Stability Of Protoplasts Research Articles

Peptidase localization in Lactobacillus casei 5 Mn373 by stable protoplasts formation

Pure subcellular fractions were obtained from a Lactobacillus casei strain, selected among numerous isolates from Grana cheese on the basis of their peptidolytic activity. Several lytic conditions for the attainement of stable protoplasts were checked and three different cellular markers were employed to assess subcellular fraction purity. The maximum yeld and the best stability of protoplasts, as followed by Scanning Electron Microscopy, were obtained using 25mM Hepes pH 7, 15 mM MgCl2 and 20 U mutanolysin/mL, without the addition of osmotic stabilizers. Peptidolytic activities determined on subcellular fractions against Leu, Arg, Pro and Arg-Pro p-nitroanilides, had a cytoplasmic location. Comparative tests performed on either intact or broken cells by polyclonal antibodies against a general aminopeptidase and a X-prolyl dipeptidyl aminopeptidase confirmed this result. On the basis of such enzymatic activities, determined either on broken or intact cells, it is suggested that chromogenic substrates must be transported inside the cells.

Effect of Pencycuron on the Osmotic Stability of Protoplasts of <i>Rhizoctonia solani</i>

Pencycuron shows a specific inhibitory activity on the mycelial growth of Rhizoctonia solani. Even in the same anastomosis groups (AGs), e.g. AG4, there are both isolates sensitive and less sensitive (inherently resistant) to pencycuron. The regeneration of colonies from protoplasts of R-C (pencycuron-sensitive isolate in AG4) significantly decreased by the osmotic shock in the presence of pencycuron, while such effect was cancelled by washing off the chemical prior to the osmotic shock. However, in Rh-131 (a less sensitive isolate in AG4), the application of the chemical appeared to stimulate the regeneration from protoplasts. Further the measurement of optical density of protoplasts suspensions was performed to elucidate the effect of pencycuron on the osmotic stability of protoplasts in a short period. The optical density of suspensions of R-C protoplasts rapidly declined in a short period of incubation by the osmotic shock in the presence of pencycuron, but the effect on Rh-131 protoplasts was not statistically significant regardless of the presence or the absence of the chemical. These results suggest that the cell membrane of the pencycuron-sensitive isolate (R-C) is specifically affected by pencycuron.

Physiological studies with the fungus Entomophaga aulicae during morphogenesis in three different media under fermentation conditions

The effects of three different media on amino acid uptake and production and glucose and oxygen utilization during protoplast growth and hyphal body production by the fungus Entomophaga aulicae under fermentation conditions were studied. The three media consisted of a basal medium plus either (i) 2.8% fetal calf serum, (ii) 0.8% tryptic soy broth plus 0.4% bovine serum albumin, or (iii) 0.8% tryptic soy broth plus 0.4% calcium caseinate. The protoplasts grew most rapidly (initial peaks on days 2 and 3) and hyphal bodies were detected first (day 3) in the media containing albumin and caseinate. The day 9 hyphal body yields were 3.1 × 107, 7.5 × 108, and 3.1 × 109/10 L in media containing the serum, albumin, and caseinate, respectively. Growth in the albumin and caseinate media also gave the first detectable glucose utilization (days 2 and 3, respectively) and this rapidly increased to 94.9 and 90.6% utilization, respectively, on day 4. Oxygen and glucose utilization were closely related. During protoplast growth prior to hyphal body production, the only common pattern detected was the initial utilization of glutamine in serum- and caseinate-containing media. During the initial period of hyphal body production, cysteic acid, threonine, serine, asparagine, leucine, tyrosine, phenylalanine, and arginine were first utilized and glycine, alanine, and ammonia were first produced in the albumin and caseinate media. At this time (days 3–5), glutamine, proline, cystine, and tryptophan were first utilized and valine and histidine were produced in the albumin medium, and methionine was first utilized and cystathionine produced in the caseinate medium. Four main patterns of overall amino acid utilization and production were identified. The delay in major protoplast growth in the basal medium plus fetal calf serum is felt to result from inhibition by free fatty acids in the serum. Protein utilization was not detected and its main function is considered to be enhancement of protoplast stability against fermentation shear forces.Key words: Entomophaga aulicae, physiology, fermentation growth, protoplasts, hyphal bodies.

