arabidopsis leaves is related to surface area regulation

Extracellular freezing in plants results in dehydration and mechanical stresses upon the plasma membrane. Plants that acquire enhanced freezing tolerance after cold acclimation can withstand these two physical stresses. To understand the tolerance to freeze-induced physical stresses, the cryobehavior of the plasma membrane was observed using protoplasts isolated from cold-acclimated Arabidopsis thaliana leaves with the combination of a lipophilic fluorescent dye FM 1-43 and cryomicroscopy. We found that many vesicular structures appeared in the cytoplasmic region near the plasma membrane just after extracellular freezing occurred. These structures, referred to as freeze-induced vesicular structures (FIVs), then developed horizontally near the plasma membrane during freezing. There was a strong correlation between the increase in individual FIV size and the decrease in the surface area of the protoplasts during freezing. Some FIVs fused with their neighbors as the temperature decreased. Occasionally, FIVs fused with the plasma membrane, which may be necessary to relax the stress upon the plasma membrane during freezing. Vesicular structures resembling FIVs were also induced when protoplasts were mechanically pressed between a coverslip and slide glass. Fewer FIVs formed when protoplasts were subjected to hyperosmotic solution, suggesting that FIV formation is associated with mechanical stress rather than dehydration. Collectively, these results suggest that cold-acclimated plant cells may balance membrane tension in the plasma membrane by regulating the surface area. This enables plant cells to withstand the direct mechanical stress imposed by extracellular freezing.     

Electron microscopic studies on protoplast release from I.C.A.-1.65 line (I.F.C.-1.65 line) of Bacillus subtilis cells.

Under lysozyme action a minicell-forming line (I.C.A.-1.65) of B. subtilis releases protoplasts. The main cytologic events which proceed protoplast releasing are described. Different areas of the cell wall prove a remarkable difference in their sensitivity to enzymatic lysozyme action. Central areas of the cell wall are most sensitive and the polar areas are most resistant. Mesosomal vesicles and tubules are extruded and released together with other cytoplasmic extrusion during the protoplasting process. The cell wall of minicells does not prove resistance particularities to lysozyme action. The minicells release protoplasts.     

Vectorial and nonvectorial transphosphorylation catalyzed by enzymes II of the bacterial phosphotransferase system

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.     

Changes in the plasma membrane of regenerating protoplasts of Candida albicans as revealed by freeze-fracture electron microscopy

Modifications occurring in the plasma membrane and their relationship to newly synthesized microfibrils were examined in regenerating protoplasts of Candida albicans by freeze-fracture electron microscopy. Freshly prepared protoplasts showed no residual wall material, and long invaginations covered the surface of the plasma membrane. Analysis of the external face (E-face) of the plasma membrane showed a significant decrease in the number of intramembranous particles (IMP) in comparison with the original cells. After 40 min incubation in regeneration medium, newly synthesized microfibrils which seemed to originate from protrusions in the plasma membrane were observed. The plasma membrane showed important modifications with respect to IMP. After 3 h 45 min, the cells were covered by an abnormal wall which showed isolated fibrils partially embedded in the matrix material. The plasma membrane of these partially regenerated protoplasts was similar to that of original cells. After 8 h, regeneration of the protoplasts seemed to be complete as no differences from the original cells were detected in the plasma membrane or the wall. Calcofluor white altered the deposition of wall polymers during regeneration, but did not modify the plasma membrane of the protoplasts.     

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

Ultrastructural Changes Associated with Spore Formation in Sporangia and Sporangiola of Thamnidium elegans Link

Fully formed pre-cleavage sporangia and sporangiola of Thamnidiumelegans Link were bounded by a primary wall plus a thick, internalsecondary wall layer. In sporangia in late pre-cleavage, Golgi-likecisternae were associated with groups of cytoplasmic vesiclesof characteristic size and appearance which were not found insporangia containing large cleavage vesicles. In both sporangia and sporangiola, protoplast cleavage was effectedby enlargement of endogenous cleavage vesicles each containinga lining layer of variable appearance, mutual fusion of cleavagevesicle membranes and fusion of cleavage vesicle membranes withthe plasmalemma. Golgi-like cisternae and small vesicular profileswere present in sporangium protoplasts at all stages of cleavagevesicle enlargement. In sporangia, the columella zone was delimitedby cleavage vesicles and separated from the sporogenous zoneby a fibrillar wall. A similar wall, which sometimes protrudedto form a small columella, was formed in sporangiola. Recently delimited spore protoplasts were bounded by plasmalemmamembrane derived from cleavage vesicle bounding membrane andsporangium or sporangiolum plasmalemma and surrounded by aninvesting layer derived from cleavage vesicle lining material.The investing layer at first appeared single, but later twoelectron opaque profiles were discernible. The spore wall wasformed between the investing layer and the plasmalemma. Wallsof sporangia and sporangiola which contained fully formed sporesconsisted of the primary layers only.     

Localization of ribonucleases in the mycelium of the fungus Aspergillus clavatus

The localization of ribonucleases in Aspergillus clavatus mycelium was studied using differential centrifugation of mycelial homogenates, preparation of protoplasts, by cytochemical and immunochemical studies. The RNase activity was represented mainly by guanylic and nonspecific RNases and was not associated with the intracellular structures. The cytochemical and immunochemical observations revealed the presence of a nonspecific RNase activity in the periplasm, mitochondria, perinuclear space and vesicular structures. The presence of this RNase in the periplasmic space and vesicular structures of A. clavatus suggests that it may be secreted through a fusion of the vesicular membranes with the cytoplasmic membrane, and then from the periplasm through the cell wall into the surrounding medium.     

