Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

Factors Influencing Protoplast Viability of Suspension-Cultured Rice Cells during Isolation Process

Callus cells of rice (Oryza sativa L.) that were actively dividing in suspension culture had lost the ability to divide during the isolation process of protoplasts. Factors influencing the protoplast viability were examined using highly purified preparations of cellulase C₁, xylanase, and pectin lyase, which were essential enzymes for the isolation of protoplasts from the rice cells. The treatment of the cells with xylanase and pectin lyase, both of which are macerating enzymes, caused cellular damage. Xylanase treatment was more detrimental to the cells. Osmotic stress, cell wall fragments solubilized by xylanase, and disassembly of cortical microtubules were not the primary factors which damaged the rice cells and protoplasts. The addition of AgNO₃, an inhibitor of ethylene action, to the protoplast isolation medium increased the number of colonies formed from the cultured protoplasts, although the yield of protoplasts was reduced by the addition. Superoxide radical (O₂-) was generated from the cells treated with xylanase or pectin lyase. The addition of superoxide dismutase and catalase to the protoplast isolation medium resulted in a marked improvement in protoplast viability especially when the non-additive control protoplasts formed colonies with a low frequency. The addition of glutathione peroxidase and phospholipase A₂, which have been known to reduce and detoxify lipid hydroperoxides in membranes, to the protoplast culture medium significantly increased the frequency of colony formation. These results suggested that some of the damage to rice protoplasts may be caused by oxygen toxicity.     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.     

Isolation of Staphylococcus aureus protoplasts using lysoamidase

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.     

Mutant strains ofStreptomyces cinnamonensis protoplasts

Mycelium of Streptomyces cinnamonensis mutant strains cultivated in a synthetic medium with glycine produced protoplasts after lysis of cell walls with lysozyme. The protoplast yield was up to 95%. The protoplasts could revert and mycelial forms were thus regenerated. In a sucrose-containing medium the protoplasts stored at 4 degrees C were stable for 2 d.     

Isolation and regeneration of Micromonospora olivoasterospora protoplasts

The conditions for preparation and regeneration of the protoplasts of M. olivoasterospora were developed. It was found that effective formation of the protoplasts required preliminary cultivation of M. olivoasterospora in the medium containing glycine in a concentration inhibiting its growth at least by 60-80 per cent. The strains studied markedly differed in their sensitivity to glycine and were highly sensitive to it. The efficacy of the protoplast formation depended on the culture age and increased with the use of the lytic enzyme 3 of Cytophaga dissolvens. The possibility and advisability of the use of prolonged lysis of the Micromonospora cell walls were shown. A rich organic medium was used for regeneration of the protoplasts.     

Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.     

Optimal Cultural and Physiological Conditions for Handling Streptomyces rimosus Protoplasts

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no alpha, epsilon-ll-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

Characteristic features of protoplast formation and regeneration in Fusidium coccineum

The methods for preparation and regeneration of protoplasts were tested with respect to the strains of F. coccineum markedly differing in their capacity for antibiotic production, sporulation and the growth rate. It was found that the substrate used for the culture growth had a significant effect on the cell wall and sensitivity of the mycelium to lytic enzymes. An enzyme from Hellix pomatia and its combination with lysozyme were used for lysing the culture. The cytological investigation of the time course of the culture lysis revealed a stage-by-stage pattern of protoplast formation by means of fragmentation of the hyphal contents till a ball was formed. Two to 4 protoplasts differing in their size and structure were formed within a cell. The pH value and osmotically stabilizing component had some effect on the rate of protoplast formation. Highly productive strains were characterized by formation of protoplasts heterogenous in their size and by decreased frequency of regenerations. The enzyme-free protoplasts preserved their viability and capacity for germination in osmotically stabilizing media for 72-96 hours of storage at 4 degrees C. On solid media the regeneration frequency reached 38 per cent. The regenerated cells formed colonies morphologically similar to those of the intact culture.     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Sporulation of protoplasts

Vegetative cells ofBacillus megaterium formed protoplasts in a sucrose-stabilized medium under the influence of lysozyme. The protoplasts sporulated during subsequent incubation. The morphological properties, germination, resistance to u. v. irradiation and thermo-resistance of protoplast spores were the same as with normal cells. It thus appears that in the later sporulation stages the spore formation occurs, without participation of the sporangium cell wall.     