Physical parameters affecting electrofusion of yeasts: ζ-potential on the surface of yeast protoplasts and osmotic pressure of the solution

Yeast electrofusion is a relatively new technique which appears to offer some advantages over conventional chemical procedures. However, various parameters were found to affect fusion efficiency. The effects of both the osmotic pressure of the solution and the ζ-potential of yeast protoplasts on electrofusion were studied in detail in this study. The optimal osmotic pressure of the sorbitol solution for stability of protoplasts was about 0.9 Osm kg −1 because over 70% of protoplasts remained intact when an intensive pulse of 7 kV cm −1 of 100 μs duration was applied. On the other hand, the optimal pressure of sorbitol for the highest fusion efficiency was 0.6-0.7 Osm kg −1 when several pulses (5.5 kV cm −1, 25 μs, 1–10 charges) were applied. In the presence of divalent cations (especially Ca 2+ and Mg 2+) at a conductivity of 40–50 μs cm −1, a remarkable increase of fusion efficiency and frequent appearance of multimembrane-fused protoplasts were observed. Under the conditions established, fusion efficiency by electrofusion was 1:50-1:100 and approximately 10,000 times higher than that achieved by chemical methods such as the polyethylene glycol procedure. The acceleration of cell fusion by both divalent cations and change of conductivity was considered due to neutralization of anion residues of suger-protein complexes on the surface of the protoplasts because the ζ-potential of protoplasts was around 0 mV under the conditions used.

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.

Stimulatory effect of manganese and glycine derivatives on production and release of β-galactosidase by alkalophilic Bacillus no. C-125

Mn2+, glycine, glycine derivatives, amino acid analogues, and D-amino acids were investigated for their role in the production and release of β-galactosidase by alkalophilic Bacillus no. C-125. The simultaneous addition of Mn2+ and either glycine methyl ester, glycine ethyl ester, or glycinamide markedly increased the production and release of the enzyme. The addition of more than 100 μM Mn2+ was indispensable for increased enzyme production and release even in a nutrient-rich medium. When both Mn2+ and 2.0% glycine ethyl ester were added simultaneously, the maximum cell mass increased 3.6-fold, the maximum total activity, 19.8-fold, and 65% of the total activity was found extracellularly. Protease production was stimulated by Mn2+ but repressed by glycine ethyl ester. The simultaneous addition of Mn2+ and 2.0% glycine ethyl ester repressed protease activity strongly. The extracellular protein: nucleic acid ratio was higher in the presence of both Mn2+ and glycine ethyl ester, indicating that this method is advantageous for practical application to extracellular accumulation of intracellular proteins. The simultaneous addition of Mn2+ and glycine ethyl ester to the medium decreased the stability of protoplasts, especially at alkaline pH.

Choline stimulates synthesis of extracellular proteins in Trichoderma reesei QM 9414