Effects of Lysozyme and Inorganic Anions on the Morphology of Streptococcus mutans BHT: Electron Microscopic Examination

The effects of hen egg white lysozyme and the inorganic salt sodium thiocyanate on the integrity of Streptococcus mutans BHT were studied by transmission electron microscopy. Both control cells and cells exposed to NaSCN possessed thick outer cell walls and densely staining inner cell walls juxtaposed to the plasma membranes. In the presence of NaSCN, however, the S. mutans BHT nucleoid was coagulated into thick electron-dense filaments. Exposure of S. mutans BHT to 150 μg of hen egg white lysozyme per ml resulted in the progressive destruction of both the cell walls and the plasma membranes. The enzyme appeared to affect the region of the cell wall septum, and exposure to 150 μg of hen egg white lysozyme per ml for as short a time as 10 min resulted in visible morphological cell wall alterations. At 30 min, ultrastructural observations revealed that the majority of the cells were in the process of expelling a portion of their cytoplasmic contents from the septal and other regions of the cells at the time of fixation. After 3 h of incubation in the presence of this high lysozyme concentration, gelled protoplasmic masses, which were free from the cells, were evident. In addition, extensive damage to the outer and inner cell walls and to the plasma membranes was apparent, although the cells maintained their shape. On some areas of the cell surface, the outer cell wall and plasma membrane were completely absent, whereas at other locations the outer cell wall was either split away from the inner cell wall and plasma membrane or distended from an area free of inner cell wall and plasma membrane. Upon addition of NaSCN to the hen egg white lysozyme-treated cells, both the gelled protoplasmic masses and the damaged cells exhibited an exploded appearance and existed as membrane ghosts, cell wall fragments, or dense aggregates of cytoplasmic components. The effects of a low lysozyme concentration (22.5 μg/ml) on S. mutans morphology were less pronounced at short incubation times (i.e., 10 and 30 min) than those that were observed with a high enzyme concentration; however, breaks in the cell walls and dissolution of the plasma membranes with resulting cell lysis were visible after a prolonged (3-h) incubation and after subsequent addition of NaSCN.     

Protoplast formation during spore germination inStreptomyces

Germ tubes from spores ofStreptomyces were very sensitive to lysozyme attack. A good yield of stable protoplasts was obtained 30 min after addition of the enzyme, making the growth of the microorganism in a high-glycine-content medium unnecessary. Physiologically unaltered, stable protoplasts, which are formed from cells in the same stage of their developmental cycle, may be obtained in the presence of lysozyme. After protoplast release, abundant membranous structures were observed inside the empty walls.     

Morphological Phenomena Associated with Penicillinase Induction and Secretion in Bacillus licheniformis

Cells of uninduced Bacillus licheniformis (strain 749) in mid-logarithmic phase have no extensive intracytoplasmic membrane. After induction with cephalosporin C, characteristic organelles that contain tubules and vesicles with single-layered membranes and no visible internal substructure can be seen in thin sections in the periplasm. A magnoconstitutive penicillinase producer (749/C) contains similar structures. It is suggested that they represent a penicillinase secretory apparatus. In the first 15 min after induction, negatively stained preparations of induced 749 show large intracellular vesicles without individual contact with the cell surface. Negatively stained 749/C and fully induced 749 contain invaginations comparable to the structures seen in thin sections. When protoplasts of induced 749 and of 749/C are prepared, vesicles and tubules similar to those seen in thin sections of whole cells are liberated from the cell. Growing protoplasts of induced 749 show massive convolutions of the peripheral membrane, multiple layers of membrane, and characteristic long, slender tubules extending from the protoplast surface. These phenomena are not observed in uninduced 749 except for the production of a relatively small number of tubules. In 749/C, there were fewer convolutions than in induced 749, although tubule production was similar. Multiple layers of membrane were not observed in 749/C. The relation of the penicillinase secretory structures to mesosomes and to secretory structures of other organisms is discussed.     

Lipoteichoic acid is a major component of the Bacillus subtilis periplasm

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism Bacillus subtilis for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending ~30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the B. subtilis periplasm.     

Trafficking of the plant potassium inward rectifier KAT1 in guard cell protoplasts of Vicia faba

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 fused to the green fluorescent protein (GFP). Confocal images and whole-cell patch-clamp measurements confirmed the incorporation of active KAT1 channels into the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier current density of the plasma membrane was much larger in transfected protoplasts than in wild-type (wt) protoplasts. This shows a coupling between K+ channel synthesis and incorporation of the channel into the plasma membrane. Pressure-driven increase and decrease in surface area led to the incorporation and removal of vesicular membrane carrying active Kin+ rectifier in wt and transfected protoplasts. These vesicular membranes revealed a higher channel density than the plasma membrane, suggesting that Kin+ rectifier remains in clusters during trafficking to and from the plasma membrane. The observed results can be explained by a model illustrating that vesicles of a pre-plasma membrane pool carry K+ channels preferentially in clusters during constitutive and pressure-driven exo- and endocytosis.     

激光诱导金盏菊原生质体融合方法初探

本文简述运用激光微束诱导金盏菊(Calendula Officinali L.)叶肉细胞原生质体融合的方法和初步结果,并就激光诱导植物原生质体融合的条件进行初步讨论。     

一种光学显微镜下观察原生质体的染色方法

Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｌａｃｔｏｆｅｒｍｅｎｔｕｍ菌液经溶菌酶处理后分别与４种微生物染色液混合,在光学显微镜下比较观察原生质体形态的效果。结果表明;染色样品中的原生质体比未经染色的形态清晰易观察,而且显微照相效果好;其中使用草酸铵结晶紫和复红染色液的效果更佳。该方法程序简单、操作方便、效果明显,还适用于悬滴法观察菌体形态和细菌运动方式。