Freezing behavior of free protoplasts of winter rye

Free protoplasts prepared from the epicotyls of nonhardened rye seedlings were subjected to fast and slow freezing on a microscope-adapted thermoelectric stage. During rapid freezing to ?12 °C, ice formation occurred inside the protoplasts causing lethal disruption of cell and membrane organization. Under slow freezing to ?12 °C, ice formation occurred outside the protoplast with accompanying dehydration and contraction of the protoplast. Complete rehydration and recovery of the protoplasts occurred upon thawing after slow freezing. Free protoplasts therefore afford a new system for the study of mechanisms of plant cell freezing injury and resistance free of the complications presented by a cell wall.     

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.     

Microbiology in the 1990s: reflections about open questions and trends

The optimal conditions for protoplast formation ofCandida apicola were by using an enzyme fromArthrobacter sp. in combination with 2-mercaptoethanol. The kinetic data support the two-layered structure model of cell wall for this yeast but the structure of the cell wall depended on the age of cells and culture conditions. To regenerate the protoplasts, the type of osmotic stabilizer was important: sorbitol gave 16 to 30% regeneration. Electron microscopy revealed the presence of vesicles in the sections of protoplasts and whole cells ofCandida apicola grown in production medium and producing glycolipids. In sections of whole cells, vesicle-like structures are located in the periplasmic space and in protoplasts they can either be attached to, or released from, the cell surface. These vesicles are thought to be involved in the transport of the surface-active glycolipids and in the protection of the cell against denaturing effects.     

Electron microscopy study of cell structures and their changes during growth and regeneration ofSchizosaccharomyces pombe protoplasts

The submicroscopic structure of growing and regeneratingSchizosaccharomyces pombe protoplasts cultivated in solid and liquid medium was studied by means of ultrathin sectioning. The protoplasts regenerate within 24 hours. Shortly before growth commences, rudiments of the new cell wall can be identified on the protoplast surface. Simultaneously, a large number of dictyosomes appears in the cytoplasm and decreases as synthesis of the new wall progresses. An increase occurs in the number of endoplasmic reticulum membranes some of which are arranged parallel with the cytoplasm membrane of the protoplast. Throughout the whole time of regeneration the protoplasts contain only one nucleus. The nucleo-cytoplasm ratio of growing and regenerating protoplasts is lower than in intact cells. The number of mitochondria falls at the outset of regeneration and does not rise again until towards the end.     

A simple system for plant cell microinjection and culture

Microinjection of plant protoplasts and cells has been recently reported, however a system that combines simplicity of design, harmless immobilization, high resolution visibility and ability to monitor individual target cells is lacking. This report describes a system which combines these features. It consists of a microinjection-microculture dish containing immobilized protoplasts and a simple chamber that maintains sterility and humidity during injection. Highly purified protoplast preparations are plated at low population density as a thin monolayer of widely separated cells embedded in agarose layered over a thicker (0.2 mm at center to 1 mm at edge) layer of agarose-solidified medium. This physical arrangement allows for rapid location, mapping and injection of the immobilized protoplasts and also their subsequent location for growth monitoring. The double layers of agarose provide adequate nutrition for culturing injected cells to the microcalli stage. In addition to protoplast injection, this system was also used to inject 3- to 4-day old nonspherical cells derived from protoplasts. Colony formation rates from injected protoplasts and cells with regenerated walls were equivalent to those of uninjected controls. Furthermore, tobacco protoplasts stored at 4°C in liquid medium for up to two weeks remained fully competent for plating and injection. These cold-stored protoplasts, when injected, formed colonies at rates similar to those from fresh preparations. The ability to store protoplasts without loss of viability considerably increases the ease and convenience of cell injection experiments.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of the other products that may also be suitable.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.