A correlation between intracellular phospholipid levels and the rate of exoprotein synthesis was investigated in the filamentous fungus Trichoderma reesei QM 9414 during growth on cellulose. When the incubation temperature was varied between 20 and 37°C, the exoprotein synthesis rate correlated with the total cellular amount of phospholipids, but not with an individual phospholipid component. In contrast, when phospholipid bases were added exogenuously, a significant stimulation of exoprotein synthesis was observed with choline. The addition of the surfactant Tween 80—which also stimulates exoprotein secretion in T. reesei QM 9414—prevented choline stimulation. Optimal stimulation occurred around 20 mM choline. Choline stimulated exoprotein synthesis in general as shown by increased activities of several extracellular enzymes. Mycelia required preincubation for at least 20 h before stimulation of choline could be seen. Mycelia pregrown in the absence or presence of choline were equally effective in formation of β-glucosidase upon induction with methyl-β-d-glucoside, and the addition of choline to the induction medium had no effect. Choline did not alter the osmotic stability of protoplasts of T. reesei. Electron microscopic examinations and analysis of chemical constituents as well as marker enzymes from choline grown and non-choline grown mycelia revealed higher contents of mitochondria and endoplasmic reticula in choline grown mycelia. The possibility is discussed that choline may stimulate exoprotein synthesis by increasing the cellular content of endoplasmic reticula.

Transformation of Streptomyces erythraeus.

Streptomyces erythraeus strains were transformed efficiently with six different plasmid DNA vectors by a protocol that uses 0.2 mM Ca2+, 5 mM Mg2+ and 10% DMSO to enhance the stability of protoplasts to storage at -80 degrees C and their transformability at 30 degrees C. The primary thiostrepton-resistant (Thior) transformants for most vectors were unstable even when grown selectively. This instability did not appear to be due to incompatibility with indigenous S. erythraeus plasmids. Conversely, stable transformation was not the result of plasmid or host mutations. Transformation instability or plasmid copy number thus prevented successful shotgun-cloning of DNA that complemented the eryD mutation, which blocks the biosynthesis of erythromycin A, because only plasmid DNA without an insert could be isolated from a Thior Ery+ clone.

Maintenance of plant cell membrane integrity and function by the immobilisation of protoplasts in alginate matrices

Isolated protoplasts (Vicia faba) suspended in a mannitol solution over a time period of about 10 d undergo a process of aging characterised by increased ethane formation, decrease in oxygen evolution and CO2 uptake, pigment degradation and a change in certain enzyme activities (ribulose-1,5-bisphosphate carboxylase and protease). The technique of immobilisation of protoplasts in a cross-linked alginate matrix offers a way to delay the appearance of the indicators mentioned. Various possible mechanisms for the increased stability of protoplasts are discussed.

Seasonal and physiological aspects of the isolation of tobacco protoplasts

The viability of tobacco (N. tabacum– L. cv. Xanthi‐nc) protoplasts isolated during the winter months is affected by membrane calcium, total calcium, plant age and supplementary lighting. Feeding calcium nitrate and calcium chloride helps to increase protoplast stability. Feeding is important because it increases membrane calcium, but it also has other beneficial effects which may be connected with increased nitrate and chloride (anion) uptake and cation/anion interaction. Whatever optimal feeding regime is chosen, it must be used in conjunction with the correct harvesting practice, which depends on whether or not supplementary light is given. Plants grown without supplementary light are best harvested between 8–9 weeks. If supplementary light is given, older plants (with and without supplementary calcium feeding) of approx. ten weeks are better for protoplast isolation. Plants grown under supplementary light have low leaf shape index (LSI) values (i.e. wider leaves) compared to plants in low light.Spring and summer calcium feeding treatments had no significant effect on increasing total or membrane calcium. Likewise, there was no significant correlation between total/membrane calcium and percentage protoplast viability from days 0–5; and protoplasts isolated from plants grown in shade and fed with calcium salts, were no more stable than protoplasts from control plants. Irrespective of the feed treatments given, spring and summer plants older than about fifty days and grown under shaded and normal light conditions, produced poor protoplasts despite the fact that there was more calcium present in the membranes compared to protoplasts of younger plants. This decrease in percentage protoplast viability appears to be associated with changes in leaf shape. As plants age from 49–63 days, the leaf shape index (LSI) values increase (i.e. leaves are becoming narrower) and % protoplast viability decreases.

Lactoperoxidase-125I localization of salt-extractable alkaline phosphatase on the cytoplasmic membrane of Bacillus licheniformis.

Previous histochemical and biochemical localizations of alkaline phosphatase in Bacillus licheniformis MC14 have shown that the membrane-associated form of the enzyme is located on the inner surface of the cytoplasmic membrane, and soluble forms are located in the periplasmic space and in the growth medium. The distribution of salt-extractable alkaline phosphatase on the surfaces of the cytoplasmic membrane of B. licheniformis MC14 was determined by using lactoperoxidase-125I labeling techniques. Cells harvested during rapid alkaline phosphatase production were converted to protoplasts or lysed protoplasts and labeled. Analysis of the data obtained indicated that 30% of the salt-extractable, membrane-associated alkaline phosphatase was located on the outer surface of the cytoplasmic membrane, whereas 70% of the membrane-associated enzyme was localized on the inner surface. Controls for protoplast integrity (release of tritiated thymidine or examination of cytoplasmic proteins for label content) indicated excellent protoplast stability. Controls indicated that chemical labeling was not a factor in the apparent distribution of alkaline phosphatase on the membrane. These results support the previously reported histochemical localization of alkaline phosphatase on the membrane inner surface. The presence of alkaline phosphatase on the membrane outer surface is reasonable, considering the soluble forms of the enzyme found in the periplasmic region and in the culture medium.

Factors that Influence the Yield, Stability in Culture and Cell Wall Regeneration of Spinach Mesophyll Protoplasts

A study has been made of factors that influence the yield, stability in culture, and ability to regenerate cell walls of isolated spinach (Spinacia olevacea L.) mesophyll protoplasts. The presence of 7 mM CaZ + or 7 mM Mg2+ in the isolation medium, which also included 1.5 % (w/v) Driselase, 0.25 % (w/v) pectinase and 0.8 M sorbitol, increased the yield and stability in culture of the protoplasts in a liquid nutrient medium. This latter medium has been used previously in the culture of spinach leaf discs, and does not support the division of spinach mesophyll cells. Protoplasts isolated in the presence of CaZ+ showed a greater capacity for cell wall regeneration compared with protoplasts isolated in the absence of ions or in the presence of Mg2+. Though darkness during culture improved the initial protoplast stability, it inhibited wall regeneration. The initial stability of protoplasts appeared to be dependent on plasma membrane stability, but after a few days in culture the most effective treatments were those which stimulated cell wall regeneration. In many cases, associated with cell wall regeneration, there was a budding off of small vesicles which sometimes contained chloroplasts. Protoplasts isolated in the presence of Ca2+ and incubated in low light had a 50% survival rate after 8 days culture and most had regenerated cell walls. Such culture of spinach protoplasts has not been shown previously and should be useful in developmental studies of spinach chloroplasts.

Protoplasts from Oat Primary Leaves as Tools for Experiments on the Compartmentation in Lipid and Flavonoid Metabolism

Abstract Mesophyll protoplasts were isolated from defined stages of developing primary leaves of Avena sativa by a simple procedure in high and reproducible yield of about 50%. Rates of photosynthesis were measured by different techniques and turned out to be lower by a factor of 2 - 5 with protoplasts as compared to parent tissues, whereas dark respiration rates were about the same. Stability of protoplasts during storage in suspension medium at room temperature was checked by various methods. In contrast to protoplast numbers and accessibility of soluble enzymes, which suggested reasonable stability, photosynthetic oxygen evolution seems to be a more sensitive criterion. This rate dropped to about 50% of its original value during storage for 3 h. The protoplasts from differ­ent developmental stages did not show significant differences with respect to these various param­eters. Mechanical rupture or chemical destruction of the plasma membrane by filipin resulted in release of chloroplasts, which according to activity profiles of soluble marker enzymes after gra­dient centrifugation were intact. Centrifugation in a vertical rotor separated mitochondria from chloroplasts. Irrespective of the developmental stage of the parent tissue each protoplast contained about 200 chloroplasts. In conjunction with chlorophyll determinations it was calculated that about 2 × 109 chloroplasts account for 1 mg of chlorophyll. Lipid mixtures from leaves, protoplasts and chloroplasts were compared. They did not contain compounds which usually indicate enzy­matic alteration due to destruction of cellular compartmentation. Flavonoid profiles obtained from mesophyll protoplasts deviate significantly from epidermal patterns and prove the occurrence of flavonoids in photosynthetically active m esophyll cells.

Effects of Physical Factors on Protoplasts of Entomophthora Egressa

The effects of various physical factors on the growth of protoplasts of Entomophthora egressa were studied. In order to obtain growth in cultures shaken at 50 and 100 rpm using Grace's liquid medium, a fetal-calfserum (FCS) supplement of 28 ml per liter (2.7%) was required. Concentrations of FCS up to 50 ml per liter failed to support growth at 150 rpm. Analysis of Grace's medium indicated that it had an osmolality of 350 mOsM. This osmolality, except where designated, was maintained at the start of experiments in all media. The optimum sucrose concentration for protoplast stability (retention of spindle shape) was 350 mM (403 mOsM); for centrifugation, a 330-mM-sucrose-10-mM-MES buffer (pH 6.2) was optimum as based upon protoplast retention of shape and subsequent viability. The optimum temperature for growth (protoplast yield) was between 17 and 21 C. Of the three buffers tested (MES, MOPS and TES), MES at 10 mM was found to provide the best balance between buffering capacity and toxicity. The protoplasts grew over the pH range 5.2-8.2. The optimum pH for growth was 6.2.

Polyamines, ribonuclease and the improvement of oat leaf protoplasts

L-arginine and L-lysine delay spontaneous lysis of oat leaf protoplasts, preserve uniformity of chloroplast distribution in protoplasts and prevent protoplast aggregation and adhesion to the substratum. Osmotic shock, exogenous RNAase and cell-free centrifugal supernatant fractions of mechanically-lysed protoplasts all induce protoplast lysis; arginine and lysine protect against these stresses as well. Arginine and lysine also produce effects at the metabolic level that may be related to their morphological stabilization of protoplasts. For example, the RNAase activity of freshly extracted oat leaf protoplasts is low, but rises progressively as the protoplasts are incubated in vitro; the same rise occurs during incubation of excised leaves prior to protoplast extraction. The RNAase increase in both systems can be prevented by 10–50 mM L-arginine or L-lysine. Concomitantly, these compounds support higher levels of incorporation of [ 3H] uridine into RNA. All the above effects can also be produced by polyamines (putrescine, cadaverine and spermidine) which are metabolically related to lysine and arginine; spermine is less effective at the concentrations tested.

Formation and reversion of Streptomycete protoplasts: cultural condition and morphological study.

SUMMARY: Cultural conditions for forming and stabilizing protoplasts of Streptomyces griseus and S. venezuelae were studied by reference to the number of protoplasts formed, leakage from protoplasts and reversion rate. Effective formation and stabilization of the protoplasts was accomplished by using a hypertonic medium containing 10 mm-MgCl2 and 25 mm-CaCl2. Electron microscopy showed that the protoplasts of S. griseus, when prepared in the above medium, formed vesicles on the cytoplasmic membrane or out of the protoplasts, but when prepared in a medium with 3 mm-MgCl2 and 3 mm-CaCl2 they provided few such vesicles. The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 or 50 mm-MgCl2, 50 or 20 mm-CaCl2, 0·44 or 0·22 mm-phosphate and 0·01 % Casamino acids. Combinations of appropriate concentrations of MgCl2 and CaCl2 were significantly effective in the reversion as well as in the stability of protoplasts. The concentration of phosphate should be adjusted to 0·44 or 0·22 mm. Casamino acids enhanced both growth and reversion rates. The reversion of protoplasts of Streptomyces griseus was followed by using phase-contrast microscopy. The protoplasts incubated on medium R1 enlarged to about 40 µm and then generated filamentous hyphae from the periphery.

Membranes of Bacillus stearothermophilus: factors affecting protoplast stability and thermostability of alkaline phosphatase and reduced nicotinamide adenine dinucleotide oxidase.

Protoplasts of Bacillus stearothermophilus NCA 1503-4R are resistant to osmotic rupture and are not sensitive to mechanical manipulation. Protoplast stability is maintained by divalent cations. The thermostability of protoplasts is enhanced when the cells are grown at elevated temperatures. The membrane content of the cell and the protein-to-lipid ratio of the membrane increases as the growth temperature is increased. The membrane-bound nicotinamide adenine dinucleotide (reduced form) oxidase system from cultures grown at 70 C was more thermostable than the same enzyme system from cultures grown at 55 C. Alkaline phosphatase was resistant to thermal inactivation in the intact protoplast. The extent of this protection is dependent on protoplast stability.

Osmotic behavior of bacterial protoplasts: temperature effects.

Among protoplasts released from cells of Bacillus megaterium grown at 20, 30, or 37 C, osmotic swelling in NaCl solution at a given external osmotic pressure was greatest for protoplasts from cells grown at 20 C and least for protoplasts from cells grown at 37 C. Protoplasts from cells grown at lower temperaturs were also less stable to osmotic shock and lysed at higher external osmotic pressures than did protoplasts from cells grown at higher temperatures. But for cells grown at any one temperature, osmotic stabilization was itself temperature dependent so that the higher the ambient incubation temperature, the higher the osmotic pressure needed to prevent lysis of a given fraction of the input protoplast population. However, comparison of the osmotic stability of protoplasts from cells grown at different temperatures at various ambient incubation temperatures revealed that, except at 5 C where no differences were discerned, protoplasts from cells grown at lower temperatures still lysed at higher osmotic pressures than did those from cells grown at higher temperatures. The apparent internal osmolality (28 to 31 atm) did not vary significantly among whole cells from the three growth temperatures. Therefore, the observed differences in osmotic behavior could not be attributed to changes in internal osmotic pressure. Rather, it seemed likely that the differences were due to changes in membrane properties.

Procedure for the isolation of mutants of Bacillus subtilis with defective cytoplasmic membranes.

A procedure has been devised to isolate mutants of Bacillus subtilis with structurally defective membranes. The procedure used to screen for the mutants involved comparison of the stability of protoplasts of the mutant with those of the wild type in a medium of sufficient osmotic strength to stabilize wild-type protoplasts. Mutagenized cells were grown as clones on agar plates, and then replicated onto plates containing 0.5 m lactose, which is sufficient to stabilize wild-type protoplasts. The colonies on the lactose-containing plates were then treated with lysozyme to convert the cells to protoplasts. Colonies of wild-type protoplasts remained opaque; however, colonies of mutant protoplasts lysed and became clear. Twenty-nine osmotically fragile mutants were isolated in this manner; the membranes of several mutants were found to contain alterations in the composition of their proteins or lipids.

Variations in the membranes of Streptococcus faecalis related to different cultural conditions.

1. Membrane ultrastructure, phospholipid composition and protoplast stability and shape were studied in cells of Streptococcus faecalis grown with or without glucose and harvested at the stationary phase. 2. When the bacteria were grown with glucose and the pH was allowed to drop to acidic values, the cells exhibited a consistently well defined triple layered cytoplasmic membrane and yielded, upon treatment with lysozyme, stable protoplasts. 3. When the pH was kept at 7.2 by continuous neutralization with KOH or, to a greater extent, when the culture was carried out without glucose, the bacteria exhibited unclear membrane profiles and yielded unstable protoplasts. 4. The presence of positively charged phospholipids was dependent on the addition of glucose, its proportion being higher at acidic pH values.